Journal: Gene Expression

Article Title: Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

doi:

Figure Lengend Snippet: Schematic of research design. (A) Plasmid of cloned human NeuroD1/GFP; (B) control plasmid, X/GFP. Paired sets of human fetal astroglial SV40 transformed (SVG) cell cultures were transfected with (A) or (B) using Fugene 6 reagent. After 24 h, paired cultures were analyzed and processed with fluorescence-activated cell sorting (FACS). GFP-expressing cells were sorted and collected to ∼98% purity. Total RNA was extracted from each set of enriched GFP-expressing cells for DNA microarray analysis. In parallel experiments, paired cultures were examined immunocytochemically at 24 and 72 h after transfection for expression of the neuronal protein β-tubulin III (TuJ-1).

Article Snippet: Cell Culture and Transfection Human fetal astroglial SV40 transformed cells (SVG p12, ATCC) were plated in 75-mm culture flasks (Costar, Corning Inc.) in modified Eagle’s MEM (DMEM, ATCC), 10% heat-inactivated FBS (Invitrogen), and penicillin (100 U/ml)/streptomycin (100 μg/ml).

Techniques: Plasmid Preparation, Clone Assay, Control, Transformation Assay, Transfection, Fluorescence, FACS, Expressing, Microarray

Journal: Gene Expression

Article Title: Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

doi:

Figure Lengend Snippet: (Panel 1) SVG cells were transfected with plasmid encoding hNeuroD1/GFP (upper two panels) and a control plasmid (lower two panels). After 24 h NeuroD1-immunoreactive cells were viewed under fluorescence microscopy. The upper panels show identical fields of an SVG cell culture containing two GFP+ cells coexpressing NeuroD1. The lower panels show cells transfected with the control plasmid. GFP+ cells are seen but they do not express NeuroD1. (Panel 2) A1, B1, C1, and D1 were from cultures transfected with control GFP vector; A2, B2, C2, and D2 were cultures transfected with NeuroD1/GFP. After 24 h in vitro, cultures were immunolabeled with antibodies to vimentin, nestin, and TuJ1 (scale bar = 50 μm in each frame). Row A: Cells expressing green fluorescence (GFP+). No antibodies were utilized. The fluorescence in the NeuroD1/GFP transfected cultures tends to be localized in the nucleus (A2) and in the control GFP transfected cells the fluorescence is more diffuse, involving the cytoplasm (A1). Row B: Vimentin is expressed in every cell regardless of transfection with NeuroD1/GFP (B2) or control GFP (B1). Row C: Nestin immunoreactivity is present in non-GFP-expressing cells in both sets of cultures. A double-labeled (GFP+/nestin+) cell is illustrated in the NeuroD1/GFP transfected cells (C2). In the control GFP transfected cultures (C1), it is rare to find double-labeled (GFP+/nestin+) cells despite the presence of nestin+ cells in non-GFP-expressing cells. Row D: The NeuroD1 transfected cultures (D2) exhibit double-labeled cells (TuJ1+/GFP+). Notice that many of the cells in the NeuroD1/GFP transfected cultures express TuJ1 even when they are not exhibiting green fluorescence. (Panel 3) Row G: Fluorescence-activated cell sorting (FACS) was performed at 24 h after transfection. An aliquot of cells was plated and viewed under phase (G1) and fluorescence microscopy (G2), revealing a highly enriched (95–98% pure) population of GFP+ cells. FACS-purified cells from paired sets of cultures were then processed for DNA microarray analysis in three separate experiments that form the basis for the transcription profiling.

Article Snippet: Cell Culture and Transfection Human fetal astroglial SV40 transformed cells (SVG p12, ATCC) were plated in 75-mm culture flasks (Costar, Corning Inc.) in modified Eagle’s MEM (DMEM, ATCC), 10% heat-inactivated FBS (Invitrogen), and penicillin (100 U/ml)/streptomycin (100 μg/ml).

Techniques: Transfection, Plasmid Preparation, Control, Fluorescence, Microscopy, Cell Culture, In Vitro, Immunolabeling, Expressing, Labeling, FACS, Purification, Microarray

Journal: Gene Expression

Article Title: Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

doi:

Figure Lengend Snippet: Typical data from FACS sort of SVG cells 24 h after transfection with NeuroD1/GFP. (A) Plot of side scatter (SSC) against forward scatter (FSC). (B) Cell counts plotted against intensity of fluorescence (FL1-H GFP).

Article Snippet: Cell Culture and Transfection Human fetal astroglial SV40 transformed cells (SVG p12, ATCC) were plated in 75-mm culture flasks (Costar, Corning Inc.) in modified Eagle’s MEM (DMEM, ATCC), 10% heat-inactivated FBS (Invitrogen), and penicillin (100 U/ml)/streptomycin (100 μg/ml).

Techniques: Transfection, Fluorescence

Journal: Gene Expression

Article Title: Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

doi:

Figure Lengend Snippet: FACS DATA OF SVGp12 CELLS AFTER 24-h TRANSFECTION WITH hNeuroD1/GFP AND CONTROL (pcDNA3.1/GFP) PLASMID CONSTRUCTS

Article Snippet: Cell Culture and Transfection Human fetal astroglial SV40 transformed cells (SVG p12, ATCC) were plated in 75-mm culture flasks (Costar, Corning Inc.) in modified Eagle’s MEM (DMEM, ATCC), 10% heat-inactivated FBS (Invitrogen), and penicillin (100 U/ml)/streptomycin (100 μg/ml).

Techniques: Transfection, Control, Plasmid Preparation

Journal: Gene Expression

Article Title: Transcriptional Profile of NeuroD1 Expression in a Human Fetal Astroglial Cell Line

doi:

Figure Lengend Snippet: (A) RT-PCR of NeuroD1 mRNA. RT-PCR was performed with human total RNA isolated from 1-day post-hNeuroD1-GFP and X/GFP transfected cells (not FACS sorted). Lane 3: 674 bp cDNA (with total RNA from X/GFP transfected cells). Lane 5: 674 bp cDNA (with total RNA from hNeuroD1-GFP transfected cells). Lanes 2 and 4 were negative controls without reverse transcriptase using RNA under the same conditions as in the next lane. This shows that we could not detect any endogenous NeuroD1 expression and transfection with our construct induces NeuroD1 transcription in this cell type. (B) RT-PCR of β-tubulin III was performed with human total RNA isolated from 3-day posttransfected and untransfected cells in the same tube and modified PCR conditions described in Materials and Methods. The products were separated with electrophoresis on 2% agarose gel. Lane 2: 838 bp for β-actin and 283 bp β-tubulin from untransfected cells. Lane 4: 838 bp for β-actin and 283 bp β-tubulin from hNeuroD1 transfected cells. Lanes 1 and 3 were negative controls without reverse transcriptase in it using RNA under the same conditions as in the next lane.

Article Snippet: Cell Culture and Transfection Human fetal astroglial SV40 transformed cells (SVG p12, ATCC) were plated in 75-mm culture flasks (Costar, Corning Inc.) in modified Eagle’s MEM (DMEM, ATCC), 10% heat-inactivated FBS (Invitrogen), and penicillin (100 U/ml)/streptomycin (100 μg/ml).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Reverse Transcription, Expressing, Construct, Modification, Electrophoresis, Agarose Gel Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Marker, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and generation of the Samd7−/− allele. (A) Northern blot analysis of mouse Samd7 in developing and adult retinas. Northern blot analysis of Samd7 transcripts was performed using mRNAs purified from retinas of mice between P1 and 4 wk of age. (Upper) A major band of ≈2.4 kb Samd7 mRNA was detected. (Lower) Ethidium bromide staining of RNAs. (B) Immunostaining of P6 WT mouse retinal sections using antibodies against Samd7 (red), Thrb2 (a cone precursor marker, green), and S-opsin (an S-cone marker, blue). Dotted lines indicate Thrb2-positive cone photoreceptor nuclei. The Samd7 signal is obviously weaker both in S-opsin–positive S-cones (arrows) and in S-opsin–negative M-cones (arrowheads) than in rods. (C) Diagram of the targeting vector and the Samd7−/− allele. Removal of exons 4–6 is predicted to result in a translational frame shift and a complete loss of Samd7 function. (D) RT-PCR analysis of the Samd7 transcript in the Samd7−/− retina. An approximately 500-bp fragment of Samd7 was amplified from cDNA prepared from the WT retina. No Samd7 transcript was detected in the Samd7−/− retina. β-Actin was used as a loading control. (E) Western blot analysis of the Samd7 protein in the Samd7−/− retina. An approximately 56-kDa Samd7 band was detected in the WT retina. No Samd7 band was detected in the Samd7−/− retina. β-Actin was used as a loading control.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Northern Blot, Purification, Staining, Immunostaining, Marker, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Immunohistochemical analysis of the Samd7−/− retina. (A) Retinal sections from 2-mo-old WT and Samd7−/− mice were immunostained with the anti–M-opsin antibody (a marker for cone outer segments, green) and PNA (a cone photoreceptor outer and inner segment marker, red). No obvious difference was observed between WT and Samd7−/− retinas. (B) The distribution of S-opsin (red) and rhodopsin (green) signals was normal in the Samd7+/− retina at 2 mo. (C) At P9, ectopic S-opsin signals in premature rod photoreceptor outer segments were observed in the Samd7−/− retina. (D) Retinal sections from dorsal and ventral regions of 2-mo-old WT control and Samd7−/− mice were immunostained using anti–S-opsin (red) and anti-rhodopsin (green) antibodies with DAPI (blue). There were fewer S-opsin–positive cones in the dorsal region than in the ventral region of the control retina. Ectopic S-opsin expression in rod outer segments was similar in the dorsal and ventral regions of the Samd7−/− retina. (E) Flat-mount immunostaining of dorsal and ventral regions of WT control and Samd7−/− retinas. M-opsin (green) and Rhodamine-PNA (red) were used for visualizing cone photoreceptor cells. The numbers of M-opsin and PNA double-positive cells decreased in the ventral region in both WT control and Samd7−/− retinas. (F) Retinal sections from WT and Samd7−/− mice were immunostained with anti-Pikachurin (a marker of photoreceptor synaptic clefts, red) and anti-Ctbp2 (a marker of synaptic ribbons, green) antibodies. No obvious difference was observed between WT and Samd7−/− retinas at 2 mo. (G) Immunofluorescent examination of WT and Samd7−/− retinal sections from 2-mo-old mice using anti-Calbindin (a marker for amacrine and horizontal cells, red), anti-S100b (a maker for Müller glia, green), anti-Chx10 (a marker for bipolar cells, red), and anti-Pax6 (a marker for amacrine and ganglion cells, green) antibodies showed no obvious difference between WT and Samd7−/− retinas. (H) Retinal sections from WT and Samd7−/− mice at 12M were stained with toluidine blue. The thickness of the ONL of WT and Samd7−/− retinas was measured. Average layer thickness in the WT retina was set to 100%. No significant change in ONL thickness was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; n.s., not significant; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Immunohistochemical staining, Marker, Expressing, Immunostaining, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. ERGs were recorded from WT (n = 5) and Samd7−/− (n = 5) mice at 12 wk. (A and B) Representative scotopic (A) and photopic (B) ERGs in WT and Samd7−/− mice elicited by white light. (C) The amplitude of the scotopic ERG a-wave as a function of the stimulus intensity (1.0 log cd⋅s⋅m−2) was significantly reduced in Samd7−/− mice. (D) The amplitude of the scotopic ERG b-wave as a function of the stimulus intensity (−1.4 log cd⋅s⋅m−2) decreased significantly in Samd7−/− mice. Error bars show the SD. **P < 0.03.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. (A) The a-wave amplitudes of scotopic ERGs from WT (n = 5) and Samd7−/− (n = 5) mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2) at 12 wk. The amplitude of the a-wave at +1.0 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (B) The b-wave amplitudes of scotopic ERG from WT and Samd7−/− mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2). The amplitudes of the b-wave at −2.6 and −1.4 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (C) The b-wave amplitudes of photopic ERG from WT and Samd7−/− mice elicited by four different white light stimuli (−0.8, −0.2, 0.4, 1.0 log cd⋅s⋅m−2). No obvious change in amplitude was observed between WT and Samd7−/− mice. Error bars show the SD.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Global change of retinal expression profile in the Samd7−/− retina. (A) Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and cone-enriched (red circle) and rod-enriched (yellow circle) genes. Microarray analysis was performed using mRNAs from the WT and Samd7−/− retinas at P12. One hundred sixty-three genes were up-regulated (signal log ratio greater than +1.0, blue circle), and 251 genes were down-regulated (signal log ratio less than −0.5, green circle) in the Samd7−/− retina compared with those in the WT retina. (B) Lists of the 33 genes that overlap between cone-enriched genes and up-regulated genes in the Samd7−/− retina (Left) and the 23 genes that overlap between rod-enriched genes and down-regulated genes in the Samd7−/− retina (Right). Cone- and rod-enriched genes (more than fourfold FPKM value) were identified using RNA-seq data from a previous study (22). (C and D) The expression levels of the selected genes were measured by qRT-PCR using mRNAs of WT and Samd7−/− retinas at P12. Up-regulation of nonrod genes (C) and down-regulation of rod genes (D) in the Samd7−/− retina by microarray analysis were confirmed. (E) Nonrod genes are ectopically expressed in the ONL of the Samd7−/− retina. In situ hybridization analysis of WT and Samd7−/− retinal sections at P12 was performed using probes for the up-regulated genes in the Samd7−/− retina: Cacna1h (a bipolar cell gene), Gngt2, Cnga3, and S-opsin (cone-specific genes). The vertical brackets indicate the extent of the ONL. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Error bars show the SD (n = 4). **P < 0.03. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Microarray, RNA Sequencing Assay, Quantitative RT-PCR, In Situ Hybridization

