Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 2. Inhibition of p38 blocks NGF-induced neurite out- growth. A and B, PC12 cells were pretreated with the indicated con- centrations of SB203580 or 30 mM PD98059 for 30 min prior to treat- ment with 100 ng/ml NGF for 60 h. Representative images under a phase-contrast microscope (A) and quantitation of the percentage of cells with neurites (B) are shown. C and D, cells were cotransfected with pEGFP-C1 together with an empty expression vector SRa (2) or an expression vector encoding kinase-negative MKK6 (KN-MKK6), wild type p38 (WT-p38), or dominant-negative p38 (AGF-p38) (15). After 12 h the cells were treated with or without 10 mM SB203580. Then, 48 h after the transfection the cells were treated with or without 100 ng/ml NGF. Representative images of the transfected cells 60 h after NGF addition identified by the fluorescence of GFP (C) and quantitation of the percentage of cells with neurites (D) are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation, Dominant Negative Mutation, Transfection, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 1. NGF induces p38 activation as well as ERK/MAPK ac- tivation. A, PC12 cells were treated with 100 ng/ml NGF or 50 mM arsenite for the indicated times (left) or with the indicated concentra- tions of NGF for 10 min (right), and the cell extracts were subjected to the immune complex kinase assay for p38 using activating transcrip- tion factor 2, ATF2, as a substrate (upper). The same cell extracts were subjected to immunoblotting with anti-phospho-p38 (middle) or anti- p38 antibodies (bottom). B, cells were pretreated with or without a p38 inhibitor SB203580 at 10 mM (lower or upper, respectively) for 30 min prior to NGF treatment as indicated, and the extracts were subjected to immunoblotting with anti-ERK/MAPK antibody. The electrophoreti- cally retarded bands represent active forms, i.e. phosphorylated forms of ERK/MAPK (ERK1 and ERK2, arrowheads) against inactive forms (arrows).

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Immune Complex Kinase Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 3. Expression of a constitutively active MAPKK/MEK (SE- SE-KK) induces p38 activation as well as ERK/MAPK, and the p38 inhibitor blocks neurite outgrowth induced by SESE-KK. A, PC12 cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or a constitutively active construct of MAPKK/MEK (SESE-KK (13); equivalent to Glu-217/Glu-221 MAPKK/ MEK in Ref. 7) expression vector and were treated with or without 10 mM SB203580. B, cells were cotransfected with HA-p38 or HA-ERK/ MAPK (MAPK) together with an empty expression vector SRa (2) or an expression vector encoding wild type MAPKK/MEK (WT-KK) or SE- SE-KK and assayed for the activity of HA-p38 or HA-MAPK. The activity of HA-p38 was also measured in cells treated with 100 ng/ml NGF for 10 min (1NGF). C, cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or an SESE-KK expres- sion vector and were subjected to immunostaining with anti-phospho- p38 antibody.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Plasmid Preparation, Construct, Activity Assay, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 4. EGF induces transient activation of p38 and, when combined with sustained activation of p38, causes neurite out- growth in PC12 cells. A, PC12 cells were treated with either 30 nM EGF (upper), 50 mM arsenite (upper), or 100 ng/ml NGF (lower) for the indicated times and assayed for p38 activity as described in the legend to Fig. 1A. B, cells were treated with EGF, NGF, or arsenite for the indicated times and subjected to immunostaining with anti-phospho- p38 antibody (lower; phase contrast, upper). C, cells were transfected with pEGFP-C1 together with either an empty expression vector SRa (2) or both wild type MKK6 and wild type p38 expression vectors (MKK6 & p38) and treated with or without 10 mM SB203580. 48 h after the transfection the cells were treated with or without EGF. Represent- ative images of the transfected cells 72 h after EGF addition identified by the fluorescence of GFP are shown. D, cells were treated with EGF, arsenite, or both for 1 h, washed, and then incubated in fresh medium. Representative images 60 h after the treatment under a phase-contrast microscope are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Immunostaining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Incubation, Microscopy

