Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b The real-time PCR analysis of SREBP1 and SREBP2 expression in control and knockdown DLD1 ( a ) and Pt130 ( b ) cells. Two different shRNA targeting sequences were used for knocking down SREBP1 or SREBP2. c , d The expression of SREBP1 and SREBP2 proteins were downregulated in stable DLD1 ( c ) and Pt130 ( d ) knockdown cells. Protein lysates from control and knockdown cells were analyzed by western blot. Both the precursor and mature forms of SREBP1 and SREBP2 proteins were detected. e , f Knockdown of SREBP1 and SREBP2 decreased the expression of downstream genes related to fatty acid synthesis and metabolism in DLD1 ( e ) and Pt130 ( f ) cells. Data represent the mean ± SD (* p < 0.001 and # p < 0.05 compared to sh-Control). g Knockdown of SREBP1 or SREBP2 decreased lipogenesis in colon cancer cells. Total cellular lipids were extracted from control and SREBP knockdown DLD1 and Pt130 cells and the levels of free fatty acids, cholesterol, and triglyceride were measured and normalized to the amount of total proteins. Data represent the mean ± SD (* p < 0.001, § p < 0.01, and # p < 0.05 compared to sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Knockdown, shRNA, Western Blot

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b Knockdown of SREBP1 or SREBP2 decreased the rate of proliferation in DLD1 ( a ) and Pt130 ( b ) cells. Equal number of control and SREBP knockdown cells were allowed to grow for 4 days and the number of cells were counted each day. Data represent the mean ± SD ( § p < 0.01 and # p < 0.05 compared to sh-Control). c , d Knockdown of SREBP1 or SRBEP-2 decreased the formation of tumor spheroids in the stem cell suspension medium. Control and SREBP knockdown DLD1 ( c ) and Pt130 ( d ) cells were seeded as single cells in the stem cell suspension medium and the number of colonies formed was determined after 6 days. Data represent the mean ± SD ( ¶ p < 0.0001 and * p < 0.001 compared to sh-Control). e Knockdown of SREBP1 or SRBEP2 reduced the expression of genes associated with colon cancer stem cells. The relative expression of CD44 , CD133 , LGR5 , and AXIN2 mRNA was determined using real-time PCR in control and SREBP knockdown DLD1 and Pt130 cells. Data represent the mean ± SD ( § p < 0.01 and # p < 0.05 compared to sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Knockdown, Control, Suspension, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a Representative OCR measurements obtained from the Mito tress test performed in control (sh-Control) and SREBP knockdown (sh-SREBP1 and sh-SREBP2) Pt130 cells using the Seahorse XF96 Extracellular Flux analyzer. Oligomycin, FCCP, and antimycin A (Anti-A) were added at the indicated points. The shaded areas indicate the OCR levels associated with basal and maximal respiration, ATP turnover and reserved capacity, respectively. b Experiments as shown in a were quantified and the relative levels of OCR associated with basal and maximal respiration, ATP turnover and reserved capacity were calculated. Data represent the mean ± SEM ( ¶ p < 0.0001 and # p < 0.05 compared to Sh-Control). c Representative ECAR measurements obtained from the glycolysis stress test performed in control and SREBP knockdown Pt130 cells using the Seahorse XF96 Extracellular Flux analyzer. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the indicated points. The shaded areas indicate the ECAR levels associated with glycolysis, glycolytic capacity, and glycolytic reserve, respectively. d Experiments as shown in c were quantified and ECAR associated with glycolysis, glycolytic capacity, and glycolytic reserve was calculated. Data represent the mean ± SEM ( ¶ p < 0.0001 compared to Sh-Control). e Knockdown of SREBP resulted in metabolic shifts in Pt130 cells. The energetic map of control and SREBP knockdown Pt130 cells was based on the measurements obtained in a and c . The OCR and ECAR data shown in the map represent the OCR of basal respiration from the Mito stress test and the glycolysis-related ECAR from the glycolysis stress test, respectively (data represent the mean ± SEM)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Control, Knockdown

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a , b Knockdown of SREBP1 and SREBP2 reduced fatty acid oxidation (FAO) in DLD1 ( a ) and Pt130 ( b ) cells. The rate of cellular FAO was determined by subjecting control and SREBP knockdown cells to the FAO assays using the Seahorse XF96 Extracellular Flux analyzer. Data represent the mean ± SEM ( § p < 0.01 compared to Sh-Control). c Knockdown of SCAP reduced FAO in DLD1 and Pt130 cells. The rate of cellular FAO was determined using Seahorse FAO assays. Data represent the mean ± SEM ( # p < 0.05, compared to Sh-Control)