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 interacts with Phc proteins in PRC1 and a rod TF Nr2e3. (A) Yeast two-hybrid assay using full-length Samd7 as the bait. Proteins containing SAM domains with high similarity to Samd7 (Samd7/11, Phc1/2/3, and L3mbtl3) were identified as Samd7 interactor proteins. Growth on selective plates lacking histidine (−His) and adenine indicates a physical interaction between the bait and prey constructs. (B) Immunoprecipitation analysis of Samd7 and Phc2. A plasmid expressing FLAG-Samd7 or FLAG-Phc2 was transfected with a plasmid expressing GFP-Samd7 into HEK293 cells. FLAG-tagged proteins were immunoprecipitated using an anti-FLAG antibody. GFP-Samd7 was coimmunoprecipitated with FLAG-Samd7 or FLAG-Phc2. (C) Immunoprecipitation was performed using mouse retinal lysate from 2-mo-old mice using an anti-Phc2 antibody. Immunoprecipitated Samd7 with Phc2 was detected by Western blot analysis using the anti-Samd7 antibody. (D) A 3D structural model of the EH and ML surfaces of the Samd7 SAM domain. The putative 3D structure of the Samd7 SAM domain was obtained by MODELLAR (https://salilab.org/modeller/) using the Phc3 SAM domain as a template [Protein Data Bank (PDB) ID code 4PZN]. Two molecules of the Samd7 SAM domain (green and blue) are shown. Amino acids essential for interaction on the EH surface (L372) and the ML surface (L358/H363) are indicated in red. (E) Immunoprecipitation of FLAG-Samd7-WT with GFP-Samd7-WT or GFP-Samd7-LR (with the L372R mutation on EH surface of the SAM domain). Reduced interaction between FLAG-Samd7-WT or FLAG-Samd7-LR and GFP-Samd7-LR was observed compared with that between FLAG-Samd7-WT and GFP-Samd7-WT. (F) FSEC analysis of the Samd7 protein. Plasmids expressing GFP-fused WT Samd7 (GFP-Samd7-WT) and Samd7 with a mutation on the EH surface of the SAM domain (GFP-Samd7-L372R) were transfected into HEK293 cells, and the fluorescent signals in lysates were analyzed by FSEC. In GFP-Samd7-WT lysates, a putative monomer peak between the 44- and 158-kDa size markers and a broad peak likely corresponding to polymers larger than 669 kDa (arrowhead) were observed. Putative monomer and oligomer peaks but no obvious peaks at a higher molecular size were detected in GFP-Samd7-L372R lysates. (G) Yeast two-hybrid assay using Samd7 with mutations in the SAM domain ML (L358R/H363R, HR) and/or EH (L372R, LR) surfaces as baits. The interaction of Samd7 constructs with SAM domain proteins (Samd7/11, Phc1/2/3, L3mbtl3) was analyzed. Samd7 with mutations both on the ML and EH surfaces (L372R/L358R/H363R, LRHR) showed no interaction with any of the SAM domain proteins tested (Samd7/11, Phc1/2/3, L3mbtl3). (H) Immunoprecipitation analysis of Samd7 with rod photoreceptor transcription factors. A plasmid expressing FLAG-Samd7 was transfected with a plasmid expressing HA-Nr2e3 or HA-Nrl into HEK293 cells. FLAG-tagged Samd7 was immunoprecipitated using an anti-FLAG antibody. The interaction of Samd7 with Nr2e3 was observed, whereas Samd7 showed no substantial interaction with Nrl.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Y2H Assay, Construct, Immunoprecipitation, Plasmid Preparation, Expressing, Transfection, Western Blot, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Partial colocalization of Samd7 and Phc2 in rod photoreceptor nuclei. Retinal sections at P6, P12, and 1 mo (A and B) and at 2 mo (A and C) were immunostained with the anti-Samd7 (red) and anti-Phc2 (green) antibodies. Small points of Samd7 and Phc2 partially colocalized (arrowheads in A) surrounding heterochromatin regions of rod photoreceptor nuclei stained with DAPI (blue). Subnuclear localization of Phc2 was unchanged in the photoreceptor nuclei in the Samd7−/− retina compared with that in the control retina (C).

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Analysis of Samd7 function in transcriptional regulation. (A) Samd7 did not affect transcriptional activation of the Rhodopsin promoter by Crx and Nrl. A luciferase reporter construct driven by the Rhodopsin promoter was cotransfected with Crx and Nrl expression plasmids into HEK293 cells, and luciferase activities of cell lysates were measured at 48 h after transfection. Cotransfection of the Samd7 expression plasmid did not affect the transactivation of the Rhodopsin promoter by Crx and Nrl. (B) Localization of Samd7 in Polycomb bodies. Phc2-dependent colocalization of Samd7 with Ring1B in Polycomb bodies in HEK293 cells. A plasmid expressing GFP-Samd7-WT was transfected with a plasmid expressing FLAG-Phc2 into HEK293 cells. Cells were stained with anti-FLAG and anti-Ring1B antibodies. Puncta of GFP-Samd7 (green) colocalized with the Ring1B signals (blue, a marker for Polycomb bodies) were observed in Polycomb bodies (arrowheads) in a Phc2-dependent manner (red). (C) Localization of Samd7 in Polycomb bodies. Subcellular localization of various Samd7-mutant proteins in U2OS cells was analyzed. U2OS cells were transfected with FLAG-tagged Samd7-mutant protein expression plasmid and were immunostained with anti-FLAG and anti-Ring1B antibodies with DAPI (blue). Puncta of the FLAG signal (green) colocalized with Ring1B signals (red) in Polycomb bodies of cells transfected with Samd7-WT or Samd7-del2 expression plasmids (arrowheads). In contrast, FLAG signals did not colocalize with the Ring1B signal in Polycomb bodies of cells transfected with the Samd7-del1, -del3, or -L372R expression plasmid. In cells with a high-level expression of Samd7-WT, -del1, or -del2 proteins, Polycomb bodies are not formed, likely due to Ring1B depletion. Dotted lines indicate cell nuclei. (D) Schematic diagrams showing the structure of Samd7-mutant proteins. Two highly conserved homology domains (HD1 and HD2) in the Samd7 protein, based on amino acid residue homology among species from mouse to zebrafish, were identified. Samd7-del1, -del2, and -del3 constructs lack the HD1, HD2, and SAM domain, respectively. The Samd7-L372R construct has a point mutation in the SAM domain disrupting its oligomerization activity. (E) Indirect physical interaction between Samd7 and Ring1B through Phc2. The immunoprecipitation assay was performed using HEK293 cells transfected with FLAG-Samd7, Myc-Ring1B, and HA-Phc2 expression plasmids. Myc-Ring1B was coimmunoprecipitated with FLAG-Samd7 in a Phc2-dependent manner. (F) Partial colocalization of Samd7 with Suz12, H2A119ub, and H3K27me3 but not with H3K4me3 marks in U2OS cells. The plasmid expressing FLAG-Samd7-WT was transfected into U2OS cells and was immunostained with anti-Suz12 (a component of PRC2), anti-H2A119ub, anti-H3K27me3, and anti-H3K4me3 antibodies. Samd7 colocalized with Suz12, H3K27me3, and H2AK119ub in some of Polycomb bodies (arrowheads). In contrast, the active histone mark H3K4me3 did not colocalize with Samd7-positive puncta in cell nuclei.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Activation Assay, Luciferase, Construct, Expressing, Transfection, Cotransfection, Plasmid Preparation, Staining, Marker, Mutagenesis, Activity Assay, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 regulates H3K27me3 in the retina. (A and B) ChIP-seq profiles of Cnga3 (A) and Cacna1h (B) loci for H3K27me3 in developing rod photoreceptor cells at three different stages (P2, P10, and P28). H3K27me3 levels at Cnga3 and Cacna1h genes, which were up-regulated in the Samd7−/− retina, increased along with rod photoreceptor development. The ChIP-seq profiles were analyzed using previous ChIP-seq data (21). (C) A Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in the developing rods (red circle). Thirty-one genes overlapped between the up-regulated genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in developing rods. (D–F) ChIP-qPCR analysis of H3K27me3 (D) and H2AK119ub (E) on the selected up-regulated genes in Samd7−/− retinas. H3K27me3 levels of the selected genes in WT and Samd7−/− retinas were analyzed by ChIP-qPCR. The H3K27me3 levels of the selected up-regulated genes (Cacna1h, Cnga3, Gngt2, En2, S-opsin, and Rxrg) in WT and Samd7−/− retinas are indicated. No significant change in H3K27me3 levels was observed for the Hoxc13 and Gnat2 promoters. The H2AK119ub levels of the selected up-regulated genes in the Samd7−/− retina (Cacna1h, Cnga3, En2, and S-opsin) significantly decreased in the Samd7−/− retina. (F) ChIP with IgG was performed as a negative control. Error bars show the SD (n = 3). **P < 0.03, *P < 0.05. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: ChIP-sequencing, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: A proposed model of the Samd7 function in rod photoreceptor cells. (Left) In WT rod photoreceptor cells the Samd7-PRC1 complex induces chromatin condensation together with PRC2 and increases H3K27me3 marks on the S-opsin promoter. Increased H3K27me3 represses S-opsin expression in rod photoreceptor cells. (Right) In the Samd7−/− retina, Samd7 deletion causes a reduction of H3K27me3 and H2AK119ub on the S-opsin promoter, resulting in the ectopic expression of S-opsin in rod photoreceptor cells.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Primer sequences

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: In Situ

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A Representative IHC analysis of mammary tumors with antibodies against Vim. Vim positive tumor cells (Black arrows) and stromal cells (Yellow arrows) are indicated. B Microarray analysis of mammary tumors. GSEA enrichment plot for a signature for Tgfβ signaling activity. NES: Normalized Enrichment Score (NES), Nominal p value (p), and False Discovery Rate q-value (FDR q) were detected comparing p18 −/− ; Brca1 +/− tumors ( n = 10) to p18 −/− and p18 +/− tumors including nine p18 −/− and one p18 +/− tumors. C Representative IHC analysis of mammary tumors from a p18 −/− and p18 −/− ; Brca1 +/− mice. To quantify the Tgfβr2 positive tumor cells, the intensity of Tgfβr2 antibody-specific staining by IHC in tumor cells were categorized into -, 1 + , 2 + , and 3 + . The representative images in the boxed area for each category were shown. D Summary of mammary tumors in mice with Balb/c background. EMT + tumors are tumors that are positive for at least two EMT markers (decreased E-Cad, increased Vim, Fn1, or CD29), and two EMT-TFs (Twist, Snail, Slug, Foxc2 or p-Fra1) in >2% tumor cells. Tgfβr2 + tumors are tumors that are positive for Tgfβr2 with 2+ or 3+ intensity in >20% tumor cells. The asterisk (*) denotes a significance from p18 −/− ; Brca1 +/− and p18 −/− tumors by two-tailed Fisher’s exact test. E Representative immunofluorescent staining of mammary tumors from a p18 −/− and p18 −/− ; Brca1 +/− mice. Note that the majority of Tgfβr2 positive p18 −/− ; Brca1 +/− tumor cells were co-stained with Ck14 (blue arrows). Ck14 singly positive basal epithelial cells (white arrows) in the normal gland are indicated.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Microarray, Activity Assay, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A, B Tumors (T) from p18 −/− and p18 −/− ; Brca1 MGKO mice were analyzed by qRT-PCR (A) and western blot ( B ). Tumor-free mammary glands (TF) from the same mouse were used as controls. Data in ( A ) represent the mean ± SD of three tumors in each group. The asterisk (*) denotes a significance from tumors and tumor-free tissues of the same genotype by a two-tailed, unpaired T test. C Four representative tumors from p18 −/− and p18 −/− ; Brca1 MGKO mice were analyzed by western blot. D Representative immunostaining of mammary tumors from a p18 −/− and p18 −/− ; Brca1 MGKO mice. E p18 −/− and p18 −/− ; Brca1 MGKO mammary tumors were analyzed by IHC, and H-scores for Tgfβr2 and p-Smad2 were calculated. The results represent the mean ± SD of five individual tumors per group for Tgfβr2 and four individual tumors per group for p-Smad2. F p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were transplanted into MFPs of NSG mice. Four weeks later, recipient mice with tumors generated (>0.5 cm 3 in size) were analyzed. # five tumor-bearing mice displayed lung metastasis by H.E. analysis. G 3 ×10 4 p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were cultured to generate primary tumorspheres in 10 days. The number of spheres large than 50 μm was quantified from triplicate experiments. The results represent the mean ± SD of three individual tumors per group. Statistical significance in ( E ) and ( G ) was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Quantitative RT-PCR, Western Blot, Two Tailed Test, Immunostaining, Generated, Cell Culture

