Journal: Pharmaceutics

Article Title: Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds

doi: 10.3390/pharmaceutics13101611

Figure Lengend Snippet: Chelidonine and Rotenone exhibit potential and selective anti-tumor activity. Data of viability and immunofluorescence assays. ( A ) Viability screening of candidate molecules. ( B ) Selective activity of Chelidonine and Rotenone against transformed fibroblast BJ-EHLT. ( C ) Quantitative analysis of fluorescence intensity of γH2AX signal in BJ-hTERT and BJ-EHLT. ( D ) Representative images of γH2AX fluorescent signal. ns, p > 0.05; *, p < 0.05; ***, p < 0.001.

Article Snippet: Human fibroblasts (BJ-hTERT) were obtained by infecting primary BJ cells with a retrovirus carrying hTERT (Addgene plasmid #1773) resulting in a telomerase immortalized cell line, while BJ-EHLT were derived from the transformation of the same BJ cells with an hTERT and SV40 early region resulting in p53 and pRB silencing [ ].

Techniques: Activity Assay, Immunofluorescence, Transformation Assay, Fluorescence

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold prediction of full-length BLTP3A with ConSurf conservation scores (top left) for each a.a. or cross-section of surface rendering of BLTP3A channel (top right) highlighting hydrophobic residues (orange). Linear representation of BLTP3A: per residue ConSurf scores (top) and RBG organization (bottom). IDR predicted intrinsically disordered region (light gray), CH C-terminal helix (dark gray). ( B ) Alignment of the a.a. of motifs important for the indicated protein interactions of BLTP3B with corresponding sequences of BLTP3A. ConSurf conservation scores for each a.a. is indicated by color (same color scheme as in ( A )). ( C ) Western blot of lysates of wild-type mouse tissues for BLTP3A and vinculin as a loading control. ( D ) Western blot of control and edited (BLTP3A^V5) cell clones for BLTP3A, V5, and alpha-tubulin as a loading control. ( E ) Fluorescence images of endogenously edited (left) or parental control (middle) A549 cells with antibodies against LAMP1 (green) and V5 (magenta). Scale bar, 10 μm. The insets are a zoom of a small region of the cell. Scale bar, 1 μm. Right: Fluorescence of exogenous BLTP3A-GFP (green) in endogenously edited A549 cell. Scale bar, 5 μm. Insets: zoom of square region of cell showing co-localization of endogenous BLTP3A^V5 signal from immunolabeling with antibodies against V5 (magenta) and BLTP3A-GFP fluorescence (green). Scale bar, 1 μm. ( F ) Fluorescence image of wild-type RPE-1 cell immunostained with antibodies against LAMP1 (green) and DAPI (blue). Scale bar, 5 μm. ( G ) Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (large field, inverted grays) and LAMP1-GFP (not shown). Scale bar, 5 μm. The area enclosed by a dotted rectangle is shown at right at high magnification with BLTP3A-mRFP in magenta and LAMP1-GFP in green (individual channels are shown as inverted grays). Red arrows indicate large BLTP3A accumulations not associated with lysosomes. Scale bar, 1 μm. ( H ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles, many of them tethered to the surface of lysosomes. Scale bar, 500 nm. ( I ) Distance between the membranes of lysosomes and tethered vesicles from EM micrographs. Mean = 10.8 nm; standard error of the mean = ±0.20 nm. ( J ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3A-mRFP (inverted grays) and immunolabeled with antibodies against VAMP7 (shown in the high mag fields at right). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3A-mRFP (magenta) and immunolabeled (green) with antibodies against the following endogenous proteins, VAMP4 and ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Residue, Western Blot, Control, Clone Assay, Fluorescence, Immunolabeling, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Live fluorescence images (inverted grays) of RPE-1 cells expressing either GFP-Rab45 (left), BLTP3A-mRFP (center), or both proteins together (only BLTP3A is shown) (right) as indicated. Scale bar, 5 μm. High-magnification scale bar, 2 μm. ( B ) Genomic sequence of the edited BLTP3A locus (insertion of the V5 epitope) in A549 cell. Blue, small Gly-Ser linkers; green, V5 epitope sequence. ( C ) AlphaFold prediction of BLTP3A. The site where the V5 epitope (V904) was inserted is indicated. The long disordered sequence and the C-terminal helix are shown in gray. ( D ) Left: Fluorescence image of an RPE-1 cell expressing exogenous BLTP3B-mRFP (inverted grays) and immunolabeled with antibodies against endogenous VAMP7 (shown at right in the high magnification of the squared region in the main field). Scale bar, 5 μm. Right: zooms of different RPE-1 cells expressing exogenous BLTP3B-mRFP (magenta) and immunolabeled with antibodies (green) against endogenous VAMP4 or ATG9A. Individual channels are shown as inverted grays. Merge of channels on bottom. Scale bar, 1 μm. ( E ) Fluorescence images of RPE-1 cells expressing the indicated BLTP3A-mRFP construct. Scale bar, 5 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Expressing, Sequencing, Immunolabeling, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images of RPE-1 cells expressing exogenous BLTP3A-mRFP (shown in inverted grays) and (not shown) GFP-tagged wild-type Rab7 (left) or dominant negative (DN) Rab7 (T22N) (right). Scale bar, 5 μm. ( B ) CLEM of a BLTP3A-mRFP positive cluster in an RPE-1 cell expressing GFP-tagged dominant negative Rab7. Left: fluorescence image of BLTP3A-mRFP (magenta). Scale bar, 1 μm. Right: EM micrograph of the field shown at left revealing that the BLTP3A-mRFP fluorescence reflects clusters of small vesicles. Scale bar, 500 nm. ( C ) BLTP3 chimeras design. Left: Surface representation of the predicted RBG core of BLTP3A. Red and blue indicate positive and negative charges, respectively, and gray indicates hydrophobic surfaces. Right: Surface representation (top) and ribbon representation (bottom) of the “untwisted” protein showing individual RBG motifs. Bottom: Cartoon of chimeras consisting of BLTP3A (dark orange) and BLTP3B (light orange) RBG motifs. ( D ) High-magnification live fluorescence images of RPE-1 cells expressing the indicated BLTP3-mRFP constructs (magenta) and LAMP1-GFP (green). Individual channels are shown as inverted grays. Scale bar, 1 μm. ( E ) Ribbon representation of the AlphaFold prediction of a.a. 1–336 of BLTP3A. Blue indicates loops connecting adjoining RBG motifs, and gray indicates the first beta-strand of the third RBG motif. ( F ) Fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-1-336-mRFP and either (not shown) GFP-Rab7 (left), or GFP-Rab7 T22N (right). Scale bar, 5 μm. A zoom of an area of the cell at left (dotted square) expressing BLTP3A-1-336-mRFP (magenta) is also shown, along with the Rab7 fluorescence (green), demonstrating the localization of BLTP3A-1-336-mRFP around the entire profile of lysosomes. Individual channels are shown as inverted grays. Scale bar, 2 μm. ( G ) Cartoon depicting the proposed association of BLTP3A vesicle clusters with the surface of lysosomes and the dependence of this association on Rab7.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Expressing, Dominant Negative Mutation, Construct