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Physical interaction between CEP290 and the BBSome. (A) Co-immunoprecipitation of CEP290 in HEK293T cells stably expressing FLAG-BBS4 and FLAG-BBS5. Lysates from stable cell lines and control (parental) cells were subjected to immunoprecipitation (IP) with the anti-FLAG antibody and precipitated proteins were analyzed by immunoblotting with indicated antibodies. Normal mouse IgG pull-down was used as a negative control. (B and C) Interaction of endogenous CEP290 and the BBSome in HEK293T cells (B) and mouse retina (C). Lysates from HEK293T cells and mouse retina were subjected to IP using antibodies against CEP290, and precipitated proteins were analyzed by immunoblotting with indicated antibodies. (D) Schematic representation of the CEP290 deletion mutants. Numbers indicate expressed portions of CEP290 in amino acid positions. Known IQCB1-, CC2D2A- and RAB8A-binding domains and the BBSome-interacting region are also summarized. SMC, structural maintenance of chromosomes; MYO-Tail, myosin-tail homology domain. (E) The BBSome binds to the N-terminal part of CEP290. CEP290 deletion mutants (FLAG-tagged) were transfected into HEK293T cells and lysates were analyzed by IP using anti-FLAG antibodies. Untransfected cells were used as a negative control. (F) BBS4 interacts with CEP290. HA-tagged, individual BBSome components were transiently transfected with FLAG-Cep_1 constructs. Lysates were subjected to IP with anti-HA antibodies. (G) PCM1-independent interaction between the BBSome and CEP290. HEK293T cells were co-transfected with Cep_1 fragment and siRNA against PCM1.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Control, Western Blot, Negative Control, Binding Assay, Transfection, Construct

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Co-localization of CEP290 and the BBSome. (A) Co-localization of GFP-tagged BBS4 and CEP290 in RPE1 cells (yellow arrowheads). Localization of CEP290 (red) was probed with the anti-CEP290 antibody in hTERT-RPE1 after 24 h of serum withdrawal, whereas BBS4-GFP was probed with the anti-GFP antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Note the variable localization of BBS4, although these cells were cultured in the same condition (from a single well). Scale bar, 5 μm. (B) Co-localization of Bbs4 and Cep290 in the mouse retina. Antibodies against Cep290 (red) and rhodopsin (green; a marker of the OS) were used in WT photoreceptors in the left panel, whereas in the middle and right panels Bbs4 (red) and rhodopsin (green) localizations were probed in WT and Bbs4−/− (4KO) photoreceptors. Both Cep290 and Bbs4 localize to the connecting cilium. CC, connecting cilium; ONL, outer nuclear layer. Scale bar, 10 μm. (C) Co-fractionation of the BBSome and Cep290 in the photoreceptor OS fraction. The photoreceptor OS was isolated from the mouse retina and probed with antibodies against multiple subcellular marker proteins, Bbs4, Bbs7 and Cep290.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Staining, Cell Culture, Marker, Fractionation, Isolation

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: The CEP290 localization in the centriolar satellite and the connecting cilium is BBSome-dependent. (A) The BBSome is required for centriolar satellite localization of CEP290 (red). RPE1 cells were transfected with siRNAs against CEP290, BBS1, BBS4, BBS9 and PCM1. Antibodies against γ-tubulin and acetylated tubulin (green) were used to mark the basal body and cilia, respectively. (B) Quantification of CEP290 mis-localization in BBSome-depleted RPE1 cells. Cells with concentrated CEP290 staining around the centrosome (within 2 μm from the centrosome) were counted as positive, whereas cells with CEP290 only in the TZ and centrosome were considered negative. At least 120 cells per experiment were counted and graphs represent averages of three independent experiments. Error bars represent SEM. One-way ANOVA followed by Tukey's post-test was used for statistical analysis. **P < 0.01 compared with Ctrl KD cells. (C) Localization of Cep290 (green; left) in WT (top), Bbs1M390R/M390R (middle) and Bbs4−/− (bottom) mouse retinas. OS localization of Prph2 (red) with respect to acetylated tubulin (green) is not affected in BBS mutant retinas (right panels). Scale bars, 10 μm.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Transfection, Staining, Mutagenesis

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Increased body weight and higher leptin levels in Bbs4+/−; Cep290+/rd16 mice. (A) Weight gains in male animals versus age (minimum of six animals per group). Values are expressed as mean + SEM. By month 3, double-heterozygous mice are significantly heavier than single-heterozygous littermates. *P < 0.05 versus single-heterozygous animals; **P < 0.01 versus single-heterozygous animals. (B) Serum leptin levels of single- and double-heterozygous mice. One-way ANOVA and t-test were used for statistical analysis. *P < 0.05 versus single-heterozygous animals. (C) STAT3 phosphorylation upon leptin administration was reduced in double-heterozygous mice. Hypothalamic protein extracts were analyzed by western blotting. (D) Quantification of STAT3 phosphorylation. Band intensities of phospho-STAT3 (P-STAT3) were normalized with those of total STAT3 and induction ratios were calculated by comparison with vehicle-injected samples. Data represent mean + SEM. n = 5 for vehicle and n = 10 for leptin. *P < 0.05.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Phospho-proteomics, Western Blot, Comparison, Injection