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A , B Representative p18 −/− and p18 −/− ; Brca1 MGKO mammary tumor cells were analyzed by western blot ( A ) or treated with vehicle or TGFβ for different time periods, and then analyzed by qRT-PCR ( B ). Data in ( B ) represent the mean ± SD from duplicates of two independent experiments from two different pairs of primary tumor cell lines. C , E MCF7 ( C ) and MDA-MB-231 ( E ) cells were infected with either pGIPZ-empty (sh-Ctrl.) or pGIPZ-shBRCA1 targeting different sequences of human BRCA1 (sh-BRCA1-B7 and sh-BRCA1-G6). Cells stably expressing sh-Ctrl or shBRCA1 were analyzed by qRT-PCR. Data represent the mean ± SD from triplicates of each of the two independent experiments. D , F mRNA levels in MCF7-sh-Ctrl and MCF7-sh-BRCA1-G6 ( D ), or MDA-MB-231-sh-Ctrl and MDA-MB-231-sh-BRCA1-G6 ( F ) cells treated with TGFβ for 10 ( D ) or 24 ( F ) hours were analyzed. Data represent the mean ± SD from duplicates of two independent experiments. The asterisk (*) in ( B ) ( D ) and ( F ) denotes a statistical significance from vehicle- and TGFβ-treated samples determined by a two-tailed, paired T test. The asterisk (*) in ( C ) and ( E ) denotes a statistical significance from sh-BRCA1 and sh-Ctrl samples determined by a two-tailed, paired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Western Blot, Quantitative RT-PCR, Infection, Stable Transfection, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A SUM149 cells were transfected with pBabe-empty (Empty) or pBabe-HA-BRCA1 (BRCA1). The expression of genes indicated was determined by western blot and/or qRT-PCR 72 h after transfection. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from empty- and BRCA1-expressing samples determined by a two-tailed, paired T test. B Diagram showing the location of putative BRCA1 binding sites (dark black bars) in the human TGFβR2 gene, and that of primers (P1 - P8) used for ChIP analysis. +1, transcription start site. C ChIP analysis of endogenous BRCA1 binding to putative BRCA1 sites on the TGFβR2 locus in T47D cells. Normal IgG was used as a negative control. The ratio of binding signal to input was compared. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from IgG and anti-BRCA1 immunoprecipitated samples by a two-tailed, unpaired T test. D ChIP analysis of exogenous BRCA1 binding to the TGFβR2 locus in HCC1937 cells transfected with pBabe-empty (Empty) or pBabe-HA-BRCA1 (BRCA1). Normal IgG was used as a negative control. The ratio of binding signal to input was compared. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from Empty- and BRCA1-expressing samples immunoprecipitated by anti-BRCA1 by a two-tailed, unpaired T test. E A schematic representation of WT and Mut TGFβR2 promoter-reporter constructs. Numbers represent a position relative to the transcription start site and a letter (X) denotes a mutated GATA3 binding site. The location of primers used for ChIP analysis is shown. F T47D-sh-Ctrl and T47D-sh-BRCA1 cells were transfected with Renilla and pGL3-TGFβR2-WT or pGL3-TGFβR2-Mut (right panel), and SUM149 cells were transfected with Renilla and pGL3-TGFβR2-WT or pGL3-TGFβR2-Mut, as well as pBabe-empty or pBabe-HA-BRCA1 (left panel), which were then collected after 48 h and assayed for luciferase activity. Data represent the mean ± SD from triplicates of two independent experiments. The asterisk (*) denotes a statistical significance from BRCA1 and empty (left panel), or sh-BRCA1 and sh-Ctrl (right panel) samples determined by a two-tailed, paired T test. SUM149, BRCA1 mutant (Mut); T47D, BRCA1 wild type (Wt).

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Negative Control, Immunoprecipitation, Construct, Luciferase, Activity Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A – C p18 −/− ; Brca1 MGKO tumor cells were transfected with Tgfβr2 (Tgfβr2 CRISPR) and Control (Ctrl CRISPR) Double Nickase plasmids then selected with puromycin for 3 days. Tgfβr2- and control-knockout cells were then analyzed by western blot ( A ), Q-RT-PCR (B), and microscope for cell morphology ( C ). The areas with epithelial-like cells are indicated in ( C ). D Tgfβr2- and Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were treated with vehicle or TGFβ for different time periods, and the expression of Fn was then determined. Data in ( B ) and ( D ) represent the mean ± SD from duplicates of two independent experiments. The asterisk (*) in ( B ) denotes a statistical significance from Tgfβr2- and Ctrl-knockout samples, and in ( D ) denotes a statistical significance from TGFβ treated and vehicle treated samples determined by a two-tailed, paired T test. E , F 6 ×10 5 Tgfβr2- and Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were inoculated into the left and right inguinal MFPs of NSG mice, respectively, in a pairwise manner. Two weeks after transplantation, mice were dissected. The volume ( E ) and expression of Vim ( F ) of the regenerated tumors were determined by measurement and IHC. Data in ( E ) represent the mean ± SD of four tumors in each group. The asterisks (*) denote a statistical significance from Tgfβr2- and Ctrl-knockout tumors determined by a two-tailed, paired T test. G–I 6 ×10 5 Tgfβr2- or Ctrl-knockout p18 −/− ; Brca1 MGKO mammary tumor cells were inoculated into the MFPs of NSG mice. When newly generated tumors reached the maximum size allowed by IACUC in 4–7 weeks, or the mice became moribund, lungs were examined for gross appearance ( G ), H.E. staining ( H ), and quantification of the number of metastatic nodules ( I ). M, metastatic nodules. Data in ( I ) represent the mean ± SD for the numbers of metastatic nodules detected in all lobes of the lungs in each group ( n = 4). Statistical significance was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Transfection, CRISPR, Control, Knock-Out, Western Blot, Reverse Transcription Polymerase Chain Reaction, Microscopy, Expressing, Two Tailed Test, Transplantation Assay, Generated, Staining

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A 18 −/− ; Brca1 MGKO tumor cells treated with ITD1 at 4 μm for 24 h were analyzed by western blot. B 10 4 cells dissociated from 18 −/− ; Brca1 MGKO primary tumorspheres were treated with ITD1. Secondary spheres formed after 6 days of treatment were counted from quadruplicate experiments. Data represent the mean ± SD from two independent primary tumorspheres. C 1000 p18 −/− ; Brca1 MGKO tumorsphere-dissociated cells pretreated with DMSO or ITD1 for 6 days were transplanted into MFPs of NSG mice. Four weeks later, mice were dissected and tumor volumes were measured. Values represent the average tumor volumes ± SD of four tumors. The asterisk (*) in ( B ) and ( C ) denotes a statistical significance from ITD1 treated and DMSO treated samples determined by a two-tailed, unpaired T test. D , E Representative tumors generated by DMSO- or ITD1-pretreated p18 −/− ; Brca1 MGKO cells were analyzed by Western blot ( D ) and IHC ( E ). The H-scores for p-Smad2, Twist, and Tgfβr2 in IHC were calculated (E, right panel). The results represent the mean ± SD of four individual tumors per group. Statistical significance was determined by a two-tailed, unpaired T test.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Western Blot, Two Tailed Test, Generated

Journal: Cell Death & Disease

Article Title: Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFβR2 signaling pathway

doi: 10.1038/s41419-022-04646-7

Figure Lengend Snippet: A Correlation analysis of the expression of BRCA1 and TGFβR2 for MetaBric breast cancer patients. B Analysis of gene expression in NKI295 breast cancer patients according to tumor subtype. BL basal-like, CL claudin-low, H2 Her2-enriched, LA luminal A, LB luminal B, NBL normal breast-like. C Representative immunostaining of ER positive and negative invasive human breast cancers with antibodies against TGFβR2. D, E Summary of expression of TGFβR2 by IHC and BRCA1 by Q-RT-PCR ( D ). The expression levels of TGFβR2 were quantified by H-scores. The expression of BRCA1 was determined by Q-RT-PCR. BRCA1 mRNA levels relative to that of T47D cells were determined as we previously reported . Analysis of the expression of TGFβR2 by IHC and BRCA1 by Q-RT-PCR in ER + and ER-invasive breast cancers ( E ). Statistical significance was determined by a two-tailed, unpaired T test. F Correlation analysis of BRCA1 mRNA levels and H scores of TGFβR2 expression for breast cancer samples.

Article Snippet: Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1.