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Top: Linear representation of BLTP3A RBG organization and C-terminal truncations indicated by arrows. Bottom left: AlphaFold-based structure of the C terminus of BLTP3A channel. Individual residues are colored by conservation scores as Fig. . Truncations are indicated by arrows. Bottom right: Fluorescence images (inverted grays) of RPE-1 cells expressing the indicated BLTP3A-mRFP constructs along with dominant negative Rab7 (not shown) demonstrating that the property of BLTP3A to bind and cluster vesicles is dependent on its region comprised between a.a. 1327 and 1364. Note that the construct 1–1327, shows a focal accumulation next to the nucleus, which is VAMP7 negative, likely reflecting its pool bound to Rab45 (see Fig. ). Scale bar, 5 μm. Zoomed images (dotted squares) are shown below the main field along with VAMP7 fluorescence. Scale bar, 1 μm. ( B ) Western blots of cell extracts (inputs) of control and edited A549 cells, and of material immunoisolated from these extracts by anti-V5 magnetic beads. Immunolabeling for BLTP3A, V5 (endogenously tagged BLTP3A), and for GAPDH as a loading control, are shown. ( C ) Scatter plot of mass spectrometry-identified proteins in immunoisolated material from either control or endogenously edited BLTP3A^V5 A549 cells using anti-V5 magnetic beads ( N = 3, biological replicates). Proteins significantly enriched in material immunoisolated form edited cells compared to wild-type cells are plotted in the right-top quadrant. Proteins of note are labeled in magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Expressing, Construct, Dominant Negative Mutation, Western Blot, Control, Magnetic Beads, Immunolabeling, Mass Spectrometry, Labeling

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-GFP and mApple-IST1 before and after addition of LLOMe. Arrowheads (magenta) point to BLTP3A accumulations shed from lysosomes upon addition of LLOMe. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker NPC1-GFP (green). Fluorescence of individual channels is shown in inverted grays. Scale bar, 2 μm.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Marker

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in wild-type A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( B ) Time-series of live fluorescence images of BLTP3A-1-336-mRFP (magenta) and the lysosomal marker LAMP1-GFP (green) expressed in VPS13C KO A549 cells before and after addition of LLOMe. Fluorescence of individual channels is shown in inverted grays. Scale bar, 5 μm. ( C ) Live fluorescence images (inverted grays) of RPE-1 cells expressing exogenous GFP-LRRK1 K746G (left) and BLTP3A-mRFP (right). A partial association of BLTP3A-mRFP and GFP-LRRK1 K746G was observed. Scale bar, 10 μm. ( D ) Western blot of lysate of RPE-1 cells expressing exogenous RFP-LRRK1 K746G or RFP-LRRK1 D1409A for RFP (to detect LRRK1 fusions), Rab7, phospho-Rab7 S72, and alpha-tubulin as a loading control. Individual lanes are biological replicates.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Marker, Expressing, Western Blot, Control

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Alignment of the region of BLTP3A orthologues from different species centered on the a.a. region required for mATG8 binding in human BLTP3A. The alignment shows a high degree of conservation of the key residues of the LC3-interacting region (LIR) motif among several chordates and also observed in flies. ( B ) Live fluorescence images (inverted grays) of RPE-1 cells expressing BLTP3A-mRFP (top) or GFP-LC3B (bottom) in either fed (left) or starved (right) conditions. Arrows (magenta) indicate GFP-LC3B positive foci. Scale bar, 5 μm. ( C ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3A-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( D ) Time-series of live fluorescence images (inverted grays) of exogenous BLTP3AΔLIR-mRFP and GFP-LC3B before and after addition of LLOMe. Arrowheads point to lysosomes where LC3B, but not BLTP3A lacking a LIR motif, decorates the entire lysosome profile upon addition of LLOMe. Scale bar, 5 μm. ( E ) Correlative fluorescence-FIB-SEM microscopy of GFP-LC3B and BLTP3A-mRFP-positive lysosomes in an RPE-1 cell 15 min after LLOMe addition. Left: Fluorescence image of an RPE-1 cell expressing GFP-LC3B (green), BLTP3A-mRFP (magenta), and mito-BFP (not shown), 15 min after addition of LLOMe. Scale bar, 5 μm. Region of cell for FIB-SEM marked by dotted line. Middle: High magnification of the RPE-1 cell at left (blue square) showing both the BLTP3A-mRFP (magenta) and the GFP-LC3B (green) channels. Individual channels are shown as inverted grays. Merged channel also shows mito-BFP (blue) used for aligning EM and fluorescence images. Scale bar, 2 μm. Right: EM reconstruction (scale bar, 1 μm) corresponding to the blue square from left. Lysosome, dark green; ER, yellow; mitochondria, blue; small vesicles, magenta.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Binding Assay, Fluorescence, Expressing, Microscopy

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) AlphaFold3 multimer prediction of full-length MAP1LC3B (green) and a.a. 1110–1150 of BLTP3A (magenta). Arrows indicate key residues of the LIR motif of BLTP3A. ( B ) AlphaFold3 multimer predictions of mATG8 proteins and a.a. 1110–1150 of BLTP3A with and without the LIR motif (ΔLIR). ( C ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B before and after addition of GPN. Arrowheads point to lysosomes where BLTP3A and LC3B decorate the entire profile upon addition of GPN. Time, seconds. Scale bar, 5 μm. ( D ) Genomic sequence of the edited BLTP3A locus in A549 cell. Blue, gRNA; green, PAM; red, indel mutations. ( E ) Quantification of relative LAMP1 expression from western blots ( N = 3, biological replicates) of Fig. . Error bars indicate the standard error of the mean (SEM).