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Impaired rhodopsin trafficking and diminished ERG responses in mice with combined loss of Cep290 and Bbs4 alleles. (A) Immunohistochemical analysis of WT, Bbs4+/+;Cep290rd/rd, Bbs4+/−;Cep290rd/rd and Bbs4−/−; Cep290rd/rd retinas at P21 with antibodies against rhodopsin. Scale bar, 5 μm. (B) DA-SCR ERG b-wave amplitudes in the indicated mouse genotypes (at the age of 1 month). Removing one or two Bbs4 alleles on a Cep290rd/rd background results in a lower ERG response. **P < 0.01 versus Bbs4+/+;Cep290rd/rd mice. Error bars are SD.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunohistochemical staining

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Antiadipogenic effect of G. cambogia extract and the related protein expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) on RPS6KA1 and STAT3 phosphorylation in MDI-induced 3T3-L1 preadipocytes (differentiation started cells) for the indicated times (n = 4 per group). Con: MDI-untreated cells, MDI: MDI-treated cells. **p < 0.01 vs. Con, ##p < 0.01 vs. MDI. (B) Kinase activity of MAPK3/ERK1 and JAK2 in response to G. cambogia extract (n = 4 per group). The active MAPK3/ERK1 and JAK2 enzymes were used to assess kinase activity in the presence or absence of G. cambogia extract at the indicated concentrations in vitro. *p < 0.05 and **p < 0.01 vs. each control. (C) Effect of G. cambogia extract (300 μg/ml), FMK (3 μM) and stattic (5 μM) on CEBPA and PPARG expression in mature 3T3-L1 adipocytes (fully differentiated adipocytes) (n = 4 per group). Con: undifferentiated cells, Diff: mature 3T3-L1 adipocytes. **p < 0.01 vs. Con, ##p < 0.01 vs. Diff. (D) Effect of G. cambogia extract (300 μg/ml) on lipid accumulation in mature 3T3-L1 adipocytes at the indicated time points. The time table (upper) and representative images of Oil red O staining (below) are presented. EGCG (50 μM) was used as a positive control (n = 15 per group). Scale bar: 50 μm. (E) Effect of G. cambogia extract (300 μg/ml) on CEBPA and PPARG expression in mature 3T3-L1 adipocytes at the indicated time points (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each group. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Activity Assay, In Vitro, Staining, Positive Control