Techniques: Expressing, Gene Expression, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: GenomeGraph of SKAR as a splice target of TDP-43. HEK293E cells were transfected with control siRNA (scrambled) or treated with siRNA against TDP-43 (siRNA TDP-43 ). Four biological replicates of each group were hybridized on a Human Exon 1.0-ST Gene Chip. Intensity values of microarray hybridizations, single values (gray), mean group intensities of scrambled siRNA (blue) and siRNA TDP-43 (green), are shown as normalized background-corrected logarithmic intensities ( A ) and RMA corrected probe-level data ( B ). Vertical lines separate the 18 individual probe sets covering the POLDIP3/SKAR gene. ( C ) Depicted are the mean group values of the FIRMA score. The fold change of the FIRMA score (FC(F)) is shown in red. ( D ) Genomic representation of the POLDIP3/SKAR gene in orange. Gray lines at the top of this panel indicate localization of the individual probe sets within the genomic coordinates. ( E ) The two Ensembl annotated alternative splice isoforms SKAR α and SKAR β are depicted in blue. SKAR exon 3 is highlighted by a box. ( F ) The SKAR α protein isoform is shown in pink, the RRM domain is shown in dark blue. Highlighted in green is the exon 3 derived part. At the bottom the amino acid sequence of exon 3 is given.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Microarray, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: Validation of SKAR alternative splicing upon transient silencing of TDP-43. TDP-43 was either silenced transiently by siRNA treatment ( A , C , E and G ) or stably by use of lentiviral particles encoding for a TDP-43-specific shRNA followed by the selection of single cell clones ( B , D and F ). For transient silencing, HEK293E cells were either mock treated (m) or transiently transfected with scrambled control siRNA (scr), with one of four different TDP-43-specific siRNAs (siRNA TDP-43 A-D) or with one of five specific siRNAs against FUS (siRNA FUS A-E), as indicated. (A–D) Total RNA was extracted and analyzed by RT–PCR. (A and B) Semi-quantitative RT–PCR was performed with primer pairs specific for TDP-43, SKAR (ex2–ex4), SKAR α (ex2|3–ex4) and SKAR β (ex2|4–ex4). (C and D) Real-time PCR was performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKARα/PBGD, SKARβ/PBGD and total SKAR/PBGD ratios were recalculated into absolute copy values and normalized to total SKAR values. Shown are the mean values of five independent experiments ± SEM. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. Original qRT–PCR data is presented in Supplementary Figure S1A and S1B , respectively. (E–G) Protein was extracted, electrophoresed and resulting western blots probed with antibodies specific for TDP-43, SKAR (both isoforms) and SKAR α. GAPDH was used as a loading control. FUS silencing efficiency was controlled by use of an anti-FUS antibody. Note, that, depending on the primer pair and antibody used, SKAR RNA and protein isoforms, respectively, are visualized as two bands with different molecular weights. The upper band represents SKAR α, the lower corresponds to SKAR β, as indicated.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Selection, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR alternative splicing is dependent on RRM1 of TDP-43. ( A ) Stably silenced HEK293E cells (shRNA TDP-43 ) or transiently silenced HEK293 cells (siRNA TDP-43 ) were transiently transfected with either control vector (−) or Flag-TDP-43 variants (wt, ΔRRM1, ΔRRM2, ΔRRM1/2, FFLL and ΔGRD or disease-associated mutations, as indicated). Parental HEK293E cells or cells treated with a scrambled siRNA (−) were used as an internal control. (A) Total RNA was extracted and subjected to semi-quantitative RT–PCR using primer pairs amplifying total TDP-43, endogenous TDP-43, total SKAR (ex2–ex4), SKAR α (ex2|3–ex4), SKAR β (ex2|4–ex4) and PBGD as a housekeeping gene. ( B and E ) RNA was extracted and real-time PCR performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKAR α/PBGD, SKAR β/PBGD and total SKAR/PBGD ratios were re-calculated into absolute copy values and normalized to total SKAR values. Original qRT data is presented in Supplementary Figure S1C and S1D , respectively. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. ( C and D ) Protein was extracted, electrophoresed and resulting western blots probed with anti-TDP-43, anti-Flag and anti-SKAR antibodies. GAPDH was used as a loading control. (D) Shown are the mean values ± SEM of densitometric analysis of three independent experiments. * P < 0.05; ** P < 0.005; ns = not significant.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: A repeat containing RNA stretch 3′ of exon 3 is necessary for TDP-43 and SKAR splicing. ( A ) Schematic representation of constructs used for refined RNA crosslinking assays. ( B ) Indicated fragments of SKAR DNA were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt or FFLL. No RNA was added to control samples. Samples were UV crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates (left panel) were probed with anti-TDP-43 and anti-Flag to visualize co-precipitated endogenous and exogenous TDP-43. Biotinylated SKAR RNAs pulled down transfected as well as endogenous TDP-43 wt but not FFLL. Protein inputs (right panel) of HEK293E lysates confirmed even transfection efficiencies. ( C ) Schematic representation of the three repeat motifs and mutagenized variants within the SKAR pre-RNA 3′ of exon 3. ( D ) Non-mutated or mutagenized variants of SKAR DNA part-5 were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt. No RNA was added to control samples. Samples were UV-crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates were probed with anti-TDP-43 and anti-Flag to visualize coprecipitated endogenous and exogenous TDP-43. ( E ) Schematic representation of the used SKAR minigene construct pTB SKAR part-3/4/5. Primer annealing sites are indicated by arrows. ( F and G ) HEK293E cells were transfected with pTB SKAR part-3/4/5 variants, as indicated. RNA was extracted and used for RT–PCR using primers for pTB and PBGD as a housekeeping gene. (F) Representative RT–PCR is shown. (G) Shown are the results (mean values ± SEM) of densitometric analysis of seven independent experiments calculated as the ratio of SKAR α to SKAR β. * P < 0.05; *** P < 0.0005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Construct, In Vitro, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR β is more active than SKAR α and leads to enhanced translation and increased cell size. ( A ) HEK293E cells were treated with control siRNA or transfected with siRNA against SKAR or TDP-43 as indicated. Stably silenced siRNA TDP-43 and transiently transfected HEK293E cells were transfected with either control vector (−) or plasmids encoding for Myc-SKAR α, Myc-SKAR β or Flag-TDP-43 wt, as indicated. Cells were serum starved for 16 h. After 6 h serum-stimulation cells were harvested, protein extracted and electrophoresed. Resulting western blots were probed with anti-SKAR, anti-phospho S6K1 (Thr389), anti-S6K1, anti-phospho S6 (Ser235/236), anti-S6, anti-phospho Akt substrate (RXRXXS/T) and anti-TDP-43 antibodies. GAPDH was used as a loading control. Transfection of SKAR β or depletion of TDP-43 results in overall stronger phospho-signal compared to SKAR α. ( B ) Schematic representation of luciferase constructs used for analysis of translation. ( C–G ) HEK293E cells were transfected with either Myc-SKAR α or Myc-SKAR β (C) or with control siRNA (scr) and individual siRNA TDP-43 A–D, as indicated (D–G). (C–E) Before DNA/siRNA transfection, cells were transfected with firefly control vector plus either intron-containing or intron-less Renilla luciferase constructs. (C and D) Luciferase activity was measured and normalized to control treated HEK293E cells. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. Western blotting confirmed equal expression of Myc-SKAR α and Myc-SKAR β (C, right panel). (E) qRT–PCR confirmed equal RNA levels of Renilla and firefly luciferase in non-silenced and silenced HEK293E cells. (F) Cells were counted and equal numbers of cells was collected. Protein amount was determined using BCA protein assay. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. (G) Cell size was analyzed by flow cytometry, monitoring the forward scatter parameter. Shown are the mean values ± SEM of five independent experiments. * P < 0.05; ** P < 0.005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Construct, Activity Assay, Expressing, Quantitative RT-PCR, Bicinchoninic Acid Protein Assay, Flow Cytometry

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Growth curves of AML cell lines grown in complete medium (CM) or equivalent medium lacking serine and glycine (-SG). B Mouse HSPCs were transduced with MLL-AF9 ( MA9 ) retroviruses, followed by cultured in CM or -SG progenitor medium for 2 days. The cells were then plated into mouse methylcellulose medium for CFA. Colony numbers and representative photos of colonies are shown. Bar = 200 μm. C A schematic diagram, created from GDP ( https://BioGDP.com , Agreement number: GDP2025UWV74M), depicting the CRISPR/Cas9-based screening strategy. Lentivirus-transduced Molm13 Cas9 cells were selected with puromycin for 5 days and collected for an initial gDNA sample (denoted as T0). Cells were further grown in CM or -SG medium for 8, 12, or 17 population doublings, and were denoted as P8, P12, or P17, respectively. For each gene, we calculated its gene score as the mean log 2 fold change (LFC) in the abundance of all the sgRNAs targeting the corresponding gene in different population doublings compared to T0. The SG-dependent score refers to the difference in gene scores in the absence versus presence of SG at the same population doubling. D The SG-dependent scores of the top 30 genes from the screening in ( C ), with the m 6 A regulatory genes IGF2BP3 and METTL14 being highlighted in red and purple, respectively. E , F The normalized abundance of the 12 sgRNAs targeting IGF2BP3 ( E ) or METTL14 ( F ) from cells cultured in CM or -SG medium and collected at P8, P12, and P17. The definition of data points is same as in ( C ). n = 12 sgRNAs. The boxes extend from the 25th to the 75th percentiles, with a distinct line marking the median. Whiskers extend to the maximum and minimum data points. G Growth curves of Molm13 Cas9 transduced with control sgRNA (sgNS) or IGF2BP3 sgRNA (sgBP3) and grown in CM or -SG medium. H Growth curves of control or IGF2BP3 KD cells in CM or -SG medium. I Growth curves of control or METTL14 KD Molm13 cells in CM or -SG medium. J Schematic illustration showing the AID2 system in inducing rapid degradation of IGF2BP3 protein. K Relative growth of IGF2BP3-mAID (clone BP3-mAID cells) cells in CM or -SG medium with or without treatment with 5’Ph-IAA (IAA) for 24 h. Mean ± SD values are shown. n = 2 biologically independent repeats in ( A , B , G , H , I , and K ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Transduction, Cell Culture, CRISPR, Control