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Sequencing, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Brightfield image of RPE-1 cells incubated with MSU crystals (200 μg/mL) for 2 h. Arrowheads indicate MSU crystals. Scale bars, 5 μm. ( B ) Live fluorescence images of RPE-1 cells expressing BLTP3A-mRFP and LAMP1-GFP (shown as inverted greys) and incubated with MSU crystals for 2 h. Scale bar, 5 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bars, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles positive for BLTP3A-mRFP and LAMP1-GFP. ( C ) Live fluorescence images of RPE-1 cells co-expressing either BLTP3A-mRFP (top row) or BLTP3AΔLIR-mRFP (bottom row) with GFP-LC3B and incubated with MSU crystals for 2 h. Individual channels are shown as inverted greys. Scale bar, 10 μm. Zoomed images (dotted rectangles) of individual channels are shown to the right. Scale bar, 2 μm. Arrowheads point to the surface of the crystal-containing vacuoles positive for GFP-LC3B to highlight the presence of BLTP3A-mRFP (top) and the absence of BLTP3AΔLIR-mRFP (bottom). ( D ) Time-series of live fluorescence images (inverted grays) of BLTP3A-mRFP and GFP-LC3B in RPE-1 cells incubated with MSU crystals. Scale bar, 2 μm. Arrowheads point to the surface of crystal-containing vacuoles.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Incubation, Fluorescence, Expressing

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Western blots of cell extracts (inputs) of control and edited A549 cells (BLTP3A KO clones) for the proteins indicated. ( B ) Fluorescence images of parental control (left) or BLTP3A KO (right) A549 cells with antibodies against LAMP1 (green). Scale bar, 20 μm. ( C ) Fluorescence images of parental control (top row) or BLTP3A KO (bottom row) A549 cells with antibodies against galectin-3 (inverted greys). Cells were treated with vehicle control (left column) or 1 mM LLOMe (right column). Scale bar, 10 μm. ( D ) Quantification of galectin-3 spots per cell from field ( C ) ( N = 3, biological replicates). Error bars report the standard error of the mean (SEM). ** P < 0.01. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Western Blot, Control, Clone Assay, Fluorescence

Journal: The EMBO Journal

Article Title: BLTP3A is associated with membranes of the late endocytic pathway and is an effector of CASM

doi: 10.1038/s44318-025-00543-9

Figure Lengend Snippet: ( A ) Fluorescence images (inverted greys) of BLTP3A KO A549 cells expressing indicated RFP protein (top row) with antibodies against galectin-3 (bottom row). Dotted magenta line indicates cell boundary. Cells were treated with vehicle control (left column) or 1 mM LLOMe (right three columns). Scale bar, 10 μm. ( B ) Quantification of galectin-3 spots per cell from field ( A ) ( N = 3, biological replicates.). Error bars report the standard error of the mean (SEM). *** P < 0.001; ** P < 0.01; * P < 0.05; n.s., not significant. Mean number of galectin-3 spots per cell and number of cells counted per condition indicated.

Article Snippet: pmCh-N1 BLTP3A 1-1364 , De Camilli lab; this manuscript , RRID:Addgene_241254.

Techniques: Fluorescence, Expressing, Control

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Characterization of Panc-1 NRF2 −/ − cells (A) Luciferase driven by the full (pL) or truncated (pS) NRF2 promoter after ectopic expression of wild-type KRAS or KRASG12D. DMNQ, 1,4-naphthoquinone, is a molecule that produces ROS. (B) Volcano plot of DEGs in NRF2 −/ − compared to WT-Panc-1 cells. (C and D) Gene Set Enrichment Analysis (GSEA) plots showing the enrichment of NRF2 signaling and antioxidant response genes in WT versus NRF2 −/ − Panc-1 cells. (E) Heatmap showing fold expression of NRF2 signaling in NRF2 −/− Panc-1 cells compared to WT cells; genes directly regulated by NRF2 through direct binding to the proximal promoter are marked in green (lane 4). (F) Log 2 (fold change) of major DEGs (p < 0.05). (G) Level of basal ROS in NRF2 −/ − and WT Panc-1 cells. (H) GSH/total glutathione and total thiol groups in NRF2 −/ − and WT Panc-1 cells; Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Luciferase, Expressing, Binding Assay

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: NRF2 depletion affects Panc-1 transcriptome and promotes a deep metabolic reprogramming (A) Functional enrichment analysis of WT cells compared to NRF2 −/− cells. NES = normal enrichment score, FDR = false discovery rate, p= p-value. (B–D) Heatmaps of DEGs relative to glycolysis, PPP and glutathione pathways. (E) DEGs of enzymes involved in glycolysis, PPP and glutathione cycle pathways. (F–H) Expression level of some key enzymes involved in the pathways determined by qRT-PCR. To demonstrate that the enzymes are controlled by the KRAS G12D - NRF2 axis, we re-expressed NRF2 in NRF2 −/− cells and obtained an expression profile similar to that of WT cells. (I) Western blot showing the level of tkt in WT, NRF2 −/− and NRF2 −/− cells in which NRF2 was re-expressed. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Functional Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Panc-1 NRF2 −/ − cells show higher mitochondrial function and a change in oncogenic program (A) Real-time oxygen consumption rate (OCR) was determined during successive treatments with oligomycin (ATP synthase inhibitor), FCCP (uncoupler of oxidative phosphorylation), rotenone (complex I inhibitor) and antimycin-A (complex III inhibitor) in WT, NRF2 −/ − and NRF2 −/ − +NRF2 cells. (B and C) The rates of basal respiration, ATP-coupled respiration, maximal respiration and OCR/ECAR ratio in NRF2 −/− and NRF2 −/ − +NRF2 cells were normalised to total protein content and quantified. (D) NRF2 −/ − cells show a 10-fold higher ROS level than WT cells. When NRF2 is re-expressed in the cells, the ROS level decreases to the level observed in WT cells. (E and F) GSEA plots showing the enrichment of KRAS in WT and MYC in NRF2 −/ − Panc-1 cells. (G) Expression of MYC in WT and NRF2 −/ − cells; XMD8-92 (5 μM) was used to inhibit ERK5 for 24 h. Densitometric analysis of 3 experiments is provided. (H) phosphorylation levels of AKT1 and ERK1/2 in WT, NRF2 −/ − and NRF2 −/ − + NRF2 cells. Densitometric analysis of 4 experiments is provided; data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Phospho-proteomics, Expressing