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of CEBPB, RPS6KA1 and STAT3 regulation on autophagy-related protein expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of Cebpb knockdown on CEBPB (LAP* and LAP), CEBPA, PPARG, BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for 72 h. (B) Effect of Rps6ka1 and Stat3 knockdown on CEBPA, PPARG, BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for 72 h (n = 4 per group). **p < 0.01 vs. each control. n.s.: not significant. (C) Effect of FMK (3 μM) and stattic (5 μM) on BECN1, ATG7, ATG3, LC3, SQSTM1, ATG4B and ATG12–ATG5 expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each control. The data are the mean ± S.D. (D) Schematic illustrating the interaction of CEBPB and autophagy with RPS6KA1 and STAT3. The dotted line means undisclosed facts.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on KLF3 expression in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (300 μg/ml) on KLF3, CEBPA and PPARG in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). **p < 0.01 vs. each control. (B) Effect of G. cambogia extract (0–300 μg/ml) on CEBPB, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells after 72 h (n = 4 per group). *p < 0.05 and **p < 0.01 vs. Con. (C) Effect of FMK (3 μM) and stattic (5 μM) on CEBPB, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). *p < 0.05 and **p < 0.01 vs. each control. (D) Effect of Cebpb, Rps6ka1 and Stat3 knockdown on KLF3 expression in 3T3-L1 differentiated cells after 72 h (n = 4 per group). **p < 0.01 vs. siCon. n.s.: not significant. (E) Effect of G. cambogia extract (300 μg/ml) on Klf3 transcript levels in 3T3-L1 differentiated cells for the indicated times (n = 9 per group). (F) Effect of G. cambogia extract (300 μg/ml) on the KLF3 protein half-life in 3T3-L1 differentiated cells after 72 h. After treatment of G. cambogia extract in cells, cycloheximide (CHX, 1.5 μg/ml) was coincubated with the indicated times (n = 4 per group). **p < 0.01 vs. each control. (G) Effect of G. cambogia extract (Ga, 300 μg/ml) for 72 h and MG132 (10 μM) and 3-MA (0.5 mM) for 24 h on SQSTM1 and KLF3 expression in 3T3-L1 differentiated cells (n = 4 per group). **p < 0.01 vs. Con. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on SQSTM1-mediated selective autophagic degradation of KLF3 in 3T3-L1 preadipocytes during differentiation. (A) Interaction of SQSTM1 and KLF3 in 3T3-L1 differentiated cells treated with G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. Coimmunoprecipitation (IP) was used to analyze the level of SQSTM1 that physically interacted with KLF3. The lysates were immunoprecipitated with anti-KLF3 and anti-IgG antibodies, and the precipitates were analyzed by western blotting using antibodies against SQSTM1 and KLF3 (n = 4 per group). (B) Effect of Sqstm1 knockdown on the interaction of SQSTM1 and KLF3 in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml). Sqstm1 knockdown cells were treated with G. cambogia extract (300 μg/ml) for 72 h (n = 4 per group). (C) Effect of Sqstm1 knockdown on LC3, SQSTM1, KLF3, CEBPA and PPARG expression in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml) for 72 h in the presence or absence of rapamycin (Ra, 10 nM) for 24 h (n = 4 per group). (D) Fluorescence photographs (left) and quantification data (below) of LC3 and KLF3 in Sqstm1 knockdown cells treated with G. cambogia extract (300 μg/ml) for 72 h in the presence or absence of rapamycin (10 nM) for 24 h. The fluorescence intensity of FITC (i.e., LC3) and TRITC (i.e., KLF3) was quantified using ImageJ software and colocalization (yellow dots indicated by white arrows) of FITC and TRITC per cell was determined and analyzed (n = 5 per group). Scale bars: 5 μm (E) Effect of Sqstm1 knockdown on G. cambogia extract- and rapamycin-mediated lipid accumulation in mature 3T3-L1 adipocytes (n = 12 per group). During full differentiation (day 0–8), cells were treated with G. cambogia extract (300 μg/ml), and rapamycin (10 nM) was added at day 3–8. Con: undifferentiated cells, Diff: mature 3T3-L1 adipocytes. *p < 0.05 and **p < 0.01 vs. each group. n.s.: not significant. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Expressing, Fluorescence, Software

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on KLF3 and CTBP2 interaction to regulate adipogenic factors in 3T3-L1 preadipocytes during differentiation. (A) Effect of G. cambogia extract (0–300 μg/ml) on CTBP2 expression in 3T3-L1 differentiated cells for 72 h (n = 4 per group). n.s.: not significant. (B) Effect of FMK (3 μM) and stattic (5 μM) on CTBP2 expression in 3T3-L1 differentiated cells for the indicated times (n = 4 per group). (C) Physical interaction of KLF3 and CTBP2 in 3T3-L1 differentiated cells treated with G. cambogia extract (Ga, 300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. Coimmunoprecipitation (IP) was used to analyze the interaction of KLF3 and CTBP2. The lysates were immunoprecipitated with anti-KLF3 and anti-IgG antibodies, and the precipitates were analyzed by western blotting using antibody against CTBP2 (n = 4 per group). (D) Fluorescence photographs (left) and quantification data (right) of KLF3 and CTBP2 in 3T3-L1 differentiated cells treated with G. cambogia extract (300 μg/ml), FMK (3 μM) and stattic (5 μM) for 72 h. The fluorescence intensity and colocalization of TRITC (i.e., KLF3) and FITC (i.e., CTBP2) were quantified using ImageJ software. Colocalization was analyzed using the Pearson correlation coefficient. Scale bars: 5 μm. (n = 6 per group). **p < 0.01 vs. Con, n.s.: not significant. (E) Effect of G. cambogia extract (300 μg/ml) on the transcript levels of Cebpa and Pparg in 3T3-L1 differentiated cells for the indicated times (n = 9 per group). **p < 0.01 vs. each control. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Software