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Bubble diagram showing the enrichment of metabolic pathways by the C 13 -labeled metabolites with reduced levels after IGF2BP3 KD in Molm13 cells. B Heatmaps showing levels of representative C 13 -labeled metabolites after IGF2BP3 KD in Molm13 cells. C The total levels and isotopolog distribution ( M + n , n refers to numbers of 13 C) of purines measured by LC-MS in Molm13 cells transduced with IGF2BP3 shRNAs or shNS and grown in medium containing U-[ 13 C]-glucose. D Schematic of the serine synthesis pathway and its downstream pathways. E The levels of C 13 -labeled serine ( M + 3) were measured by LC-MS in Molm13 cells transduced with IGF2BP3 shRNAs or shNS and grown in medium containing U-[ 13 C]-glucose. F – H Intracellular serine level measured by fluorescence-based serine detection assays in Molm13 cells transduced with shRNAs ( F ) or sgRNAs ( G ) targeting IGF2BP3 or shRNAs targeting METTL14 ( H ), compared to those with negative control (shNS). I The Venn diagram showing the overlap of significantly downregulated genes (adjusted P < 0.05, fold change <0.67) in Molm13 cells with IGF2BP3 KD, as well as in U937 BP3-mAID cells treated with 5’Ph-IAA for 6 or 24 h to induce degradation of the IGF2BP3 protein. J Pie chart showing numbers of transcripts with or without m 6 A modifications among the 61 overlapping genes in ( H ), and heatmap showing expression changes of the 48 m 6 A-containing transcripts in IGF2BP3 KD Molm13 cells or IGF2BP3-depleted U937 cells. K GO enrichment analysis of the 48 candidate targets of IGF2BP3 in ( I ). The degrees of interactions were generated by STRING. Mean ± SD values are shown. n = 2 biologically independent repeats in ( C ) and ( E ), while n = 3 biologically independent repeats in ( F – H ). P -values were calculated with one-sided Hypergeometric test ( A , K ); two-tailed Wald test adjusted with Benjamini-Hochberg Procedure ( I ); two-tailed student’s t -test ( F – H ); Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Transduction, Fluorescence, Negative Control, Expressing, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Western blot after IGF2BP3 KD in various AML cell lines. GAPDH was used as a loading control. B Western blot of U937 BP3-mAID cells after treatment with 5’Ph-IAA for the indicated time periods. ACTB was used as a loading control. C Western blot of Molm13 cells after overexpression of wild-type (BP3-WT) or KH3-4 mutated (BP3-KH34) IGF2BP3. D Western blot of Molm13 cells after METTL14 KD. E IGV tracks showing the distribution of m 6 A (GEO: GSE97408 ) or IGF2BP3 binding sites (GEO: GSE90639 ) in target mRNAs. Gray shadow depicts high-confidence m 6 A regions for qPCR validation in ( G ) and ( H ). F RIP assays using an IGF2BP3 antibody were performed in Molm13 cells, followed by qPCR to detect direct binding of IGF2BP3 to target mRNAs at regions indicated in ( E ). G MeRIP-qPCR was performed in control and METTL14 KD Molm13 cells to detect the change of m 6 A abundance at indicated regions of ATF4 , PHGDH , and PSAT1 transcripts. H Bst DNA polymerase-mediated cDNA extension and qPCR assays to evaluate relative m 6 A abundance changes at specific sites within ATF4 , PHGDH , and PSAT1 transcripts in control and METTL14 KD Molm13 cells. I Control and IGF2BP3 KD Molm13 cells were treated with actinomycin D for indicated time periods and the RNA level of target genes was examined by qPCR. 18S rRNA was used as a loading control. The mRNA half-life ( t 1/2 ) was calculated and shown. ( J , K ) Live cell counting ( J ) and cytometric analysis of Annexin V + apoptotic cells ( K ) in Molm13 cells upon IGF2BP3 KD and ATF4, PHGDH, or PSAT1 overexpression. EV, empty vector. ( L , M ) Molm13 cells were co-transduced with control or IGF2BP3 shRNAs and ATF4/PHGDH/PSAT1 overexpression vectors and subjected to colony-forming assays ( L ) and serine level detection ( M ). Representative results from one of the two independent experiments were shown ( A – D ). Mean ± SD values are shown. n = 3 biologically independent repeats in ( F , G , H , I , K , and M ), while n = 2 biologically independent repeats in ( J ) and ( L ). Two-tailed student’s t -test ( F – H ), two-way ANOVA ( K , M ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Western Blot, Control, Over Expression, Binding Assay, Biomarker Discovery, Cell Counting, Plasmid Preparation, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Schematic illustration of the in vivo primary BMT assay and in vitro CFA with HSPCs co-transduced with MA9 retroviruses and Igf2bp3 shRNA (or shNS) lentiviruses. B Kaplan–Meier curves showing the effect of Igf2bp3 KD on MA9 -induced leukemogenesis. C Wright-Giemsa staining of BM cells and PB blood smear, and hematoxylin and eosin (H&E) staining of livers and spleens of the representative primary BMT mice from ( B ) at the endpoint. Scale bars from left to right: 20 μm, 50 μm, 200 μm, 300 μm. D , E CFA using mouse HSPCs transduced with MA9 ( D ) or PML-RARa ( E ) plus shNS, Bp3-sh1, or Bp3-sh2 viruses. F HSPCs from FLT3-ITD/NPM1-mut mice were transduced with shNS, Bp3-sh1, or Bp3-sh2 viruses and seeded for CFA. Bar = 200 μm. G HSPCs from Mettl14 fl/fl -CRE ERT mice were transduced with MA9 retroviruses and seeded for CFA. Mettl14 KO was induced by addition of 4-Hydroxytamoxifen (4-OHT, 1 mM) during plating. H Schematic illustration of the in vivo primary BMT assay and in vitro CFA with HSPCs from Igf2bp3 WT and KO mice. I Colony numbers and representative photos of colonies in the CFA assays illustrated in ( H ). Bar = 200 μm. J Flow cytometric analysis of CD45.2 + donor cell percentages in PB of recipient mice 8 weeks after BMT. n = 7 mice. K Kaplan–Meier curves showing the effect of Igf2bp3 KO on MA9 -induced leukemogenesis. L , M Representative images of liver ( L ) and spleen ( M ) tissues from the primary BMT mice in ( K ) at the endpoint. N Wright-Giemsa staining of BM cells and PB blood smear, and H&E staining of livers and spleens of representative primary BMT recipient mice from ( K ) at the endpoint. Scale bars are same as in ( C ). O HSPCs from WT or Igf2bp3 KO mice were co-transduced with MA9 and ATF4 -, PHGDH -, or PSAT1 -overexpressing viruses and seeded for CFA. P Schematic illustration of the secondary BMT assay and CFA with MA9 -induced leukemic cells transduced with Igf2bp3 shRNA (or shNS) lentiviruses. Q CFA assays showing the effect of Igf2bp3 KD on BM cells from MA9 leukemia mice. Bar = 200 μm. R Kaplan–Meier curves showing the result of secondary BMT using cells collected from the first plating of ( O ). Representative results from one of at least two mice were shown ( C , N ). Mean ± SD values are shown. n = 2 biologically independent repeats in ( D , E , F , G , I , O , and Q ). Statistical analysis: Two-tailed student’s t -test ( J ); log-rank test ( B , K , and R ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: In Vivo, In Vitro, Transduction, shRNA, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Expression of IGF2BP3 mRNA in AML patient samples and healthy donors from the TNMplot database. Box plot, center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. n = 407 healthy samples; n = 151 AML samples. B Expression of IGF2BP3 in CD34 + and CD34 - cells from BM of healthy donors (normal) or AML patients as detected by microarray ( GSE30029 ). n = 31 CD34 + normal samples; n = 46 CD34 + AML samples; n = 44 CD34 - AML samples. C Expression of IGF2BP3 in blast (LSC − , non-engrafting) or LSC (LSC + , engrafting) cells from AML patients ( GSE199452 ). n = 40 LSC − samples; n = 70 LSC + samples. D Experimental scheme for ( E – G ). Patient-derived CD34 + cells were transduced with shNS or shRNAs targeting IGF2BP3 and subjected to different assays. E – G Growth curves ( E ), percentages of Annexin V + apoptotic cells ( F ), and colony numbers ( G ) of patient-derived CD34 + leukemia cells with or without IGF2BP3 KD. Bar = 200 μm. H The percentage of L-GMP population (CD45.2 + Lin - c-kit + Sca1 - CD34 + CD16/32 + ) in BM of the primary BMT mice. n = 6 mice per group. I , J Statistics of the percentage of Annexin V + ( I ) or Ki67 + ( J ) L-GMPs of mice in ( H ). n = 6 mice. K In vitro LDA assays using MA9 -transduced HSPCs from Igf2bp3 WT and KO mice. Logarithmic plots show the percentage of nonresponding wells (those without any colonies) at different doses of cells seeded. The estimated LSC/LIC frequency is calculated by ELDA and shown. L , M In vitro LDA assays of mouse HSPCs co-transduced with MA9 plus shRNA targeting Igf2bp3 ( L ) or its target genes ( M ). N HSPCs collected from Igf2bp3 WT and HO mice were co-transduced with MA9 and target gene-overexpressing viruses and selected in methylcellulose medium with G418 and puromycin for 7 days before seeded for LDA assays. Mean ± SD values are shown. n = 2 biologically independent repeats in E , G while n = 3 biologically independent repeats in ( F ). Statistical analysis: Two-tailed student’s t -test ( A , C , F , H – J ); two-way ANOVA ( B ); One-sided Chi-squared test ( K – N ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Expressing, Microarray, Derivative Assay, Transduction, In Vitro, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Schematic outline of experimental strategy testing the effect of Igf2bp3 KO on mouse static normal hematopoiesis. n = 7 mice per group. B – D PB analysis of Igf2bp3 WT and KO mice. The density of white blood cells (WBC), lymphoma cells (LYM), red blood cells (RBC) ( C ), palates (PLT) ( D ), neutrophils (NEUT), monocytes (MONO), and eosinophils (EO) ( E ) are shown. E Flow cytometric gating strategies for HSC and progenitors. F , G Frequencies of various hematopoietic progenitors in the BM of Igf2bp3 WT and KO mice as examined by flow cytometry. LSK, Lin - Sca1 + cKit + cells; MPP, multipotent progenitor (Lin - Sca1 + cKit + CD48 + CD150 - ); LT-HSC, long-term HSC (Lin - Sca1 + cKit + CD48 - CD150 + ); ST-HSC, short-term HSC (Lin - Sca1 + cKit + CD48 - CD150 - ); LMPP, lymphoid-primed multipotent progenitor (Lin - Sca1 high cKit high CD127 + CD135 + ); CLP, common lymphoid progenitor (Lin - Sca1 low cKit low CD127 + CD135 + ); CMP, common myeloid progenitor (Lin - Sca1 - cKit + CD34 int CD16/32 low ); GMP, granulocyte-monocyte progenitor (Lin - Sca1 - cKit + CD34 + CD16/32 high ); MEP, megakaryocyte–erythroid progenitor (Lin - Sca1 - cKit + CD34 - CD16/32 - ). H Flow cytometric gating strategies for mature cells. I Frequencies of various mature cells in the BM of Igf2bp3 WT and KO mice as examined by flow cytometry. J Schematic outline of competitive repopulation assay. n = 7 mice per group. K Flow cytometric gating strategy for BM cells derived from Igf2bp3 WT or KO donor mice in the competitive repopulation assay. L Flow cytometry analysis for different donor-derived cells (CD45 + ) in PB of recipient mice 4, 8, 12, and 16 weeks after BMT. M , N Percentage of donor-derived stem cell ( M ) and progenitor ( N ) compartments in the bone marrow of recipients 16 weeks after BMT. Mean ± SD values are shown. Statistical analysis: Two-tailed student’s t -test ( B – D , F , G , I , L – N ); n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Experimental scheme for ( B ) and ( C ). M-NSG immunodeficient recipient mice were transplanted with AML PDX cells (FLT3-TKD mutation) transduced with shNS or shRNA targeting Igf2bp3, and fed with a regular (control) or SG-free (-SG) diet. B Colony numbers of AML PDX cells (FLT3-TKD) are shown to reflect the effect of SG deprivation (-SG) on control (NS) or Igf2bp3 KD (shBp3) cells compared to complete medium (CM). C Kaplan–Meier curves showing survival of recipient mice fed with a regular (control) diet or an equivalent diet lacking SG (-SG). n = 7 mice per group. D Experimental scheme for ( E ) and ( F ). BM cells from MA9 leukemic mice were transduced with shNS or shRNA targeting Igf2bp3 and selected with puromycin before subjected to CFA assays ( E ) or BMT assays ( F ). E Colony numbers in CFA assays are shown to reflect the effect of SG deprivation (-SG) on control (NS) or Igf2bp3 KD (shBp3) cells compared to complete medium (CM). F Kaplan–Meier curves showing survival of recipient mice fed with a regular (control) diet or an equivalent diet lacking SG (-SG). n = 8 mice per group. G Experimental scheme of the xenotransplantation assays in ( H ). M-NSG immunodeficient recipient mice were transplanted with U937 BP3-mAID cells and fed with a regular (control) or SG-free (-SG) diet. 5’Ph-IAA (3 mg/kg) or vehicle control (DMSO) were given via intraperitoneal injection (i.p.) every day for 7 consecutive days starting from the 8th day after transplantation. H Kaplan–Meier curves showing survival of the M-NSG recipient mice in ( G ). n = 7 mice per group. I Schematic illustration of the working model and therapeutic strategy proposed in the study, created with the assistance of GDP (Agreement number: GDP2025KABQ7W). Mean ± SD values are shown. n = 2 biologically independent repeats in ( B ) and ( E ). Statistical analysis: log-rank test ( C , F , and H ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Mutagenesis, Transduction, shRNA, Control, Injection, Transplantation Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) The left graph shows the X-ray crystal structure of a p50 / p65 heterodimer bound to DNA as published in (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. (B) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wildtype (wt) were mutated to Asp (FL / DD) for dimerization-deficient p65 or E39 to Ile (E / I) for DNA-binding-deficient p65. (C) Principle of proximity-based biotin tagging. (D) Pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI)) with the constructs shown in (B) and their expression was induced with doxycycline (1 µg / ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 minutes as indicated. Additionally, half of the samples were treated with IL-1α (10 ng / ml) for the last 60 minutes. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti p65 and anti HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). (E) Biotinylated proteins from the experiment shown in (C) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X-axes obtained with wild type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (F) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, -log10 p ≥ 1.3). Venn diagrams show the numbers of p65 / RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. (G) The six protein sets shown in (E) were subjected to parallel overrepresentation pathway analysis using Metascape software . The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate total numbers of unique pathways. (H) The table shows the most strongly enriched pathway categories associated with the p65 / RELA wild type or mutant interactomes. Numbers in brackets indicate the total numbers of p65 / RELA interactors per condition that were subjected to overrepresentation analysis according to (E, F). The mass spectrometry data and bioinformatics analysis results are provided in Supplementary Table 1. See also and . rtTA, reverse tetracycline-controlled transactivator.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Parental HeLa cells or pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with empty vector (EV) encoding HA-miniTurbo (HA-mTb) or with p65 / RELA wild type (wt) fused C-terminally to HA-mTb (p65(wt)-HA-mTb) as described in the legend of . The expression of the constructs was induced with increasing concentrations of doxycycline for 17 h as indicated. At the end of the incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cell extracts were analyzed by Western blotting for the expression of the p65-HA-mTb fusion protein or HA-mTb using polyclonal antibodies raised against the C-terminus of p65 / RELA (sc-372) or a monoclonal antibody raised against N-terminal amino acids 1-286 of p65 / RELA (sc-8008), or an anti HA antibody, respectively. Note that the fusion protein is better recognized with the N-terminal antibody preparations. (B) HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with the indicated constructs and their expression was induced with doxycycline at 1 µg / ml for 17 h. On the next day, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Total RNA was isolated and analyzed by RT-qPCR for expression of the indicated genes. Bar graphs show means ± s.d. from two biologically independent experiments. (C) Cells were transfected as in (A) and expression of the p65 / RELA fusion protein was induced 20 h later with doxycycline (10 ng / ml) for 4 h. In last period of this incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cells were lysed and cytosolic (C), soluble nuclear (N1) and insoluble, chromatin nuclear fractions (N2) were analyzed by Western blotting for the expression and distribution of p65(wt)-HA-mTb. Antibodies against RNA polymerase II, tubulin and β-actin were used to control purity of fractions and equal loading.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Construct, Incubation, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Biotinylated proteins from the experiments shown in and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X-axes show mean ratio value and Y-axes show p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (B) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in (A). Venn diagrams show the total numbers of specific p65 / RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65 / RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Protein interaction network of the 46 known p65 / RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database . Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. (B) Venn diagram of p65 / RELA interactors in IL-1α or untreated cells revealing a total of 366 unique p65 / RELA interactors, of which 320 (87.4 %) have no documented protein interaction entries in STRING. (C) Overlap of the RELA interactome with 1639 human TFs and 801 epigenetic regulators . (D) Graphs visualizing the top 10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 / RELA (wt) or with p65 / RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. (E) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by . Numbers in brackets show identified components per complex. (F) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. (G) Volcano plots visualizing all TFs significantly enriched with wt p65 / RELA (LFC ≥ 2, -log 10 p ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal conditions. (H) Distribution of TF families found to be associated with p65 / RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (I) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. (J) The top 10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways data base entries and the top 10 subcellular localizations associated with the 366 p65 / RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape . The mass spectrometry data sets and bioinformatics analysis results are provided in Supplementary Table 1.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Labeling, Transformation Assay, Plasmid Preparation, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Final list of top ranking high confidence interactors p65 / RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. (B) Graph showing that the top 38 p65 / RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65 /RELA. Node borders visualize the main functional annotations. (C) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65 / RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated (untr.). Half of the cells per plate were treated for 1 h with IL-1α (10 ng / ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-kB target genes, two housekeeping genes and all 38 HCI p65 / RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65 / RELA interactors selected for further analysis. (D) The miniTurboID enrichment of six p65 / RELA interactors (green colors) chosen from (C) is shown. The complete set of data of the screen is provided in Supplementary Table 2. See also .

