Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: UHRF1 is required for 3T3-L1 adipogenesis. ( A ) Western blot analysis of UHRF1 protein expression in NT, UHRF1 knockout G1 (UHRF1 KO G1), UHRF1 knockout G2 (UHRF1 KO G2), shScramble, shUHRF1 C1 and shUHRF1 C2 3T3-L1 cells, with Actin as the loading control. ( B , C ) Graph representing the growth curve of NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 preadipocytes at different time points. ( D , E ) Cell count of NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 cells during adipogenic differentiation. ( F ) Oil Red O staining of adipocytes at day 10 post-differentiation in NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 cells visualized by light microscopy at ×20 magnification (scale bar, 200 μm). The percentage of adipocytes in each group is indicated in the image. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Western Blot, Expressing, Knock-Out, Control, Cell Counting, Staining, Light Microscopy

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Transcriptomics analysis of UHRF1 KO G1 (pre)adipocytes reveals DEGs and significantly altered pathways, including inhibition of adipocyte differentiation. ( A ) Schematic representation of transcriptomics analysis of whole RNA extracted from the following samples: UHRF1 KO G1 versus NT at preadipocyte stage (day 0) and UHRF1 KO versus NT at adipocyte stage (day 10) of 3T3-L1 cells. ( B ) The number of statistically significant DEGs in both datasets at the indicated cut-off P -value < 0.05 and FDR < 0.05. Upregulated and downregulated genes are highlighted in red and green, respectively. ( C – H ) Expression log ratio of top upregulated genes in both data sets of UHRF1 KO cells and its qPCR validation. ( I , J ) IPA prediction of the top 20 affected canonical pathways in both datasets, and those highlighted in red and green are the percentage of upregulated and downregulated genes in each pathway, respectively. ( K ) Summary graph of adipocyte dataset that shows the interaction network between the most significant predicted pathways and molecules. The orange line indicates activated entities with a positive z-score, and the blue line indicates inhibited entities with a negative z-score, respectively, and estimated edges are shown as dotted lines. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Inhibition, Expressing, Biomarker Discovery

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Adipogenic differentiation is suppressed in UHRF1-depleted cells. ( A ) Western blot analysis of UHRF1 and adipocyte markers C/EBP-β, PPAR-γ, and C/EBP-α in NT and UHRF1 KO G1 at different time points during adipogenic differentiation. ( B – E ) Western blot quantification of UHRF1, C/EBP-β, PPAR-γ, and C/EBP-α in NT and Uhrf1 KO G1 in which relative protein level was normalized to the loading control, Actin, using ImageJ software. ( F – J ) Western blot analysis of UHRF1 and adipogenic markers C/EBP-β, PPAR-γ, and C/EBP-α and its quantification graph in shScramble and shUHRF1 C2 during adipogenic differentiation. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Western Blot, Control, Software