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: NRF2 depletion leads to the activation of alternative energy supply pathways (A and B) Heatmaps showing DEG clusters involved in arginine/proline and glutamine metabolism. (C) Metabolic network showing the fate of arginine in Panc-1 cells lacking the KRAS G12D -NRF2 axis. The cells respond to NRF2 deletion by channeling arginine toward creatine, polyamines and nitric oxide metabolism. Enzymes involved in the metabolic pathways are shown, ↓ = downregulated, ↑ = upregulated. (D) Log 2 (fold change) of some key DEGs (p < 0.05) involved in arginine metabolism. (E and F) Expression level of some key enzymes determined by qRT-PCR. As a control, we re-expressed NRF2 in NRF2 -/- cells. The enzymes CKM1A and CKM1B are ckb isozymes localized in the mitochondria. (G) Expression of GLUL was measured by western blot. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Arginine catabolism meets the energy needs of NRF2 −/ − cells (A) Creatine biosynthesis intermediates in WT, NRF2 −/ − and NRF2 −/ − added with NRF2 cells. (B) Creatine kinase enzyme activity in WT, NRF2 −/ − and NRF2 −/ − added with NRF2 cells. (C) Western blot showing suppression of CKB with esiRNAs against CKB. (D) ATP levels in WT, NRF2 −/ − and NRF2 −/ − cells re-expressing NRF2. Suppression of CKB leads to a strong decrease in ATP levels. (E) Urea cycle intermediates in WT, NRF2 −/ − and NRF2 −/ − re-expressing NRF2. Data represent the mean ± s.d. of 5 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn’s multiple comparison test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Activity Assay, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Depletion of NRF2 makes Panc-1 cells addicted to amino acids, especially arginine, but less sensitive to glucose restriction (A) % Cell viability relative to DMEM of WT, NRF2 −/− and NRF2 −/− +NRF2 Panc-1 cells in medium without glutamine (-Gln), low glucose (LG), LG without glutamine and 2-deoxyglucose (2DG). (B) Amount of ATP (pmol ATP/cell) in WT and NRF2 −/− cells, clone KO-134 and KO-16 cells in DMEM (high glucose) and LG. (C) % colonies in WT, NRF2 −/− and NRF2 −/− re-expressing NRF2 cells cultured in DMEM, LG without arginine and LG with added arginine. (D) Confocal microscopy images of WT and NRF2 −/− spheroids embedded in Matrigel and cultured in DMEM (high glucose) or LG for 4 days. Length scale bar = 10μm. (E and F) Growth of WT, NRF2 −/− and NRF2 −/− +NRF2 spheroids in DMEM or LG. The percentage of WT and NRF2 −/− spheroids showing invasive/branching structures when grown in DMEM and LG for 4 days; Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn multiple comparisons test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Expressing, Cell Culture, Confocal Microscopy, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Effect of cyclocreatine on PDAC growth (A) Epifluorescence microscopy images of Hoechst + WT and NRF2 −/ − cells invading a 3D matrix after exposure to 20% FBS as chemoattractant for 16 h in the presence or absence of cyclocreatine. (B) Matrigel invasion rate in the absence and presence of CCr of 3 × 10 5 WT and NRF2 −/− cells grown in 0.1% FBS/DMEM or 0.1% FBS/DMEM + 5mM CCr resuspended and seeded in 24mm Transwell with 8.0 μm pore polycarbonate membrane coated with Matrigel solution. Matrix invasion into the lower chamber containing 20% FBS/DMEM was measured 16 h after seeding by staining the invading cells with DAPI. For each experiment, the invasion rate of 5 randomly selected high-power fields was evaluated. (C) ATP production (pmol ATP/cell) in WT and NRF2 −/ − cells treated with 1 and 5 mM cyclocreatine or 5 μM homoarginine for 48 h. (D) Effect on colony formation of anthrathiophenedione 1.6 μM 2a in the absence or presence of 5 μM cyclocreatine (CCr) by WT and NRF2 −/ − cells. (E) Immunoblot evaluation of NRF2 levels in WT (clone 202), NRF2 +/ − (clone 201) and NRF2 −/ − (clone 239) MIA PaCa-2 cells. (F) mRNA expression level of the indicated genes in MIA PaCa-2 clones compared to WT cells. (G) MIA PaCa-2 clones were treated with the indicated mM concentrations of sodium oxamate for 60 h and subjected to a resazurin/resorufin assay. IC50 was calculated using an unadjusted third-degree polynomial regression curve. (H) histogram showing the ATP levels of the indicated cells treated or not with 5 mM CCr for 72 h. Data refer to untreated WT cells. (I) Percentage of colony formation of WT and NRF2 −/ − MIA-PaCa-2 cells in the presence and absence of arginine. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn multiple comparison test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Epifluorescence Microscopy, Membrane, Staining, Western Blot, Expressing, Clone Assay, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: The KRAS/NRF2 axis is switched off and arginine metabolism is switched on in PDAC developing resistance to FOLFIRINOX (A) GSEA plot using as geneset genes downregulated in ID211 compared to ID188 ( Table S2 ) and our RNA-seq dataset. Functional enrichment was performed on core-enriched genes and expressed as FDR (False Discovery Rate). (B) GSEA plot obtained by using as geneset the top 500 genes downregulated in NRF2 −/ − Panc-1 cells and GSE193389 as the dataset. Functional enrichment was performed on the core-enriched genes and expressed as FDR. (C) GSEA plot obtained by using as geneset genes downregulated in PDOs obtained from FOLFIRINOX-treated patients and our RNA-seq dataset. Functional enrichment was performed on the core-enriched genes and expressed as FDR. (D) Heatmap of NRF2-regulated arginine-auxotrophic genes. (E) GSEA plot obtained by using as geneset the “NRF2-regulated arginine-auxotrophic signature” and GSE193389 as the dataset. (F) Heatmap of 177 TCGA-PDAC patients ranked accordingly to the expression of the seven indicated genes representing the “NRF2-regulated arginine-auxotrophic signature”. For each sample, the Winter hypoxia index (W.H.I.) and the fraction of the genome altered (F.G.A.) are indicated above the heatmap. (G) Dot plots showing the expression levels, expressed as z-scores, of NRF2 signaling ( G6PD, GSTM4, NQO1, GCLM, HMOX1, PRDX1 ), LDHA and Winter Hypoxia score in patients with increased expression of the “NRF2-regulated arginine auxotrophic signature” (z > 3, indicated in red) compared to other patients (indicated in gray). (H) Left, Kaplan-Meier survival analysis in 168 TCGA-PDAC patients characterized by high (above the median, in red) or low (below the median, in blue) expression of the “NRF2-regulated arginine auxotrophic signature”; right: histogram representing the frequency of genetic alterations of the indicated oncogenes and tumor suppressors in the two identified patient groups.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: RNA Sequencing, Functional Assay, Expressing