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Scheme illustrating the arrangement of siRNAs and controls on individual cell culture plates and the performance of RT-qPCR measurements in cell extracts without prior RNA purification. A linear PCR amplification step was included to pre-amplify specific transcripts. (B) Confirmation of knockdown of 38 HCI and of RELA mRNAs by RT-qPCR as shown in (A). Bar graphs show mean changes ± s.d. relative to the luciferase siRNA controls (siLuci) from three biologically independent experiments.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Cell Culture, Quantitative RT-PCR, Purification, Amplification, Knockdown, Luciferase

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Proximity-ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65 / RELA to demonstrate interactions of p65 / RELA with TFE3 (A), TFEB (B), GLIS2 (C) and ZBTB5 (D) using pairs of antibodies as indicated. PLA-spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst (blue signals). The images show representative fluorescence raw data and the violin plots on the right show quantification from the numbers of cells indicated in brackets. Samples omitting one of the two antibodies or both primary antibodies (ctr) served as negative controls. Solid lines indicate medians and dashed lines indicate 1 st and 3 rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (****p ≤ 0.0001). obtained by one-way ANOVA.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Ligation, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on basal p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 hours with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in untreated, basal conditions, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all constitutively expressed genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on IL-1α-regulated p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65 / RELA or its interactors in IL-1α-stimulated cells into combined functional networks. (B) Table summarizing the numbers of mapped IDs (= nodes) corresponding to the gene groups shown in , their protein interactions (= edges) and the protein interaction network enrichment p values as derived from STRING. (C) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65 / RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65 / RELA, S100A8 / 9, and TFE3 / TFEB. No interactions were found for ZBTB5 and GLIS2.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to use p65 / RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. (B) Windows of 1000 base pairs surrounding experimentally determined p65 / RELA ChIPseq peaks were searched for motifs of RELA and REL using matrices from the JASPAR data base. P values indicated significant enrichment compared to the whole genome. The Venn diagram shows the overlap and inserts show motif compositions. (C) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions. (D) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in or were collected and were examined for their association with a p65 / RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65 / RELA peak in their promoters or enhancers. (E) Overlap of all genes with a p65 / RELA peak in promoters or enhancers and at least one motif for the indicated transcription factors in IL_1a-stimulated conditions. (F) Genome browser view of the TNFAIP3 locus with p65 / RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 ( ; ). p65 / RELA binding regions of 1000 bp under p65 / RELA peaks and identified TF motifs are indicated by horizontal lines. (G) HeLa cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng / ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in ). Floating bar plots show percent input plus the mean of all values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Supplementary Table 4.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Venn diagrams indicating the overlap of RELA motifs with motifs of ZBTB factors that were found by miniTurboID to interact with RELA, in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques:

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: High EREG expression predicts a poor prognosis in HNSCC patients. (A) The microarray analysis between HN4 and HOK cells. (B) RT-PCR analysis of EREG mRNA levels in HOK and five other HNSCC cell lines. (C) Densitometric EREG mRNA data in B were normalized to GAPDH mRNA levels. (D) EREG expression from 7 paired cases of fresh-frozen HNSCC tumors was examined by Western blotting. (E) Densitometric EREG protein data in D were normalized to GAPDH protein levels. Significant differences were detected using a Wilcoxon signed-rank test (P < 0.005) in EREG expression between adjacent normal oral tissues and cancer tissues. (F) Representative images of EREG expression in normal tissues and HNSCC tissues via immunohistochemical (IHC) staining. (G) IHC scores of EREG expression in HNSCC tissues (n = 80) and paired adjacent normal tissues (n = 80). Significant differences were detected (P < 0.005) in EREG expression between adjacent normal tissues and HNSCC tissues. (H) EREG gene mutations in HNSCC tissues according to the cBioPortal for Cancer Genomics. (I) High EREG expression significantly correlates with the poor survival rate of HNSCC patients. The survival rates of patients with EREG-positive and EREG-negative tumors (P< 0.01) were determined using the Kaplan-Meier survival test

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG promotes HNSCC tumorigenicity in vitro and in vivo . (A) Effect of EREG on the cell growth of 3D-cultured HNSCC cancer cell lines. Three HNSCC cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2, 4, 6, 8, 10 and 12. Each cell line was treated with or without EREG from day 1 through day 11. Scale bars indicate 100 µm. (B) The growth of 3D-cultured CAL27, HN6 and HN13 cells treated with or without EREG was analyzed. Each data point represents the mean value and standard deviation of 3 replicate wells. (C) HN4 cells stably transfected with control or EREG-specific shRNAs were injected into nude mice. Tumor growth was monitored every 3 days; tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice. *P < 0.05 and **P < 0.01 for vector control cells compared with their EREG-knockdown clones. (D) HN6 cells stably transfected with control or Flag-EREG constructs were injected into nude mice. Tumor growth was monitored every 3 days, and tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice. *P < 0.05 and **P < 0.01 for the vector control cells compared with the Flag-EREG clones.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: In Vitro, In Vivo, Cell Culture, Standard Deviation, Stable Transfection, Transfection, Control, Injection, Plasmid Preparation, Knockdown, Clone Assay, Construct

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG associates with EGFR and triggers EGFR signaling. (A) Immunoblot (IB) of HN13 and HN6 cells treated with epiregulin (50 ng/ml) at the indicated time points and probed with an anti-phosphotyrosine (p-Tyr) antibody. (B) IB of SACC and HNSCC cancer cells treated with epiregulin (50 ng/ml) for 5 min and probed with an anti-p-Tyr antibody. (C) Human phospho-RTK antibody array analysis of HN6 cells serum starved for 24 hr, followed by epiregulin (50 ng/mL) treatment for 5 min. (D) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at various time points. (E) Immunofluorescence staining for EGFR in HN6 cells treated with or without 50 ng/mL epiregulin.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Western Blot, Ab Array, Immunofluorescence, Staining

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG triggers EGFR downstream signaling in an EGFR kinase-dependent manner. (A) HN13 and HN6 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and IB with the indicated antibodies. (B) IB of HN6 and HN13 cells transfected with individual small interfering RNAs (siRNAs) against EGFR in the presence or absence of epiregulin (50 ng/ml). (C) IB of HN6 and HN12 cells treated with or without epiregulin (50 ng/ml). (D) Human phosphokinase antibody array analysis of HN6 cells treated with or without epiregulin (50 ng/ml) for 5 min. (E) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at different time points. (F) HN6 and HN13 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and immunoblotting (IB) with the indicated antibodies.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Transfection, Ab Array, Western Blot

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG binds to EGFR via N57 and requires the EGFR domains I and III. (A) HEK293 cells were transiently coexpressed with FLAG-EREG and HA-tagged EGFR. Cell extracts were immunoprecipitated separately with anti-FLAG or anti-HA antibodies, and the associated EGFR and EREG proteins were examined by Western blotting. (B) Endogenous EGFR and EREG were immunoprecipitated from HN4 cells, and bound endogenous EREG and EGFR were examined by Western blotting. (C) The cellular location of EGFR (red) and EREG (green) was examined by immunofluorescence staining (nuclei were stained with DAPI; blue). Scale bar, 50 μm. (D) Schematic diagram of the WT, domain I deletion (∆D1), and domain III mutation (D355T/F357A) constructs of EGFR (FLAG-EGFR-ECD). The numbers represent amino acid residues. (E-F) FLAG-tagged WT or deletion mutants of EGFR were coexpressed with HA-EREG in HEK293 cells. Extracts were immunoprecipitated with an anti-FLAG or anti-HA antibody, and bound EREG or EGFR was examined by Western blotting using the anti-HA or anti-FLAG antibody (for input control, see Figure S4C ). (G) Sequence alignment of EREG from different species. (H) Schematic diagram of various EREG NQ mutants used in this study. The numbers indicate amino acid positions on the EREG. (I-J) HA-tagged WT or NQ mutants of EREG were coexpressed with FLAG-EGFR in HEK293 cells. EREG and EGFR were immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and analyzed by Western blotting.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Mutagenesis, Construct, Control, Sequencing

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG-induced C-Myc expression is required for EREG-promoted oncogenesis in HNSCC. (A) EREG-related gene enrichment analysis from TCGA using the cBio Cancer Genomics Portal. (B) Western blot analysis for C-Myc from three different HNSCC cell lines treated with 50 ng/mL epiregulin as indicated. (C) Western blot analysis of EREG and C-Myc expression in HN4 and HN30 cells after transfection with siEREG or siNC siRNAs. (D) RT-PCR analysis of C-Myc mRNA levels in HN13 and HN6 cells treated with 50 ng/mL epiregulin as indicated. (E) CAL27 and HN13 cells were pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h. The level of C-Myc was examined by Western blot analysis. (F) 3D culture of HN6 cells treated with or without epiregulin and BET BD inhibitors. Scale bar=100 μm. (G) A correlation was found between EREG and C-Myc at the mRNA level in four gene expression data sets.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG-induced C-Myc expression depends on EGFR activity. (A) Western blot analysis of C-Myc, p-EGFR, and EGFR from tumor cell lines pretreated with various EGFR inhibitors for 1 h followed by stimulation with epiregulin for 2 h. (B) OncoPrint of EREG-EGFR-MYC pathway alterations in HNC. Genomic alterations of different members of this pathway are mutually exclusive. (C) Western blot analysis of C-Myc, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, and STAT3 from CAL27 and HN6 cells pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EGFR-Erk activation by epiregulin is sustained. (A) Representative time courses of EGFR phosphorylation at Y1086 in SACC-83 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (B) Representative time courses of EGFR phosphorylation at Y1173 in CAL27 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (C-D) Quantification of EGFR phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantitating phosphorylation at Y1068 and Y1173. (E-F) Representative time courses of Erk phosphorylation in SACC-83 and CAL27 cells induced by different EGFR ligands. (G-H) Quantification of Erk phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantifying Erk phosphorylation.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Activation Assay, Phospho-proteomics, Control

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: High EREG expression predicted a better sensitivity to erlotinib treatment in HNSCC. (A) Cell extracts were prepared from 6 HNC cell lines, which were divided into two groups based on EREG expression, and EREG expression was analyzed by Western blotting. (B-C) Colony formation was assessed in many cancer cell lines based on EREG expression levels with erlotinib (2 µM) treatment. The number of colonies was calculated. (D-E) Effect of erlotinib on the cell growth of 3D-cultured HN4 and HN12 cells. High-EREG-expressing HN4 and low-EREG-expressing HN12 cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2 and 12. Each cell line was treated with or without erlotinib from day 1 through day 11. Scale bars indicate 100 µm. (F-G) Colony-formation analysis was performed on SACC-83 and HN30 cells. The low-EREG-expressing SACC-83 cells were treated with erlotinib, epiregulin or erlotinib plus epiregulin, and the high-EREG-expressing HN30 cells stably transfected with control or EREG-specific shRNAs were treated with or without erlotinib. The number of colonies was calculated. (H-I) Effect of erlotinib on the cell growth of 3D-cultured high-EREG-expressing HN30 cells stably transfected with control or EREG-specific shRNAs. Scale bars indicate 100 µm. * P<0.05. (J-K) Effect of epiregulin and erlotinib on the cell growth of 3D-cultured CAL27 cells. Low-EREG-expressing CAL27 cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2 and 12. Each cell line was treated with epiregulin, erlotinib or epiregulin plus erlotinib from day 1 through day 11. Scale bars indicate 100 µm. (L-O) HN30 cells stably transfected with control or EREG-specific shRNAs were injected into nude mice followed by treatment with or without erlotinib. Tumor growth was monitored every 3 days; tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Expressing, Western Blot, Cell Culture, Stable Transfection, Transfection, Control, Injection

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: The proposed model showing that EGFR domains I and III and the N57 residue of EREG are required for EREG-induced EGFR-Erk-C-Myc signaling activation, which in turn promotes oncogenesis and increases erlotinib sensitivity in HNSCC patients.

Article Snippet: Recombinant human epiregulin, EGF, AREG, and TGF-α were purchased from R&D Systems (MN, USA).