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Effect of G. cambogia extract on the identified targets in HFD-induced adipose tissues. (A and B) Effect of G. cambogia extract on phospho- and total-RPS6KA1, phospho- and total-STAT3, LC3, SQSTM1, KLF3, CEBPA and PPARG expression of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg) (n = 6 per group). (C) Correlations between phospho-RPS6KA1, phospho-STAT3 and KLF3 protein expression in eWAT and iWAT (n = 6 per group). Each point represents one sample. (D and E) Immunofluorescence analysis of phospho-RPS6KA1, phospho-STAT3 and KLF3 expression in eWAT and iWAT (n = 4 per group). Nuclei were stained with DAPI (blue), and white arrows indicate phospho-RPS6KA1, phospho-STAT3 and KLF3 colocalization with DAPI. Scale bars, 50 μm. **p < 0.01 vs. ND-fed mice, #p < 0.05 and ##p < 0.01 vs. HFD-fed mice. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Autophagy

Article Title: Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression

doi: 10.1080/15548627.2021.1936356

Figure Lengend Snippet: Analysis of the adipose tissues in the animal model. (A) Correlations between LC3-I, LC3-II, SQSTM1 and KLF3 protein expression in eWAT and iWAT (n = 6 per group). Each point represents one sample. (B) Lc3, Sqstm1, Klf3, Cebpa and Pparg transcript levels of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg) (n = 6 per group). (C) Correlations between Klf3, Lc3 and Sqstm1 transcript levels in eWAT and iWAT (n = 6 per group). Each point represents one sample. (D) Representative transmission electron micrographs of eWAT and iWAT in ND-fed, HFD-fed and HFD-fed mice administered a high dose of G. cambogia extract (400 mg/kg). The lower images are the enlarged representations of the boxed regions of the upper images. Scale bars: upper, 0.5 μm; lower, 200 nm. Autophagic vesicles are highlighted by white arrows and were quantified by counting the number of vesicles per 2 μm2 microscopic field in 4 randomly selected fields (n = 4 per group). Blue arrow: lipid droplet; green arrow: mitochondria. (E) Proposed mechanism for the antiobesity effect of G. cambogia extract in adipose tissue. **p < 0.01 vs. ND-fed mice, #p < 0.05 and ##p < 0.01 vs. HFD-fed mice. n.s.: not significant. The data are the mean ± S.D.

Article Snippet: Anti-PPARG (2443), anti-CEBPA (8178), anti-phospho-RPS6KA1 (Ser380; 9341), anti-RPS6KA1 (8408), anti-phospho-STAT3 (Tyr705; 9145), anti-STAT3 (9132), anti-phospho-CREB (Ser133, 9198), anti-CREB (9197), anti-BECN1 (3495), anti-ATG7 (8558), anti-ATG3 (3415), anti-MAP1LC3/LC3 (12741), anti-ATG4B (13507), anti-ATG12 (4180), anti-CTBP1 (8684), anti-CTBP2 (13256) and anti-rabbit (7074) were purchased from Cell Signaling Technology.

Techniques: Animal Model, Expressing, Transmission Assay

Journal: eLife

Article Title: A B-cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation

doi: 10.7554/eLife.72805

Figure Lengend Snippet:

Article Snippet: Antibody , Goat anti-mouse IgM, μ-chain-specific, polyclonal , Jackson Immuno Research , Cat# 115-005-020; RRID: AB_2338450 , Coverslip coating 2.5 μg/cm 2.

Techniques: Recombinant, Fluorescence, Labeling, Clinical Proteomics, Membrane, Staining, Cell Culture, Injection, Immunofluorescence, Western Blot, Blocking Assay, Sequencing, Negative Control, CRISPR, Cloning, Antibody Labeling, Software