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet:

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Recombinant, GSH Assay, esiRNA

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of MEN1- modulated genes in breast cancer cells. ( A ) RT-qPCR of MEN1 in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( B ) Quantitative Western immunoassays (WES) of menin expression in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( C ) Venn diagrams of differentially expressed genes (fold change ≥1.5 or ≤0.66) in T47D or MCF-7 cells after sh MEN1 knockdown compared with vehicle controls ( n = 2). ( D ) Pathway annotation analysis of MEN1 -upregulated and MEN1 -downregulated genes in T47D or MCF-7 cells using DAVID including cancer hallmark pathways. ( E ) Schematic illustration of five major metabolic pathways. ( F ) Expression heat maps of oxidative phosphorylation (OXPHOS) and glycolytic genes in both MEN1 knockdown T47D and MCF-7 cells (fold changes relative to vehicle controls). ( G ) Bar charts of the expression levels of representative OXPHOS and glycolytic genes affected by MEN1 knockdown in T47D or MCF-7 cells using RT-qPCR. Data are presented as mean ± S.D. Unpaired two-tailed Student’s t -test was used for statistics. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Quantitative RT-PCR, shRNA, Western Blot, Expressing, Knockdown, Phospho-proteomics, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of menin-associated proteins (MAPs) in breast cancer cells. ( A ) and ( B ) WES of BirA-Menin fusion proteins ( A ) and biotin-labeled proteins ( B ) in total lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after incubating with or without doxycycline and biotin. ( C ) Schematic purification and proteomic identification of MAPs using LC–MS/MS. ( D ) Heatmap of the quantification of 35 MAPs commonly shared in T47D and MCF-7 cells. MAPs further verified by WES immunoassays were indicated by arrows. ( E ) Network analysis of 35 MAPs in MCF-7 cells. The distance between menin and MAPs represented the quantitative ratio of each MAP and menin. MAPs marked in blue were further assayed by WES. ( F ) Nuclear or cytoplasmic lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after streptavidin beads pull-down were detected by WES with antibodies against menin, KMT2A, MED12, WAPL, GATA3, LaminA/C, or GAPDH. FL, full length; SP, spliced form.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Labeling, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. ( A ) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. ( B ) Heatmaps of the expression correlation between MEN1 /selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1 –OXPHOS genes or MEN1 –glycolytic genes in breast tumors. ( C ) and ( D ) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes ( C ) or glycolytic genes ( D ) in normal and tumor samples. ( E ) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where ≥median is “high” and

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, In Silico, Gene Expression

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Bioenergetic dynamics are regulated by menin and MAPs in T47D and MCF-7 cells. ( A ) and ( B ) Glycolytic and OXPHOS ATP productions in T47D ( A ) or MCF-7 ( B ) cells infected with vehicle, sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. Statistics represented the difference of glycolytic or OXPHOS ATP production between shRNA knockdown and vehicle controls. ( C ) and ( D ) Bar charts representing mitochondrial functions in the single knockdown of MEN1 , KMT2A , MED12 , WAPL , or GATA3 and their vehicle control in T47D ( C ) or MCF-7 ( D ) cells. ( E ) Schematic summary of mitochondrial dynamics affected by the knockdown of MEN1 or MAPs. ( F , G ) Bar charts represented the glycolytic functions in T47D ( F ) or MCF-7 ( G ) cells subject to gene knockdown by sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. ( H ) Schematic summary of glycolytic functions affected by the knockdown of MEN1 or MAPs. Data are presented as mean ± S.D. ( n = 15–20 technical-replicate wells). Statistical significance was performed by an unpaired two-tailed Student’s t -test between treated groups and corresponding controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Infection, shRNA, Knockdown, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Integrity of the menin–KMT2A complex is required for OXPHOS functions. ( A ) WES of T47D or MCF-7 cells treated with DMSO or 1 μM MI-503 for 3 days (left). Relative protein expression normalized to the average of LaminA/C in WES (right). FL, full length; SP, spliced form. ( B ) Nuclear lysates of T47D or MCF-7 cells treated with DMSO or 1 μM of MI-503 for 3 days were immunoprecipitated with the menin antibody or IgG, and assayed by WES (upper). Relative protein expression in WES (lower). The protein expression in DMSO treated input was normalized as 1. FL, full length; SP, spliced form. ( C ) Glycolytic or OXPHOS ATP production in T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO control for 72 h. ( D , E ) Bar charts of the Seahorse mitochondrial stress test ( D ) and glycolytic stress test ( E ) on T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO for 72 h. Data are presented as mean ± S.D. ( n = 10–15 technical-replicate wells). An unpaired two-tailed Student’s t -test was used to determine statistical significance for the difference between MI-503-treated groups and its controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: MEN1 and OXPHOS expression are increased in breast circulating tumor cells (CTCs). ( A ) t-SNE profile plots and cell clustering of 93 CTCs from 5 breast cancer patients based on the single cell RT-qPCR expression profiling of 11 OXPHOS genes ( NDUFA7 , NDUFA11 , NDUFA13 , NDUFB7 , NDUFS7 , NDUFS8 , NDUFV1 , SDHA , SDHB , SDHC , and SDHD ). ( B ) Violin plots of MEN1 or selected MAPs expression, mean expression of 7 glycolytic genes ( ALDOA , ALDOC , ENO1 , PFKL , PFKP , PGK1 , and TPI1 ) or mean expression of 11 OXPHOS genes (aforementioned) in the five cell clusters. Statistical significance among clusters was carried out using the Duncan multi-range test. ( C ) Mean expression of 7 glycolytic genes and 11 OXPHOS genes in these 93 breast CTCs or in the TCGA primary breast cancer cohort. ( D ) Glycolytic and OXPHOS ATP productions of T47D or MCF-7 cells after circulation ( n = 6–10 technical replicates). ( E , F ) Mitochondrial ( E ) and glycolytic ( F ) functions of T47D or MCF-7 cells after circulation ( n = 5–9 technical-replicate wells). Statistics represented the difference between no circulating control and each treatment. Data are presented as mean ± S.D. An unpaired two-tailed Student’s t -test was used for statistical significance determination. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Quantitative RT-PCR, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: Reciprocal Interaction of Wnt and RXR-α Pathways in Hepatocyte Development and Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0118480

Figure Lengend Snippet: (A) Clustering of RXR-α and Wnt pathway gene expression profiles of normal mouse liver samples taken from four different periods of development. (B) Reporter assays for Wnt and RXR pathways comparing hepatoblasts (E14) to immature hepatocytes (E18). Both TCF (p = 0.016) and (C) RXR-α activities (p = 0.021) were significantly different (D) RNA and (E) protein expression of RXR-α in hepatoblasts compared to immature hepatocytes.