Techniques: Residue, Activation Assay

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a WT and TG2 −/− mice ( n = 11/group) were treated with IMQ for four consecutive days. Cytokine and chemokine mRNA levels in the back skin epidermis were measured by RT-PCR. b , c Primary keratinocytes prepared from WT and TG2 −/− mice were treated with IMQ (200 μM) or Aldara cream (25 μM) for 3 h. Ccl20 , Il6 , and Cxcl1 mRNA ( b ) and protein levels in the media ( c ) were measured by RT-PCR and a multiplex cytometric bead array, respectively ( n = 3/group). d , e HaCaT cells stably transfected with control and TG2 shRNA were treated with IMQ (200 μM) or Aldara cream (25 μM). IL6 , CXCL8 , and CCL20 mRNA ( d ) and protein levels in the media ( e ) were measured by RT-PCR after 3 h and a multiplex cytometric bead array after 6 h, respectively. Protein levels were normalized to total cellular soluble protein ( n = 3/group). f Human primary keratinocytes stably transfected with control or TG2 shRNA were treated with IMQ (200 μM; n = 3). IL6 , CXCL8 , and CCL20 mRNA levels were measured by RT-PCR after 3 h. Data represent the mean ± SEM ( n = 3/group). * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Reverse Transcription Polymerase Chain Reaction, Cream, Multiplex Assay, Stable Transfection, Transfection, Control, shRNA

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a HaCaT cells stably transfected with control and TG2 shRNA were treated with IMQ, and TG2 mRNA levels were determined by RT-PCR. b HaCaT cells were treated with IMQ in the presence of 5-(biotinamido) pentylamine (BP), and their lysates were subjected to western blot analysis with peroxidase-conjugated streptavidin to detect BP-incorporated proteins. In situ TG activity was measured according to band intensity by ImageJ software ( n = 3/group). c HaCaT cells co-transfected with NF-κB-Luc reporters and phRL-TK control vectors were treated with IMQ for 6 h. NF-κB activity was represented by dual luciferase activity normalized to the control ( n = 3/group). d Schematic representation of WT and mutated CCL20-promoter reporter plasmids. Transcription factor-binding sites are indicated. e HaCaT cells co-transfected with reporter constructs and phRL-TK control vectors for 48 h were treated with 200 μM IMQ for 12 h. CCL20-promoter activity was estimated by normalizing firefly luciferase luminescence to phRL-TK vector Renilla luciferase. Data are relative to the pGL3 control ( n = 4/group) and represent the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Stable Transfection, Transfection, Control, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, In Situ, Activity Assay, Software, Luciferase, Binding Assay, Construct, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a , b Effect of DMSO- or IMQ-treated WT- or TG2 −/− -CM on CCR6 + γδT-cell ( a ) and CD11b + Ly-6G + cell ( b ) migration according to Transwell migration assays. Flow cytometric analysis of the percentage of cells migrating toward the CM in the presence or absence of anti-CXCL1 ( a ) and anti-CCL20 ( b ) antibodies ( n = 3/group). c , d WT and TG2 −/− mouse-derived CCR6 + γδT-cell ( c ) and CD11b + Ly-6G + cell ( d ) migration in response to recombinant mouse CCL20 and CXCL1, respectively. Data represent the mean ± SEM ( n = 3/group). * p < 0.05; ** p < 0.01. e , f Cells were isolated from the back skin of WT and TG2 −/− mice treated with IMQ for four consecutive days and analyzed by flow cytometry. Representative flow cytometric profiles of CCR6 + γδTCR low cells ( e ) and CD11b high Ly-6G + cells ( f ). The percentage of cells is shown. Data represent the mean ± SEM ( n = 9/group). g Skin sections were immunostained with CCR6 (green)- and IL-17 (red)-specific antibodies. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. h – j Back skin was separated into epidermis and dermis and analyzed by RT-PCR for IL-17-producing γδT-cell markers ( h ) ( Ccr6 and Il23r ), IL17 and related cytokines ( i ) ( Il17a , Il17f , and Il22 ), and myeloid cell-derived cytokines ( j ) ( Il12a and Il23a ). Data represent the mean ± SEM ( n = 11/group). * p < 0.05; ** p < 0.01 vs. WT mice.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Migration, Derivative Assay, Recombinant, Isolation, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Keratinocyte transglutaminase 2 promotes CCR6 + γδT-cell recruitment by upregulating CCL20 in psoriatic inflammation

doi: 10.1038/s41419-020-2495-z

Figure Lengend Snippet: a – d Gene-expression profile and TG2 correlation were analyzed using reported microarray data from 64 normal controls (NN) and normal skin lesions (PN) and psoriatic lesions (PP) from 58 psoriatic patients. a TG2 mRNA levels in NN, PN, and PP groups. * p < 0.05; ** p < 0.01; *** p < 0.001. b Correlation between TG2 levels and 23,520 genes was analyzed by Spearman’s rank correlation. The ranking and correlation coefficient of the indicated genes are presented in the plots. Scatter plots of correlation between TG2 expression and IL6, CXCL8 , and CCL20 expression ( c ) and IL17F, IL17A , and IL22 expression ( d ) in NN, PN, and PP groups.

Article Snippet: Recombinant mouse CCL20 (BioLegend, 582304), CXCL1 (BioLegend, 573704), or conditioned media collected from WT or TG2 −/− mouse primary keratinocytes treated with IMQ for 24 h with or without 1 μg/ml of anti-CCL20 (R&D Systems, MAB7601) or anti-CXCL1 (R&D Systems, MAB453) neutralizing antibody were placed on the lower chamber.

Techniques: Gene Expression, Microarray, Expressing

Journal: eLife

Article Title: Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation

doi: 10.7554/eLife.71929

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , SNU5 , ATCC , ATCC-CRL-5973 , Cultured in IMDM, 20%FBS, and 1% GlutaMAX..

Techniques: Expressing, Reverse Transcription, Cell Culture, Recombinant, Plasmid Preparation, Retroviral, Construct, Control, shRNA, Luciferase, Sequencing, Modification, Transfection, Negative Control, Microarray, Methylation, Imaging, Isolation, SYBR Green Assay, DNA Methylation Assay, Glutathione Assay, Software, Staining

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Graphical representation of Esrp1 (blue) and Esrp2 (red) expression in mouse tissues and cells (BioGPS) . Relatively overlapping expression patterns for Esrp1 and Esrp2, except in the Liver where Esrp2 is expressed and Esrp1 is not. ( B ) (Panel A ) Array of whole-body sections with e10.5 (#1–#3), e12.5 (#4 and #5), e15.5 (#6 and #7) and postnatal (#8 and #9) mice following staining with cresyl violet. (Panel B ) X-ray film autoradiography detection of Esrp2 mRNA, seen as bright labeling. The labeling is clearly detectable at stage e15.5 in the skin. Pronounced signal is detectable in postnatal mice skin, olfactory neuroepithelium, esophagus, stomach and rectum. Moderate labeling is seen in submaxillary gland, liver, lung and kidney. (Panel C ) Control (sense) hybridization in an adjacent section comparable to (panel B ). ( C ) (Panel A ) Whole-body sections of adult mouse (male) following staining with cresyl violet. (Panel B ) X-ray film autoradiography detection of Esrp2 mRNA. The expression pattern encompasses skin, stomach, intestine and gut-associated salivary glands and liver. In the stomach, Esrp2 is expressed in glandular epithelium, but not in non-glandular epithelium. Non-specific labeling is seen in bone (*) and thymus (**). (Panel C ) Control (sense) hybridization. Abbreviations: AT—adipose tissue; Br—brain; C —colon; Es—esophagus; H—heart; K—kidney; Li—liver; Lu—lung; Mu—skeletal muscles; NC—nasal chamber; ONE—olfactory neuroepithelium; Pa—pancreas; R(embryo)/Re(adult)—rectum; Re (embryo)—retina; Sm—submaxillary gland; St—stomach; Tc—telencephalon; Th—thymus; UB—urinary bladder; (as)—antisense; (s)—sense (Magnification: 2.4×). ( D ) Digoxigenin-UTP-labeled in situ hybridization of Esrp1 in P0 epidermis of WT ( Esrp1 +/+ , Esrp2 +/+ ) and KO ( Esrp1 −/− , Esrp2 +/+ ). Esrp1 expression in restricted to the basal keratinocyte (including epithelial cells of the hair follicle) to the granular layer of the epidermis, and absence of signal in the Esrp1 KO epidermis. E: Epidermis, D: Dermis, HF: Hair follicle. ( E ) Expression of Esrp1 and Esrp2 from published microarrays. Esrp1 and Esrp2 are enriched in the epithelial compartments of the skin and associated appendages: Epidermis, Matrix, Outer Root Sheath (ORS), Bulge cells, and Hair Germ (HG) while absent in the non-epithelial cells: Dermis, Dermal Papilla (DP), and Melanocytes. Graphs from left to right: ( , GSE10773), ( , GSE3142), and ( , GSE15185) represent Esrp1 and Esrp2 expression from publically available microarray data. DOI: http://dx.doi.org/10.7554/eLife.08954.004

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Expressing, Staining, Autoradiography, Labeling, Control, Hybridization, Muscles, In Situ Hybridization, Microarray

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. ( B ) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. ( C ) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. ( D ) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. ( E ) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented. DOI: http://dx.doi.org/10.7554/eLife.08954.005

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Knock-In, Southern Blot, Biomarker Discovery, Injection, Sequencing, Construct, Generated, Knock-Out

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: Sagittal sections were generated from WT, KO, and DKO E15.5 embryos. Indicated organs were evaluated for gross morphological defects by H&E staining of sections. Esrp1/Esrp2 DKO embryos do not form lungs (*) and salivary glands (arrow). Thoracic, lung, and kidney images were taken at 4×. Thymus and salivary images were taken at 10× (except for the DKO salivary section for orientation and clear indication of salivary gland agenesis) (n = 3 for each genetic group). DOI: http://dx.doi.org/10.7554/eLife.08954.007

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Generated, Staining

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Representative H&E stained sections of dorsal skin from control Esrp1 +/+ , Esrp2 −/− and Esrp DKO ( Esrp1 −/− , Esrp2 −/− ) E18.5 embryos used in B – E . ( B – D ) Metrics of epidermal thickness measured from basal keratinocyte layer to granular layer ( B ), hair follicle number measured over 9 fields of view ( C ), and hair follicle stages from Esrp DKO (n = 6) and control (CON) (n = 8) littermates. Two-tailed Student's t -test was used for B , C and 2way ANOVA multiple comparisons test for D . ( E ) Immunofluorescence of skin differentiation markers for basal keratinocytes (Krt14 and p63), spinous layer (K10), cornified layer (Loricrin (Lor)), and granular layer (Filaggrin (Fil)). β-catenin and its transcriptional target Lef1 are markers of developing hair follicles (n = 3). DOI: http://dx.doi.org/10.7554/eLife.08954.008

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Staining, Control, Two Tailed Test, Immunofluorescence

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) qRT-PCR expression of Esrp1 and Esrp2 in purified E18.5 epidermis from the designated genetics of Esrp1 and Esrp2 KO embryos (n = 3). Western blot confirmation of Esrp1 and Esrp2 KO in purified epidermis from E18.5 embryos (n = 2). ( B ) Graphical representation of the epithelial -IIIb exon inclusion rates for Fgfr1 , Fgfr2 , and Fgfr3 in epidermis (n = 3). ( C ) Esrp regulated splicing events in Enah and Arhgef11. Graphical representation of Percent Spliced in (PSI) are presented (n = 3). Two-way ANOVA multiple comparisons tests statistical analysis was used and all groups were compared to Esrp1 +/+ , Esrp2 +/+ (WT). Statistical indications for p-values, *<0.05, **<0.01, ***<0.001, ****<0.0001. DOI: http://dx.doi.org/10.7554/eLife.08954.010

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Quantitative RT-PCR, Expressing, Purification, Western Blot

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Heatmap representing the predicted ΔPSI values for skipped exon (SE) events from KO, KH, and DKO epidermis compared to WT. Summary table of total detected splicing events in KO, KH, and DKO strand-specific RNA-seq. Venn diagram depicting overlap in detected SE events in KO, KH, and DKO datasets. Splicing events detected by rMATS at a FDR < 5% and |deltaPSI| ≥ 5% are depicted here. ( B ) Graphs for PSI for six Esrp targets are shown. Representative radioactive RT-PCR PAGE gels are presented. All events measured are from three independent biological samples. Two-way ANOVA multiple comparisons tests statistical analysis was used and all groups were compared to Esrp1 +/+ , Esrp2 +/+ (WT). ( C ) Representative UCSC custom genome browser snapshots of Esrp1 KO full switch ( Fam213b ), gradual ( Lsm14b ), and DKO only ( Arhgef10l ) SE splicing events. Negative strand transcripts are shown in faded colors compared to bold colored positive strand transcripts. ( D ) Graphical representation of the predicted deltaPSI of 25 ssRNA-seq targets from WT vs DKO rMATS analysis, compared to the RT-PCR validated deltaPSI. Pierson Correlation with r- and p-values is indicated. DOI: http://dx.doi.org/10.7554/eLife.08954.011 10.7554/eLife.08954.012 Figure 4—source data 1. rMATS analysis of Esrp deficient epidermis. DOI: http://dx.doi.org/10.7554/eLife.08954.012 10.7554/eLife.08954.013 Figure 4—source data 2. RNAseq and RT-PCR validated SE splicing events. DOI: http://dx.doi.org/10.7554/eLife.08954.013