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: GPNMB is induced and secreted in UHRF1-lacking 3T3-L1 preadipocytes. ( A ) Venn diagram showing 846 common upregulated genes between preadipocyte and adipocyte data sets. ( B ) The Expression log ratio of GPNMB in both datasets. ( C , D ) Relative mRNA expression of GPNMB in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 at different time-points during adipocyte differentiation by qPCR. ( E , F ) GPNMB protein levels in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 by Western blot analysis with Actin as the loading control. ( G , H ) ImageJ software-based Quantitation of GPNMB protein in Western blot results by normalizing to Actin. Fold change values were calculated relative to the D0 control. ( I ) Immunostaining of GPNMB in NT, UHRF1 KO G1 preadipocytes where nuclei are stained in blue (DAPI), GPNMB stained in green (FITC). Images were taken at 63 × magnification. ( J ) Quantification of GPNMB fluorescence intensity in NT and UHRF1 KO G1 preadipocytes immunostaining by ImageJ software. ( K ) ELISA detected the secreted level of GPNMB in the conditioned media of NT and UHRF1 KO G1 preadipocytes. ( L , M ) Western blot analysis of GPNMB and its quantification graph in the conditioned media of NT and UHRF1 KO G1 preadipocytes. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Expressing, Western Blot, Control, Software, Quantitation Assay, Immunostaining, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: TGF-β and its targets are induced in UHRF1 KO cells during differentiation. ( A ) Upstream regulators of common upregulated genes predicted by IPA analysis. Upregulated targets are represented in red color, and downregulated targets are shown in green color. ( B ) Upregulated TGF-β targets predicted in adipocyte dataset. ( C , D ) Western blot analysis showing TGF-β induction in NT and UHRF1 KO G1 during differentiation and its quantification graph. ( E – P ) qPCR analysis of MMP3, MMP13, COL1A1, COL6A3, MCP1, Fibronectin, and MITF in NT, UHRF1 KO G1 and shUHRF1 C2 cells during differentiation. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Recombinant GPNMB induces TGF-β and suppresses adipogenesis in 3T3-L1 cells. ( A – C ) Western blot analysis of secreted TGF-β and MMP13 in conditioned media collected from NT, UHRF1 KO G1, GPNMB-treated NT, and GPNMB-treated UHRF1 KO G1 cells and its quantification data. ( D ) Oil Red O images of control and recombinant GPNMB (100 ng/ml) treated 3T3-L1 cells on day 8 post-induced 3T3-L1 cells (scale bar, 200 μm). ( E , F ) Western blot analysis showing induction of TGF-β in recombinant GPNMB-treated 3T3-L1 during adipocyte differentiation and its quantification graph. ( G ) Expression log ratio of TGF-β targets in adipocyte data set. ( H ) Western blot analysis of adipogenic markers such as PPAR-γ, C/EBP-α, and Adiponectin in control and GPNMB-treated 3T3-L1 cells. ( I ) Western blot quantification graph of PPAR-γ, C/EBP-α, and Adiponectin in control and GPNMB-treated 3T3-L1 cells. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Recombinant, Western Blot, Control, Expressing

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Overexpression of UHRF1 reverses the phenotype observed in UHRF1-depleted cells. ( A ) Representative fluorescence and brightfield microscopy images of lentivirus-mediated expression of GFP control or UHRF1-GFP in HEK293T cells (Scale bar, 100 μm). GFP-positive cells in the total cell population are shown in the merged image. ( B ) Representative confocal microscopy images of GFP control and UHRF1-GFP in HEK293T cells. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm. ( C – H ) Representative western blot data and quantification of changes in the expression levels of UHRF1-GFP, TGF-β, GPNMB, MMP3 and MMP13 levels in UHRF1-GFP HEK293T cells versus GFP control. Target protein levels were normalized to Actin using ImageJ software. ( I ) Fluorescence and brightfield microscopy images of lentivirus-mediated expression of GFP control and UHRF1-GFP in SW872 cells (Scale bar, 100 μm). GFP-positive cells in the total cell population are shown in the merged image. ( J ) Representative confocal microscopy images of GFP control and UHRF1-GFP in SW872 cells. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm. ( K – N ) Representative western blot data and quantification of changes in the expression levels of UHRF1-GFP, PPAR-γ and TGF-β in UHRF1-GFP SW872 cells versus GFP control. Target protein levels were normalized to Actin using ImageJ software. ( O , P ) Oil Red O staining images of oleic acid (50 µM)-induced differentiation of SW872 cells overexpressing GFP control or UHRF1-GFP on day 4 post-differentiation. Scale bar 100 μm. ( P ) Bar graph showing the total number of lipid droplets present in the entire 40 × field Oil Red O images of GFP control and UHRF1-GFP. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: Antibodies used were: Uhrf1 (D6G8E, Rabbit mAb#12,387, cell signaling Techno.), β-Actin (8H10D10, Mouse mAb#3700, cell signaling Techno.), C/EBPβ (Rabbit, ab32358, Abcam), PPAR-γ (Rabbit#2443, Cell Signaling Techno.), C/EBPα (Rabbit #2295, Cell Signaling Techno.), Glut 4 ((1F8) Mouse Ab#2213, Cell Signaling Techno.), GPNMB ((E7U1Z) Rabbit mAb#90205, Cell Signaling Techno.), Adiponectin (Rabbit, C45B10, Cell signaling Techno.), TGF-β (Rabbit, 3711S, Cell signaling Techno.), MMP13 (ab39012, RRID: AB_776416, Abcam), Anti-rabbit IgG (7074, RRID: AB_2099233, Cell Signaling Techno.), and Anti-mouse IgG (7076, RRID: AB_330924, Cell Signaling Techno.).