Article Snippet: Human RXRα cDNA was PCR amplified from the pBABE-hygro human RXRα (plasmid 11440 from Addgene) using primers with engineered BglII and XhoI restriction site sequences.

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Reciprocal Interaction of Wnt and RXR-α Pathways in Hepatocyte Development and Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0118480

Figure Lengend Snippet: HCC samples in the Boyault dataset were clustered based on expression of 138 RXR-α and Wnt pathway genes. Beneath the heatmap are four rows, showing for each HCC sample (1) cluster assignment to the two major groups found by unsupervised clustering of all genes (2) relative prognosis based on the 65-gene signature of Kim et al., ranging from red = poor, white = neutral, green = good; (3) grey bars indicate activating mutation in CTNNB1 ; (4) average expression of the 5 genes known to be overexpressed in CTNNB1 -mutant HCC cells; red = expression, green = less expression of the 5-gene signature associated with CTNNB1 mutation.

Article Snippet: Human RXRα cDNA was PCR amplified from the pBABE-hygro human RXRα (plasmid 11440 from Addgene) using primers with engineered BglII and XhoI restriction site sequences.

Techniques: Expressing, Mutagenesis

Journal: PLoS ONE

Article Title: Reciprocal Interaction of Wnt and RXR-α Pathways in Hepatocyte Development and Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0118480

Figure Lengend Snippet: (A) Validation of two independent shRNAs for their ability to lower RXR-α protein levels in hepatoctyes as determined by immunoblotting using Ras Gap protein expression as a loading control and shRNA directed against luciferase as a vector control. B) Tumor growth following subcutaneous injection in nude mice of MYC/p53 -/- ; E18 hepatocytes infected with either shluc (red column), RXRA sh1 (purple column), or RXRA sh2 (blue column). Error bars indicate standard deviations. Tumor incidence is noted above columns for each condition. C ) Survival curves of nude mice after intrasplenic injections of MYC/p53 -/- ; E18 hepatocytes transfected with either shluc (red line), or RXRA sh1 (purple line), or RXRA sh2 (line), n = 10 injections. D) Images of livers taken from mice following transplantation of MYC/p53 -/- E18 hepatocytes transfected with shluc, RXRA sh1, or RXRA sh2. The five panels are from left to right, intact livers, GFP-imaging of livers, hematoxylin and eosin staining of liver tissue sections, PCNA immunohistochemical staining, GFP immunohistochemical staining. Size bar = 200 μm.

Article Snippet: Human RXRα cDNA was PCR amplified from the pBABE-hygro human RXRα (plasmid 11440 from Addgene) using primers with engineered BglII and XhoI restriction site sequences.

Techniques: Biomarker Discovery, Western Blot, Expressing, Control, shRNA, Luciferase, Plasmid Preparation, Injection, Infection, Transfection, Transplantation Assay, Imaging, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Non-centromeric CENP-A epigenetically regulates epithelial-mesenchymal plasticity and heterogeneity in human cells

doi: 10.1101/2025.09.09.672651

Figure Lengend Snippet: EMT programs impacted by increased CENP-A levels and CENP-A mislocalization. (A) mRNA expression analysis of EMT genes in MCF10-2A cells at day 24. Bar plots show log 2 fold change relative to the p53-WT non-induced condition (TMM-normalized). Genes are grouped by signaling pathway (indicated above) and functional category (inflammation or development). Colored bars indicate statistically significant changes (adjusted p-value < 0.05). (B) Western blot analysis of total protein extracts from MCF10-2A cells (-Dox or +Dox), with vinculin as loading control. Primary antibodies are indicated on the right. *IL-6 detection as multiple bands, consistent with its presence as differentially modified (glycosylated) isoforms, with apparent molecular weights from 23 to 30 kDa. (C) β-catenin localization in p53-DN MCF10-2A cells at days 10 and 24. Left: Epifluorescence images showing β-catenin (green), vimentin (red) and DAPI (blue). Scale bars = 40 µm. Right: Proportions of cells with positive β-catenin nuclear signal. Plots show mean ± 95% confidence interval for 4 or 5 independent experiments. Dots indicate mean per experiment. n ≥ 600 cells/state across ≥ 3 fields for each replicate. Statistical significance tested by two-tailed Welch’s t test. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001. (D) ChIP-seq analysis of CENP-A at transcription start sites (TSS) on days 10 and 24 post-induction (relative to non-induced). Top left: Heatmap of CENP-A enrichment at gene TSS. Columns represent protein-coding genes, clustered by changes in CENP-A and H3.3 occupancy at their TSS. Rows indicate relative gain or loss of CENP-A (log 2 -transformed, input-normalized, sequencing depth-normalized, and mean-centered). Corresponding H3.3 heatmap shown in Figure S3. Bottom left: Gene ontology (GO) enrichment analysis for three gene clusters. Resp., Response; Cardiomyo. Adren. Sign., Cardiomyocyte adrenergic signaling; Neuroact., Neuroactive. Right: Relative gain in CENP-A at EMT gene TSS. Colored dots indicate genes significantly upregulated upon CENP-A induction (from panel A).

Article Snippet: For generation of p53-WT and p53-DN cells, we transduced cells with an empty vector (pWZL Hygro; Scott Lowe, Addgene plasmid #18750) or a vector containing a dominant-negative TP53 construct (pBABE-Hygro p53DD, Bob Weinberg, Addgene plasmid #9058) as in Jeffery et al. .