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Strand-specific RNA-seq custom UCSC genome browser snapshots of Fgfr1- , Fgfr2- , and Fgfr3-IIIb and -IIIc mutually exclusive splicing events. These tracks confirm the variable sensitivity of this family of mutually exclusive splicing events to loss of Esrp1 and Esrp2. All three Fgfr transcripts include the mesenchymally expressed -IIIc isoform when both Esrp1 and Esrp2 are knocked out. Fgfr2 shows no switch in splicing until both Esrps are ablated, whereas both Fgfr1 and Fgfr3 show variable sensitivity to Esrp1 and Esrp2 loss. Negative strand transcripts are shown as faded colors compared to bold colored positive strand transcripts. ( B ) Strand-specific RNA-seq custom UCSC genome browser snapshot of the Cd44 gene. Inclusion of the variable region (10 exons) results in the multiple isoforms termed the Cd44v which are expressed in epithelial cells. KO of Esrp1 and Esrp2 results in full conversion of Cd44v to the mesenchymally expressed Cd44s (short) isoform. This confirms the Esrps also regulate the complex splicing of 10 consecutive exons in Cd44 in vivo. Representative gel of an ethidium bromide stained agarose gel of a Cd44 RT-PCR is shown (n = 3). DOI: http://dx.doi.org/10.7554/eLife.08954.014

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: RNA Sequencing, In Vivo, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Haematologica

Article Title: The Sox4/Tcf7l1 axis promotes progression of BCR-ABL -positive acute lymphoblastic leukemia

doi: 10.3324/haematol.2014.104695

Figure Lengend Snippet: Identification of Sox4-regulated genes in ALL. (A) Genes differentially expressed in transformed Sox4fl/flSE-Cre and Sox4fl/+SE-Cre cells. Fetal liver (FL) or bone marrow (BM) pro-B cells from Sox4fl/fl or Sox4fl/+ mice were transformed with p190 BCR-ABL and transduced with SE-Cre. Gene expression microarray experiments were performed and results were compared between the two types of cells. Genes with a Sox4fl/flSE-Cre/Sox4fl/+SE-Cre signal intensity ratio of <1/3 and P<0.0001 were listed. (B) Analysis of Sox4 mRNA expression by real-time RT-PCR (left) and protein by western blotting (right) in transformed Sox4fl/+SE-Cre, Sox4fl/flSE-Cre, and Sox4fl/flSE-Cre;Sox4 pro-B cells. (C) Ratio (Sox4fl/flSE-Cre;Sox4 to Sox4fl/flSE-Cre) of mRNA expression levels of differentially expressed genes in both bone marrow (BM) and fetal liver (FL) derived transformed cells by real-time RT-PCR. The relative mRNA levels of specific genes were normalized to the level of Gapdh mRNA. Note that the ratios for most genes tested were substantially above 1, suggesting that expression of these genes was reversed upon ectopic Sox4 expression. (D) Scatter diagram demonstrating the correlation between Tcf7l1 and Sox4 expression. Tcf7l1 mRNA level is correlated with Sox4 mRNA level in leukemic cells from patients with ALL (n=11; r=0.666; P=0.0253). Expression of Gapdh was used for normalization of the RT-PCR results. (E) Enrichment of Tcf7l1 promoter sequence in Sox4-specific ChIP DNA by quantitative PCR in a bioChIP analysis. Biotin-conjugating enzyme, BirA ligase, and biotin acceptor peptide (BAP)-tagged Sox4 (BAP-Sox4) (BAP served as control) were introduced into the p190 BCR-ABL-transformed pro-B cells that had Sox4fl/fl deletion (Sox4fl/flSE-Cre). Chromatin was pulled down by magnetic beads conjugated with streptavidin (Dynabeads® MyOne™ Streptavidin T1; Invitrogen, Grand Island, NY, USA). The Sox4-specific and the control ChIP DNA was purified and subjected to real-time PCR for expression of the Tcf7l1 promoter sequence. (F) Mutational analysis of a potential Sox4 binding sequence in the Tcf7l1 promoter. The 460 bp fragment upstream of the transcription start site contains the potential Sox4 binding sequence in which mutations were introduced (-28 to -23bp: ‘ctttgt’ to ‘tgctag’) by PCR (Online Supplementary Methods). This fragment was used to construct mutant reporter pSIN-luc plasmid (MU). BCR-ABL-transformed BAP and BAP-Sox4 pro-B cells were transduced with retrovirus carrying wild-type sequence (WT), MU vector, or empty vector and luciferase activities were determined 2 days after transduction using empty vector as the background control. Data are representative of three independent experiments. Values are means ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: In the presence of biotin, BirA catalyzes conjugation of biotin to BAP-Sox4 which can then be specifically pulled down, together with bound DNA fragments (Sox4 specific ChIP DNA), by magnetic beads conjugated with streptavidin (Dynabeads ® MyOneTM Streptavidin T1; Invitrogen, Grand Island, NY, USA).

Techniques: Transformation Assay, Transduction, Gene Expression, Microarray, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Control, Magnetic Beads, Purification, Binding Assay, Construct, Mutagenesis, Plasmid Preparation, Luciferase

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 is up-regulated in Lung adenocarcinoma (LUAD) and correlates with a poor prognosis for LUAD patients. (A~C) SAM68 mRNA levels was up-regulated in LUAD compared to normal lung tissue based on the Oncomine, GEO and TCGA database. (D~G) From TCGA LUAD specimen cohorts, compared with the patients with low expression level of SAM68 , the patients with high mRNA expression of SAM68 had higher recurrence rates and death rates, shorter RFS, and OS. (H~J) The SAM68 mRNA and protein levels were detected by qPCR, RT-PCR and Western blot in the LUAD tissues (T) and their corresponding adjacent non-tumoral tissues (N). (K) IHC analysis of the expression of SAM68 protein was represented in LUAD tissues and the corresponding adjacent normal lung tissues. (L) Differences in expression levels of SAM68 protein in LUAD tissues (n = 50) and adjacent normal lung tissues (n = 50). Two-tailed t-tests were used B, C, H and L. Pearson Chi-Square tests were used in D and F.Log rank tests were used in E and G.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Downregulation of SAM68 inhibited the malignant phenotypes of LUAD cells in vitro and tumorigenesis and progression in vivo . (A and B) NCI-H1975 and A549 cells were transfected with anti-SAM68 siRNAs, SAM68 mRNA (A) and protein levels (B) level were determined by qPCR and Western blot, respectively. (C) After SAM68 silencing, SAM68 was immuno-stained with anti-SAM68 antibody in NCI-H1975 cells. (D-F) The effects of SAM68 silencing on NCI-H1975 and A549 cells growth (D), colony formation (E), and migration and invasion (F) were detected. (G) CRISPR-Cas9 mediated knockout (KO) of SAM68 in NCI-H1975 cells as detected by immunofluorescence. (H) The in vivo growth of SAM68 KO (sgSAM68) NCI-H1975 cellswas detected (n = 6). (I)The in vivo lung metastasis of SAM68 KO NCI-H1975 cells was examined (n = 5). (J and K) The hematoxylin and eosin (HE) staining and Kaplan-Meier curves are shown for two cohorts of transplanted mice carrying SAM68 KO cells and control groups. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-tailed t-tests were used A, E, F and I. Two-way ANOVA was used in D. Log rank tests was used in L.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: In Vitro, In Vivo, Transfection, Western Blot, Staining, Migration, CRISPR, Knock-Out, Immunofluorescence, Control, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 binds to the Glycine residues in RGG box of hnRNP A1. (A) HEK293T cells were transfected with Flag-SAM68. After Co-IP, proteins that interacted with Flag-SAM68 were identified using silver staining combined mass spectrometry. (B) The SAM68-interacting proteins were performed by GO analysis. (C and D) The unique peptide of SAM68 (C) and hnRNP (D) A1 were identified by mass spectrometry. (E and F) Flag-SAM68 and HA-hnRNP A1 plasmid were transfected into NCI-H1975 cells, cellular lysates were treated with 10 mg/mL RNase A (Thermofisher, EN0531) for 1 h or no treatment, Flag-SAM68 complexes were co-immunoprecipitated by anti-Flag antibody, then hnRNP A1 was detected (E), and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-HA antibody, then SAM68 was detected (F). (G) Confocal images of endogenous SAM68 and hnRNP A1 in NCI-H1975 cells. (H) Wild-type (WT) and indicated mutations with the different domain of hnRNP A1 were constructed. (I and J) The indicated HA-hnRNP A1 WT and mutation constructs and Flag-SAM68 were co-transfected into HEK293T cells, Flag-SAM68 and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-Flag and HA antibody, respectively; HA-hnRNP A1 mutants were detected using anti-HA antibodies, and Flag-SAM68 were detected using anti-Flag antibodies. (K and L) The HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 were co-transfected into HEK293T cells, and the interactions of hnRNP A1 RAA mutant with Flag-SAM68 were detected as described in (I) and (J). (M and N) The Flag-SAM68 WT or its indicated mutation with HA-hnRNP A1 were co-transfected into HEK293T cells, the interactions of Flag-SAM68 indicated mutation with HA-hnRNP A1 were detected as described in (I) and (J).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Plasmid Preparation, Immunoprecipitation, Construct, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Silencing hnRNP A1 antagonized the enhancement of malignant phenotypes induced by SAM68 overexpression. (A~D) The anti-hnRNP A1 siRNAs were transfected into NCI-H1975 and A549 cells, hnRNP A1 protein levels (A) level was detected by Western blot, and the effects of silencing hnRNP A1 on NCI-H1975 and A549 cells growth (B), colony formation (C), migration and invasion (D) were detected. (E~H) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975 and A549 cells, the indicated protein levels (E), cell growth (F), colony formation (G), migration and invasion (H) were detected. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-way ANOVA were used in B and F, two-tailed t-tests were used C, D, G and H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Over Expression, Transfection, Western Blot, Migration, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 favors the binding of the RGG motif of hnRNP A1 to the intronic sequences flanking exon 9 (EI9) by binding to the Glycine residues in RGG box of hnRNP A1. (A) Quantification of SAM68-regulated AS events in each category was measured RNA sequencing. (A3SS/A5SS, alternative 3'/5' splice sites, MXE, mutually exclusive exons, RI, retained introns, SE, skipped exons) (B) Changes in PSI values of SAM68-regulated AS events were shown. (C) SAM68 regulated PKM pre-mRNA splicing and promoted PKM2 isoform formation. (D) CLIP assay of SAM68 and hnRNP A1 binding to the PKM pre-mRNA. NCI-H1975 cells were UV-crosslinked and immunoprecipitated with control IgGs or antibodies, as indicated. (E) RNA affinity purification followed by Western blot showed in vitro binding of the indicated biotin-labeled RNAs with endogenous hnRNP A1 or SAM68. (F) The Flag-SAM68 plasmid at the indicated doses was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (G) NCI-H1975 cells were co-transfected with Flag-SAM68 plasmid and the indicated hnRNP A1 mutations, and in vitro binding of EI9 (50-68) RNA probes with WT or the indicated hnRNP A1 mutations was detected. (H) The HA-hnRNP A1 WT or its RAA mutation was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (I) HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 plasmids were co-transfected into NCI-H1975 cells, and in vitro binding of EI9 (50-68) RNA probes with WT or its RAA mutation was detected. (J) The WT SAM68 or its indicated mutant with WT hnRNP A1-HA plasmids were cotransfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Binding Assay, RNA Sequencing, Immunoprecipitation, Control, Affinity Purification, Western Blot, In Vitro, Labeling, Plasmid Preparation, Transfection, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Sam68 and hnRNP A1 cooperated in PKM splicing to decrease PKM1 isoform formation and increase PKM2 isoform formation, then promoting the aerobic glycolysis of LUAD cells. (A and B) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids (A) or anti-SAM68 siRNAs (B), followed by the PKM splicing assay. (C) PKM splicing was performed in SAM68 KO NCI-H1975 cells. (D) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, followed by the PKM splicing assay. (E) PKM splicing was performed in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression. (F) The ratio of PKM2/PKM1 is calculated in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression (G and H) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids, glucose uptake and lactate production were measured. (I and J) Glucose uptake and lactate production were measured in SAM68 KO NCI-H1975 cells. (K and L) NCI-H1975 cells were transfected with anti- hnRNP A1 siRNAs, then glucose uptake (K) and lactate production (L) were detected. (M and N) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, and glucose uptake (M) and lactate production (N) were detected. (O and P) The Flag-SAM68 plasmid and anti-PKM2 siRNAs were co-transfected into NCI-H1975, and glucose uptake (O) and lactate production (P) were detected.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Splicing Assay, Plasmid Preparation, Expressing

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 increases PKM2 isoform formation, and promotes malignant phenotypes and aerobic glycolysis by cooperating to hnRNP A1 . The WT SAM68 or its indicated mutant was transfected into SAM68 KO NCI-H1975 cells; the indicated proteins were detected by Western blot (A), and cell growth (B), colony formation (C and D), migration and invasion (E), PKM splicing (F), glucose uptake (G), and lactate production (H) were detected. (I) A regulatory model of SAM68 on tumorigenesis proposed in this study. Data are represented as mean ± SEM. **p < 0.01 or ***p < 0.001. Two-way ANOVA was used in B; two-tailed t-tests were used in D~ H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Mutagenesis, Transfection, Western Blot, Migration, Two Tailed Test