Techniques: Over Expression, Fluorescence, Microscopy, Expressing, Control, Confocal Microscopy, Staining, Western Blot, Software

Journal: Epigenetics & Chromatin

Article Title: Independent functions of DNMT1 and USP7 at replication foci

doi: 10.1186/s13072-018-0179-z

Figure Lengend Snippet: Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test

Article Snippet: Antibodies used: rabbit polyclonal to DNMT1 (Cell Signaling Technology, 5032 (D63A6) 1:2500 dilution), rabbit polyclonal to USP7 (Bethyl Laboratories, IHC00018; 1:5000 dilution), rabbit polyclonal to HA tag (Abcam, ab9110, 1:5000), rabbit polyclonal to UHRF1 (Bethyl Laboratories, A301-470A, 1:1000), mouse monoclonal to alpha-tubulin (Abcam, ab7291, 1:10,000).

Techniques: DNA Methylation Assay, Expressing, shRNA, Methylation, Two Tailed Test

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: UHRF1 is required for 3T3-L1 adipogenesis. ( A ) Western blot analysis of UHRF1 protein expression in NT, UHRF1 knockout G1 (UHRF1 KO G1), UHRF1 knockout G2 (UHRF1 KO G2), shScramble, shUHRF1 C1 and shUHRF1 C2 3T3-L1 cells, with Actin as the loading control. ( B , C ) Graph representing the growth curve of NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 preadipocytes at different time points. ( D , E ) Cell count of NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 cells during adipogenic differentiation. ( F ) Oil Red O staining of adipocytes at day 10 post-differentiation in NT, UHRF1 KO G1, UHRF1 KO G2, shScramble, shUHRF1 C1, and shUHRF1 C2 cells visualized by light microscopy at ×20 magnification (scale bar, 200 μm). The percentage of adipocytes in each group is indicated in the image. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Western Blot, Expressing, Knock-Out, Control, Cell Counting, Staining, Light Microscopy

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Transcriptomics analysis of UHRF1 KO G1 (pre)adipocytes reveals DEGs and significantly altered pathways, including inhibition of adipocyte differentiation. ( A ) Schematic representation of transcriptomics analysis of whole RNA extracted from the following samples: UHRF1 KO G1 versus NT at preadipocyte stage (day 0) and UHRF1 KO versus NT at adipocyte stage (day 10) of 3T3-L1 cells. ( B ) The number of statistically significant DEGs in both datasets at the indicated cut-off P -value < 0.05 and FDR < 0.05. Upregulated and downregulated genes are highlighted in red and green, respectively. ( C – H ) Expression log ratio of top upregulated genes in both data sets of UHRF1 KO cells and its qPCR validation. ( I , J ) IPA prediction of the top 20 affected canonical pathways in both datasets, and those highlighted in red and green are the percentage of upregulated and downregulated genes in each pathway, respectively. ( K ) Summary graph of adipocyte dataset that shows the interaction network between the most significant predicted pathways and molecules. The orange line indicates activated entities with a positive z-score, and the blue line indicates inhibited entities with a negative z-score, respectively, and estimated edges are shown as dotted lines. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Inhibition, Expressing, Biomarker Discovery