Journal: JID Innovations

Article Title: A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

doi: 10.1016/j.xjidi.2021.100076

Figure Lengend Snippet: Inducible gene modulation in murine melanoma cell lines. ( a ) Schematic depicting the generation of stable cell lines expressing inducible Cas9 and sgRNA targeting GFP or Mafg . Mouse melanoma cell lines were infected with a lentiviral construct expressing Dox-inducible Cas9 and selected with blasticidin. Subsequently, TRE-Cas9‒harboring cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1 and selected with hygromycin, and polyclonal populations were used for experiments. ( b ) TRE-Cas9‒harboring mouse melanoma cell lines were infected with a GFP-U6-sgGFP reporter construct. Western blot showed that Dox treatment resulted in Cas9 (Flag) expression and a decrease in GFP levels. ( c ) TRE-Cas9‒harboring M10M3 and M167M1 cells were infected with GFP-U6-sgGFP or GFP-U6-sgMafg1. Cells were then treated with Dox, and Flag-Cas9, GFP, and MAFG expressions were analyzed by western blot. Cas9 was expressed on Dox treatment, GFP was decreased in sgGFP-expressing cells, and MAFG was decreased in sgMafg1-expressing cells. ( d ) M10M3 and ( e ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated at a low density, and colony-forming ability was examined, which is shown as percent surface area covered by colonies. ( f ) M10M3 and ( g ) M167M1 cells harboring TRE-Cas9 and sgGFP or sgMafg1 were plated in soft agar, and anchorage-independent growth was examined. Dox, doxycycline; sgGFP, sgRNA targeting GFP; sgMafg1, sgRNA targeting Mafg .

Article Snippet: Antibodies against FLAG (1:1,000, catalog number 14793S; Cell Signaling Technology, Danvers, MA), GFP (1:2,000, catalog number 2956S; Cell Signaling Technology), MAFG (1:1,000, catalog number ab154318; Abcam, Cambridge, United Kingdom), p16INK4a (1:2,000, catalog number ab211542; Abcam), p19ARF (1:1,000, catalog number ab80; Abcam), p53 (1:1,000, catalog number 3036-100; BioVision, Milpitas, CA), phosphorylated AKT S473 (1:2,000, catalog number 9188S; Cell Signaling Technology), phosphorylated AKT T308 (1:500, catalog number 13038T; Cell Signaling Technology), AKT (1:5,000, catalog number 4691T; Cell Signaling Technology), phosphorylated ERK T202/Y204 (1:1,000, catalog number 9101S; Cell Signaling Technology), ERK (1:1,000, catalog number 4695S; Cell Signaling Technology), S100B (1:1,000, catalog number ab52942; Abcam), MART1 (1:1,000, catalog number SAB4500949-100UG; Sigma-Aldrich), Cre (1:1,000, catalog number ab190177; Abcam), MITF (1:1,000, catalog number 12590S; Cell Signaling Technology), and β-actin (1:10,000, catalog number AM4302; Thermo Fisher Scientific, Waltham, MA) were used.

Techniques: Stable Transfection, Expressing, Infection, Construct, Western Blot

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Intact protein LC–MS spectra of Pin1 (black) directly identify covalent binders (blue) in the electrophilic library screen (200 μM compound for 24 h). Madduct indicates the mass of the expected adduct for the indicated example. b, Distribution of hits in the Pin1 screening campaign and their corresponding labeling (%). Nine hits (18.75%) out of the 48 top hits that labeled Pin1 at >75% (dark and light blue) share sulfolene or sulfolane moieties. Labeling percentage calculated as previously described28. c, 2D analysis of the top ten optimized binders (structures shown in f); labeling percentage in the LC–MS assay plotted against reactivity (log (K)) suggests Sulfopin for further biological evaluation. d, Fluorescence polarization assay with the top ten binders, including juglone and a nonreactive control (Sulfopin-AcA), after 14 h of preincubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. See Supplementary Table 3 for apparent Ki. mP represents the polarization value. e, PPIase substrate activity assay of Pin1 with Sulfopin (n = 3) and juglone (n = 2). Data points are plotted as the average of independent experiments ± s.e.m. for Sulfopin. f, Structures of the top ten binders in the Pin1-labeling LC–MS assay, the nonreactive control Sulfopin-AcA and juglone. g, X-ray crystal structure of Pin1 in complex with Sulfopin (1.4-Å resolution, PDB code 6VAJ). Pin1 (white) with relevant side chains in stick representation; Sulfopin is shown in pink. Hydrogen bonds are depicted as dashed lines. AU, arbitrary units.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Fluorescence, Activity Assay