Techniques: Expressing, Functional Assay, Western Blot, Control, Modification, Two Tailed Test, ChIP-sequencing, Transformation Assay, Sequencing

Journal: bioRxiv

Article Title: Non-centromeric CENP-A epigenetically regulates epithelial-mesenchymal plasticity and heterogeneity in human cells

doi: 10.1101/2025.09.09.672651

Figure Lengend Snippet: Single-nucleus multi-omic profiling of EMT programs and trajectories. (A) Experimental workflow to profile snRNA-seq and snATAC-seq simultaneously within the same cells. We collected p53-DN MCF10-2A cells at days 10 and 24 post-induction (-Dox and +Dox). Isolated nuclei were tagmented, then associated with gel beads containing a poly(dT) sequence (for production of barcoded cDNAs) and a Spacer sequence (for barcode attachment to transposed DNA fragments). Incubation of the GEMs and reverse transcription (RT) produced 10x barcoded DNA from the transposed DNA and 10x barcoded cDNA from poly-adenylated mRNAs, that were sequenced with 10X Chromium Multiome. Data integration was performed using Mowgli (Non-negative Matrix Factorization with Optimal Transport). (B) Uniform Manifold Approximation and Projection maps (UMAPs) of Mowgli embeddings. Left and middle: Epithelial and mesenchymal scores based on CDH1 and EPCAM (epithelial), and VIM, FN1 and ZEB1 (mesenchymal) expression. Right: Mesenchymal and non-mesenchymal subpopulations among the samples: non-induced at days 10 (blue) and 24 (green) and induced at days 10 (orange) and 24 (red). (C) Top: UMAP with Leiden clustering applied to Mowgli embeddings. Bottom: GO enrichment analysis of DEGs in clusters 7 and 12, relative to the other clusters. Reg. nuclear division, Regulation of nuclear division; Reg. chr. segregation; Regulation of chromosome segregation; Reg. mit. cell cycle progr., Regulation of mitotic cell cycle progression; Reg. mit. nuclear division, Regulation of mitotic nuclear division; Reg. n. chr. segregation, Regulation of nuclear chromosome segregation; Reg. mit. phase transition, Regulation of mitotic cell cycle phase transition; Ameboidal-type cell migrat., Ameboidal-type cell migration; Reg. of phase transition, Regulation of cell cycle phase transition; Reg. of sister chr. segr., Regulation of sister chromatid segregation; Pos. reg. of cell migration, Positive regulation of cell migration; Pos. reg. of apoptotic proc., Positive regulation of apoptotic processes; Pos. ref. of prog. cell death, Positive regulation of programmed cell death; Reg. mit. sister chr. segr., Regulation of mitotic sister chromatid segregation; Morphogenesis of an epith., Morphogenesis of an epithelium; Pos. reg. of cell migration, Positive regulation of cell migration. (D) UMAP showing inferred cell cycle phases. (E) Cell fate trajectories inferred by RNA velocity. The streamline plot projected onto the UMAP depicts the dynamical modelling. Arrows (gene-averaged velocity vectors) indicate the main directional flow of cell states over time.

Article Snippet: For generation of p53-WT and p53-DN cells, we transduced cells with an empty vector (pWZL Hygro; Scott Lowe, Addgene plasmid #18750) or a vector containing a dominant-negative TP53 construct (pBABE-Hygro p53DD, Bob Weinberg, Addgene plasmid #9058) as in Jeffery et al. .

Techniques: Isolation, Sequencing, Incubation, Reverse Transcription, Produced, Expressing, Sublimation, Migration

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Volcano plot of ATAC-seq peaks identified in BAP1 KOs (n=4) compared to MCF10A-Cas9 controls (n=2) cultured as mammospheres. Blue and red-colored dots represent peaks with loss and gain of ATAC-seq accessibility (log2FC < 0 and log2FC > 0), respectively. (B) Genomic compartment and (C) chromatin state distribution of differential ATAC-seq peaks (FDR < 0.01) identified in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Promoters are defined as regions 500 base pairs (bp) upstream of transcription start sites (TSSs). Kb: kilobases. Pc: Polycomb. (D) Gene ontology enrichment of genes associated with differential ATAC loss peaks (FDR < 0.01, log2FC < 0) falling 2kb upstream from TSSs in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Top 10 enriched ontologies are shown (p adjusted < 0.05). (E) Differential transcription factor binding in BAP1 KO clones compared to MCF10A-Cas9 (Ctrl) cells cultured as mammospheres. Blue: 224 underrepresented motifs (differential binding score < -0.1, p < 0.01); red: 64 overrepresented motifs (differential binding score > 0.1, p < 0.01); and gray: motifs with no representative alteration (-0.1 ≤ differential binding score ≤ 0.1) in ATAC-seq peaks from BAP1 KOs. (F) H2AK119ub1 levels in MCF10A-Cas9 control cells (Ctrl) and two BAP1 knockout (KO) clones (Cl1 and Cl2) cultured in attachment. H3 levels were assessed as an internal control. (G) Distribution of H2AK119ub1 ChIP-seq peaks (attached cells, FDR < 0.05) in regions of ATAC loss (mammosphere cultures, FDR < 0.01, log2FC < 0) identified in BAP1 KOs compared to MCF10A-Cas9 cells. Regions within 2kb upstream and downstream of TSSs and transcription end sites (TESs), respectively, are shown. ChIP-seq reads were normalized with RPGC (reads per genome coverage) per bin method. ChIP-seq peak distribution was analyzed for MCF10A wildtype (MCF10A-WT, n=2), MCF10A-Cas9 controls (MCF10A-Cas9, n=2), and both BAP1 KO clones (BAP1 KO, n=4).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, Binding Assay, Clone Assay, Control, Knock-Out, ChIP-sequencing

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Volcano plot of differential expression analysis in BAP1 KOs compared to MCF10A-Cas9 control cells cultured as mammospheres (n=2 for MCF10A-Cas9 control cells and n=4 for BAP1 KOs). Blue and red-colored dots represent differentially expressed genes (DEGs, FDR < 0.01) with reduced or increased expression (log2FC < 0 and log2FC > 0), respectively. (B) Gene ontology enrichment of downregulated (left) and upregulated (right) DEGs (FDR < 0.01) in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Top 10 enriched gene ontologies are shown (p adjusted < 0.05). (C) Gene set enrichment analysis (GSEA) plots of the transcriptome of BAP1 KOs compared to MCF10A-Cas9 control cells cultured as mammospheres, using the indicated gene sets. NES: normalized enrichment score. (D) Cell cycle analysis by propidium iodide staining in unsynchronized MCF10A-Cas9 and BAP1 KO mammospheres. Each bar graph shows the percentage distribution of cells in each cell cycle phase: G0-G1, S and G2-M. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Expressing, Control, Cell Culture, Cell Cycle Assay, Staining