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Adipogenic differentiation is suppressed in UHRF1-depleted cells. ( A ) Western blot analysis of UHRF1 and adipocyte markers C/EBP-β, PPAR-γ, and C/EBP-α in NT and UHRF1 KO G1 at different time points during adipogenic differentiation. ( B – E ) Western blot quantification of UHRF1, C/EBP-β, PPAR-γ, and C/EBP-α in NT and Uhrf1 KO G1 in which relative protein level was normalized to the loading control, Actin, using ImageJ software. ( F – J ) Western blot analysis of UHRF1 and adipogenic markers C/EBP-β, PPAR-γ, and C/EBP-α and its quantification graph in shScramble and shUHRF1 C2 during adipogenic differentiation. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Western Blot, Control, Software

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: GPNMB is induced and secreted in UHRF1-lacking 3T3-L1 preadipocytes. ( A ) Venn diagram showing 846 common upregulated genes between preadipocyte and adipocyte data sets. ( B ) The Expression log ratio of GPNMB in both datasets. ( C , D ) Relative mRNA expression of GPNMB in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 at different time-points during adipocyte differentiation by qPCR. ( E , F ) GPNMB protein levels in NT, UHRF1 KO G1, shScramble, and shUHRF1 C2 by Western blot analysis with Actin as the loading control. ( G , H ) ImageJ software-based Quantitation of GPNMB protein in Western blot results by normalizing to Actin. Fold change values were calculated relative to the D0 control. ( I ) Immunostaining of GPNMB in NT, UHRF1 KO G1 preadipocytes where nuclei are stained in blue (DAPI), GPNMB stained in green (FITC). Images were taken at 63 × magnification. ( J ) Quantification of GPNMB fluorescence intensity in NT and UHRF1 KO G1 preadipocytes immunostaining by ImageJ software. ( K ) ELISA detected the secreted level of GPNMB in the conditioned media of NT and UHRF1 KO G1 preadipocytes. ( L , M ) Western blot analysis of GPNMB and its quantification graph in the conditioned media of NT and UHRF1 KO G1 preadipocytes. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Expressing, Western Blot, Control, Software, Quantitation Assay, Immunostaining, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: TGF-β and its targets are induced in UHRF1 KO cells during differentiation. ( A ) Upstream regulators of common upregulated genes predicted by IPA analysis. Upregulated targets are represented in red color, and downregulated targets are shown in green color. ( B ) Upregulated TGF-β targets predicted in adipocyte dataset. ( C , D ) Western blot analysis showing TGF-β induction in NT and UHRF1 KO G1 during differentiation and its quantification graph. ( E – P ) qPCR analysis of MMP3, MMP13, COL1A1, COL6A3, MCP1, Fibronectin, and MITF in NT, UHRF1 KO G1 and shUHRF1 C2 cells during differentiation. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Recombinant GPNMB induces TGF-β and suppresses adipogenesis in 3T3-L1 cells. ( A – C ) Western blot analysis of secreted TGF-β and MMP13 in conditioned media collected from NT, UHRF1 KO G1, GPNMB-treated NT, and GPNMB-treated UHRF1 KO G1 cells and its quantification data. ( D ) Oil Red O images of control and recombinant GPNMB (100 ng/ml) treated 3T3-L1 cells on day 8 post-induced 3T3-L1 cells (scale bar, 200 μm). ( E , F ) Western blot analysis showing induction of TGF-β in recombinant GPNMB-treated 3T3-L1 during adipocyte differentiation and its quantification graph. ( G ) Expression log ratio of TGF-β targets in adipocyte data set. ( H ) Western blot analysis of adipogenic markers such as PPAR-γ, C/EBP-α, and Adiponectin in control and GPNMB-treated 3T3-L1 cells. ( I ) Western blot quantification graph of PPAR-γ, C/EBP-α, and Adiponectin in control and GPNMB-treated 3T3-L1 cells. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Recombinant, Western Blot, Control, Expressing