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Fluorescence polarization assay showing that the DTB-labeled probe, Sulfopin-DTB, binds Pin1 with similar potency to Sulfopin following 14 h of incubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. b, Chemical structure of Sulfopin-DTB. c, Sulfopin shows time-dependent engagement in PATU-8988T cells. PATU-8988T cells were treated with Sulfopin (1 μM) for the indicated time points followed by cell lysis, incubation with Sulfopin-DTB (1 μM), streptavidin pulldown and immunoblot analysis. d,e, Sulfopin shows long-term engagement of Pin1. PATU-8988T (d) or HCT116 (e) cells were incubated with or without Sulfopin for the indicated time points, followed by cell lysis, incubation with DTB probe, streptavidin pulldown and immunoblot analysis. Substantial engagement (>50%) was still evident after 72 h. f,g, Sulfopin fully engages Pin1 in PATU-8988T cells at 1 μM and in HCT116 cells at 0.5 μM (see Supplementary Fig. 9b for the structure of BJP-DTB). PATU-8988T (f) or HCT116 (g) cells were incubated with Sulfopin at the indicated concentrations for 5 h, followed by cell lysis, DTB probe incubation (1 h, 1 μM), streptavidin pulldown and immunoblot analysis. The noncovalent control, Sulfopin-AcA, is unable to outcompete Pin1 pulldown. c–g, Results are representative of n = 2 independent experiments. h, Sulfopin engages Pin1 in vivo. Mice were treated by oral gavage with the indicated amounts of Pin1 over 2 days for a total of three doses. Following this treatment, spleens were lysed for a competition pulldown experiment with Sulfopin-DTB. Results are representative of n = 2 independent pulldown experiments, starting from the same spleen lysates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Fluorescence, Labeling, Incubation, Lysis, Western Blot, In Vivo

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, CITe-Id profiling results showing Sulfopin-DTB-labeled cysteine sites, rank ordered by competitive dose response to Sulfopin. Out of 162 cysteine residues reproducibly labeled by Sulfopin-DTB in n = 2 independent experiments, Pin1 C113 was the only site identified with a competitive dose response >2 s.d. from the mean value of the null. (see Supplementary Dataset 3a for a full list of identified peptides, and Supplementary Fig. 10 for results with 12/24-h treatment). b, Waterfall plot showing competitive dose dependency of Pin1 C113 labeling in the CITe-Id experiment. Bars represent mean of n = 2 independent experiments. c, Out of 2,134 cysteines identified in the rdTOP-ABPP experiment, only two showed a light/heavy ratio of >2.5 and, of these, one did not replicate and only Pin1 C113 showed the maximal ratio of 15 in both replicates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Labeling