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) CRISPR/Cas9 screening approach used for the identification of epigenetic regulator genes (ERGs) involved in the acquisition of mesenchymal breast cancer stem cell (BCSC) markers in non-tumorigenic breast cells. Adapted from Halaburkova et al. 2020 . gRNA: guide RNA. (B) Representation of enriched ERG gRNAs (false discovery rate [FDR] < 0.05) identified in the mesenchymal BCSC-like population of MCF10A cells infected with the ERG gRNA library compared to the bulk of cells on the day of sorting (n=2 MCF10A-Cas9 expressing clones). (C) Venn diagram of ERGs showing single nucleotide alterations in BC patients (TCGA-BRCA) of different molecular subtypes. Top 7 mutated ERGs identified in the TNBC subtype (proportion of SNAs (pSNA) > 0.019) are highlighted. (D) Kaplan-Meier analysis of disease-free survival in BC patients (TCGA-BRCA) divided in high and low BAP1 gene expression groups. * P < 0.05, ** P < 0.01.

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: CRISPR, Infection, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) BAP1 protein expression levels in MCF10A-Cas9 control cells (Ctrl), a heterozygous BAP1 deletion clone (HET), and two BAP1 knockout (KO) clones (Cl1 and Cl2). GAPDH levels were assessed as an internal control. (B) Representative images at 10x magnification of single, size-normalized MCF10A-Cas9 control and BAP1 KO mammospheres cultured for 72h. Scale bars: 100 µm. (C) Representative images at 20x magnification of single, size-normalized MCF10A-Cas9 control and BAP1 KO mammospheres stained with hematoxylin and eosin show mammosphere architecture differences and cellular changes between BAP1 KOs and controls. BAP1 KOs show disorganized architecture and a variable amount of intracytoplasmic vacuolization and cellular decohesion, all noted at the periphery of the mammospheres. Scale bars: 100 µm. See also Supplementary Figure S1. (D) Top: distribution of CD44 high and low populations in MCF10A-Cas9 controls and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Fisher’s exact test on absolute cell counts (n=3, **** P < 0.0001). Bottom: proportion of BCSC- and epithelial-like populations based on expression of CD44, CD24 and EpCAM in MCF10A-Cas9 controls and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, ns: not significant). (E) Expression of EMT-associated genes in MCF10A-Cas9 and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Expressing, Control, Knock-Out, Clone Assay, Cell Culture, Staining

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Proportion of DEGs (FDR < 0.01) showing differential (FDR < 0.01) ATAC-seq loss (log 2 FC < 0), gain (log 2 FC > 0) or neutral signal (no loss or gain) within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9. Down: downregulated; up: upregulated. (B) Gene ontology enrichment of downregulated genes (FDR < 0.01, log 2 FC < 0) showing ATAC loss (FDR < 0.01, log 2 FC < 0) within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9. Top 10 enriched ontologies are shown (p adjusted < 0.05). (C) Diamond plot of glycosylation-associated DEGs (FDR < 0.01, GO: 0070085) showing changes in chromatin accessibility within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9 controls. Red: ATAC gain; blue: ATAC loss. y axis: RNA-seq gene expression (log2FC). (D) and (E) Genome browser snapshot of ATAC-seq and H2AK119ub1 peaks in mammosphere-cultured BAP1 KOs and MCF10A-Cas9, at the MGAT4A and ST6GALNAC1 genes. Horizontal bars represent differential ATAC-seq (FDR < 0.01) and/or ChIP-seq (FDR < 0.05) peaks. (F) Heatmap of glycosylation genes showing consistent alteration in chromatin accessibility (ATAC-seq, 2kb upstream of TSSs, left), RNA expression (RNA-seq, middle) and protein expression (proteomics, right) in BAP1 KO mammospheres compared to MCF10A-Cas9 controls. Colors and numbers on the heatmap represent the log2FC between BAP1 KOs and controls for each omics analysis (blue: ATAC loss or downregulation), and asterisks indicate that the change is significant (* FDR < 0.01). Where more than one ATAC-seq peak was annotated to a specific gene, only the closest peak to its TSS is shown in the heatmap.

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, RNA Sequencing Assay, Expressing, ChIP-sequencing, RNA Expression

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Relative abundance of chromatographic peaks identified by N -glycan profiling of mammosphere-cultured MCF10A-Cas9 and BAP1 KO cells. Significance analysis performed by Dunnett’s test (n=4, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant). Top most abundant peaks across samples are shown. GP: glycan peak. (B) O -linked N -acetylglucosamine (O-GlcNAc) expression levels in MCF10A-Cas9 controls (Ctrl) and two BAP1 knockout (KO) clones (Cl1 and Cl2) cultured as mammospheres. GAPDH levels were assessed as an internal control. (C) Top: schematic representation of the constructs used for BAP1 re-expression, containing an HA tag and hygromycin (Hygro) resistance. Representations of the wildtype (WT) and mutant BAP1 (C91S mutation) constructs are shown. Bottom: BAP1 protein expression levels in MCF10A-Cas9 controls (Ctrl), and two BAP1 knockout (KO) clones (Cl1 and Cl2) infected with BAP1 wildtype (BAP) and BAP1 C91S mutant (MUT) rescue plasmids. GAPDH levels were assessed as an internal control. (D) Left: distribution of CD44 high and low populations in BAP1 KO Cl1 and BAP1 rescue cells (wildtype: WT; and mutant: MUT) cultured as mammospheres. Significance analysis performed by Fisher’s exact test on absolute cell counts (n=2, **** P < 0.0001). Right: proportion of BCSC-like populations based on expression of CD44, CD24 and EpCAM in BAP1 KO Cl1 and BAP1 rescue cells (wildtype: WT; and mutant: MUT) cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=2, * P < 0.05, ns: not significant). (E) Expression of glycosyltransferase genes C1GALT1 , GALNT3 , GCNT1 , MGAT4A and ST6GALNAC1 in MCF10A-Cas9 (Ctrl), BAP1 KO cells and BAP1 rescues (wildtype, +; and mutant, mut) cultured as mammospheres. Significance analysis performed by Dunnett’s test compared to control (Ctrl) sample (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, Expressing, Knock-Out, Clone Assay, Control, Construct, Mutagenesis, Infection