Journal: Scientific Reports

Article Title: The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-β signaling

doi: 10.1038/s41598-024-62508-y

Figure Lengend Snippet: Overexpression of UHRF1 reverses the phenotype observed in UHRF1-depleted cells. ( A ) Representative fluorescence and brightfield microscopy images of lentivirus-mediated expression of GFP control or UHRF1-GFP in HEK293T cells (Scale bar, 100 μm). GFP-positive cells in the total cell population are shown in the merged image. ( B ) Representative confocal microscopy images of GFP control and UHRF1-GFP in HEK293T cells. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm. ( C – H ) Representative western blot data and quantification of changes in the expression levels of UHRF1-GFP, TGF-β, GPNMB, MMP3 and MMP13 levels in UHRF1-GFP HEK293T cells versus GFP control. Target protein levels were normalized to Actin using ImageJ software. ( I ) Fluorescence and brightfield microscopy images of lentivirus-mediated expression of GFP control and UHRF1-GFP in SW872 cells (Scale bar, 100 μm). GFP-positive cells in the total cell population are shown in the merged image. ( J ) Representative confocal microscopy images of GFP control and UHRF1-GFP in SW872 cells. Nuclei were stained with Hoechst (blue). Scale bar, 50 μm. ( K – N ) Representative western blot data and quantification of changes in the expression levels of UHRF1-GFP, PPAR-γ and TGF-β in UHRF1-GFP SW872 cells versus GFP control. Target protein levels were normalized to Actin using ImageJ software. ( O , P ) Oil Red O staining images of oleic acid (50 µM)-induced differentiation of SW872 cells overexpressing GFP control or UHRF1-GFP on day 4 post-differentiation. Scale bar 100 μm. ( P ) Bar graph showing the total number of lipid droplets present in the entire 40 × field Oil Red O images of GFP control and UHRF1-GFP. Data are mean ± SEM of 3 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA, * P < 0.05.

Article Snippet: GFP Control (PS100093) and GFP-tagged UHRF1 (MR226251L4) plasmids were purchased from Origene.

Techniques: Over Expression, Fluorescence, Microscopy, Expressing, Control, Confocal Microscopy, Staining, Western Blot, Software

Journal: Stem Cell Research & Therapy

Article Title: Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq

doi: 10.1186/s13287-018-1027-6

Figure Lengend Snippet: Identification of uhrf1 as a uniquely expressed proliferation gene using in vitro comparative RNA-seq. a UHRF1 immunofluorescence staining in regenerating deer antler tissue. b RM cells cultured with 30 nM uhrf1 siRNAs for 3 days exhibited decreased proliferation relative to mock-transfected control. c C3H10T1/2 cells stably transfected with uhrf1 exhibited increased proliferation relative to untransfected control and empty plasmid control. C3H10T1/2 cells stably transfected with uhrf1 maintained contact inhibition. Representative growth curves are shown. d C3H10T1/2 cells stably transfected with uhrf1 and cultured with 100 ng/mL BMP-2 for 6 days exhibited increased ALP activity relative to untransfected control and empty plasmid control. Scale bars as indicated. Data were from n = 3 isolates (an independent herd for antler immunofluorescence studies) or n = 3 independent experiments with nine replicates per group for uhrf1 knockdown and overexpression proliferation and osteoblast differentiation studies. Gray circles indicate observed data points. Error bars indicate SEM. Statistical significance as indicated

Article Snippet: Cells were transfected with 30 nM uhrf1 siRNA A and E (siRNAs were custom-designed by Santa Cruz Biotechnology Inc., Dallas, TX, based on bovine uhrf1 sequence) according to the manufacturer’s instructions (Polyplus, France) for 72 h.

Techniques: In Vitro, RNA Sequencing, Immunofluorescence, Staining, Cell Culture, Transfection, Control, Stable Transfection, Plasmid Preparation, Inhibition, Activity Assay, Knockdown, Over Expression