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, HeLa cells were treated with either DMSO, Sulfopin, or Go6976 (a Chk1 inhibitor) and exposed to 7.5 Gy IR 1 h after drug treatment. Viability was assessed 3 days post-IR. Sulfopin shows a dose dependent sensitization of the cells to irradiation (n=3; data are represented as mean values with standard deviation). b, Western blot analysis was performed 24 h post-IR, showing Sulfopin blocked phosphorylation of Thr209 of IRAK1. c, A shorter exposure shows that Sulfopin inhibits IRAK1 phosphorylation already at concentrations of 0.1 μM. d, A scheme for testing the effect of Sulfopin in vivo on germinal center B cells in response to immunization. e, Representative flow cytometric plots with Vehicle and Sulfopin (left) and quantification (right) of FASHi CD38− germinal center (GC) cells in WT mice 11 days after immunization with NP-OVA. ** p<0.01, two tailed Student’s t test.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Knock-Out, Irradiation, Standard Deviation, Western Blot, In Vivo, Two Tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, We previously27 generated a PATU-8988T Pin1 knockout (KO) cell line (Supplementary Fig. 12a). Sulfopin (1 μM) had a significant effect on cellular viability after 6 and 8 days (P = 0.01 and P = 0.01, respectively) in WT PATU-8988T cells (left), but showed no significant effect on viability in Pin1 KO cells (right); day 0-normalized growth rate for n = 3 biologically independent samples. b, Relative viability of PATU-8988T WT and Pin1 KO cells grown in 100% Matrigel domes following treatment with either Sulfopin (1 μM; n = 9 biologically independent samples; P = 1.24 × 10−18) or the noncovalent negative control, Sulfopin-AcA (1 μM; n = 9 biologically independent samples). Sulfopin-AcA showed no effect in any of the tested systems. c, Proportion of cells in various cell cycle stages as a function of Sulfopin treatment. The viability effects of Sulfopin are mediated by delayed cell cycle. PATU-8988T cells were treated with either DMSO, 2.5 μM Sulfopin or Sulfopin-AcA for 4 days. Cell cycle analysis was performed by BrdU and propidium iodide staining, followed by FACS analysis. Sulfopin treatment reduced the percentage of cells in S phase (P = 0.0004) and, in turn, increased the number of cells found in G1 phase (P = 0.003), while the noncovalent Sulfopin-AcA did not show this effect (n = 4; see Extended Data Fig. 3 for representative FACS analysis graphs and quantification of the results from two independent experiments). d, Cell culture growth curves. Sulfopin showed variation in antiproliferative effects across cancer cell lines Kuramochi, MDA-MB-468, NGP and NBL-S, with the most pronounced sensitivity observed in MDA-MB-468 cells (day 0-normalized growth rate for n = 3 biologically independent samples; P values for 2.5 μM Sulfopin after 4, 6 and 8 days were 0.007, 0.004 and 0.0004, respectively). Importantly we noted significant viability effects in Myc-high neuroblastoma cell lines NGP and NBL-S (P = 0.018 and 0.002, respectively for 2.5 μM Sulfopin after 8 days). Data points were plotted as the average of n = 3 biologically independent samples ± s.e.m. Statistical significance for all panels was calculated using one-tailed Student’s t-test with unequal variance (NS, not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Generated, Knock-Out, Negative Control, Cell Cycle Assay, Staining, Cell Culture, One-tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, PATU-8988T cells were treated for 5 or 6 days with either DMSO (0.1%), Sulfopin (1 μM, 2.5 μM) or the non-covalent control Sulfopin-AcA (2.5 μM). The cells were lysed and activation of caspase 3 and Pin1 levels were analysed by Western blot. As a positive control for caspase 3 activation the cells were treated with Staurosporin (1 μM, 4h; STS). See Supplementary Fig. 13a for the results of an additional independent experiment. Caspase 3 was not activated and Pin1 levels were not changed by the treatment with Sulfopin. b, PATU-8988T cells were treated in triplicates for 6 days with either DMSO (0.1%), Sulfopin (1 M, 2.5 M) or the non-covalent control Sulfopin-AcA (2.5 M). The cells were then stained with AnnexinV-FITC/ 7AAD and analysed by FACS. Staurosporin treatment (1 M, 4h) was used as a positive control for apoptosis. Representative FACS analysis graphs and a quantification of the results (n=3; data are represented as mean values with standard deviation). See Supplementary Fig. 13b for the results of an additional independent experiment. Live cells were defined as AnnexinV−/7AAD−, early apoptosis AnnexinV+/7AAD− and late apoptosis AnnexinV+/7AAD+.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Activation Assay, Western Blot, Positive Control, Staining, Standard Deviation

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Results of an RNA-seq experiment comparing changes in RNA levels between Mino B cells treated with either Sulfopin (1 μM, 6 h, in triplicate) or DMSO. Each dot represents log2 fold change of a transcript (x axis) versus the P value for significance of that change (y axis; Wald test, as implemented in DESeq2). The dotted line indicates P = 0.05; 206 genes were significantly downregulated. b, Results of gene set enrichment analysis using Enrichr against the ENCODE TF chromatin immunoprecipitation–sequencing set. Two of the sets most enriched were Myc target genes from different cell lines. c, HEK293 cells were transfected with 4× E-box luciferase reporter for Myc transcriptional activity levels. Cotransfection with Pin1 increased reporter activity, while 48-h treatment with Sulfopin significantly (one-tailed Student’s t-test) reduced this activity compared to DMSO (n = 3; error bars indicate s.d.).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Transfection, Luciferase, Activity Assay, Cotransfection, One-tailed Test

Journal: Molecules

Article Title: Curcumol Synergizes with Cisplatin in Osteosarcoma by Inhibiting M2-like Polarization of Tumor-Associated Macrophages

doi: 10.3390/molecules27144345

Figure Lengend Snippet: Curcumol enhanced CDDP-induced cell apoptosis in K7M2 WT osteosarcoma cells. ( A ) Cell morphologies were shown in bright-field images after treatment with curcumol, CDDP or a combination of both for 48 h. ( B ) Curcumol, CDDP, or both were employed to treat K7M2 WT cells for 48 h. The cells were then labeled with DAPI, and fluorescence microscopy was used to analyze the nuclear alterations. ( C ) Cells were harvested after being exposed to drugs as described in ( B ), and a PI (propidium iodide) staining experiment was performed and evaluated by flow cytometry. ( D ) Statistical analysis of apoptosis cells in ( C ). ** p < 0.01; Student’s t test. ( E ) Western blotting for cleaved caspase-3 and cleaved PARP in treated K7M2 WT cells.

Article Snippet: Cleaved PARP and cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry, Western Blot