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Knockdown, Control

Journal: Cell Death & Disease

Article Title: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

doi: 10.1038/s41419-018-0330-6

Figure Lengend Snippet: a Control, SREBP1, and SREBP2 knockdown Pt130 cells were injected subcutaneously into NSG mice. The size of the tumors was measured every 3 days starting at day 14. Data represent the mean ± SEM ( ¶ p < 0.0001 compared to Sh-Control group). b On day 29, tumors were excised and weighted. Data represent the mean ± SEM ( ¶ p < 0.0001 and § p < 0.01 compared to Sh-Control group). c Detection of proliferating cells in tumors with IHC staining using the anti-Ki67 antibody. Scale bar, 25 μm. d Numbers of Ki67-positive cells were quantified in 500 randomly chosen tumor cells. Data in the graph represent the mean ± SD ( ¶ p < 0.0001 compared to Sh-Control group). e Detection of apoptotic cells in tumors with IHC staining using the anti-cleaved caspase-3 antibody. Scale bar, 25 μm. f Numbers of cleaved caspase-3-positive cells were quantified in 500 randomly chosen tumor cells. Data in the graph represent the mean ± SD ( ¶ p < 0.0001 compared to Sh-Control group). g Knockdown of SREBP reduced the expression of genes associated with colon cancer stem cells. The relative expression of CD44 , CD133 , LGR5 , and AXIN2 mRNA was determined using real-time PCR in tumors derived from control, SREBP1 and SREBP2 knockdown Pt130 cells. Data represent the mean ± SD (* p < 0.001, § p < 0.01, and # p < 0.05 compared to Sh-Control group). h Tumor initiation experiments were performed using control, SREBP1, and SREBP2 knockdown Pt130 cells. The cells were mixed with Matrigel and subcutaneously inoculated into NSG mice at 1000 cells per site and total of eight injections were used for each cell line. The number of tumors formed was determined 3 months post inoculation.

Article Snippet: The SREBP1 antibody was from BD Biosciences (CA, USA), whereas the SREBP2 antibody was from R&D Systems (MN, USA).

Techniques: Control, Knockdown, Injection, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: OncoTargets and Therapy

Article Title: Investigating the Diagnostic and Therapeutic Potential of SREBF2-Related Lipid Metabolism Genes in Colon Cancer

doi: 10.2147/OTT.S428150

Figure Lengend Snippet: Correlation plot of the expression of SREBF2 -related lipid metabolism genes. The colour of each cell represents the correlation between the expression levels of two genes, with red indicating a positive correlation and black indicating a negative correlation. An “X” in a cell indicates a p value greater than 0.001.

Article Snippet: Immunohistochemical staining was performed using an anti-SREBP2 polyclonal antibody (1:1000, R&D Systems, MAB7119) or an anti-DHCR7 antibody (1:1200, Abmart, PHY2844) at 4 °C overnight, followed by incubation with polyclonal peroxidase-conjugated anti-rabbit IgG (Zhongshanjinqiao, Beijing, China) at room temperature for 20 min, according to the manufacturer’s instructions.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title: Investigating the Diagnostic and Therapeutic Potential of SREBF2-Related Lipid Metabolism Genes in Colon Cancer

doi: 10.2147/OTT.S428150

Figure Lengend Snippet: Association between SREBF2 and DHCR7 expression ( A and B ) MA plot and volcano plot generated after knockdown of SREBF2 . ( C ) Correlation analysis between SREBF2 and DHCR7 expression in the TCGA cohort. ( D ) Correlation analysis between SREBF2 and DHCR7 expression across cancers in the TCGA database. ( E and F ) GSEA enrichment analysis pathway diagram of the DHCR7 high and low expression groups. The dominant genes upregulated in the HALLMARK MYC TARGETS V2 pathway.

Article Snippet: Immunohistochemical staining was performed using an anti-SREBP2 polyclonal antibody (1:1000, R&D Systems, MAB7119) or an anti-DHCR7 antibody (1:1200, Abmart, PHY2844) at 4 °C overnight, followed by incubation with polyclonal peroxidase-conjugated anti-rabbit IgG (Zhongshanjinqiao, Beijing, China) at room temperature for 20 min, according to the manufacturer’s instructions.

Techniques: Expressing, Generated, Knockdown

Journal: OncoTargets and Therapy

Article Title: Investigating the Diagnostic and Therapeutic Potential of SREBF2-Related Lipid Metabolism Genes in Colon Cancer

doi: 10.2147/OTT.S428150

Figure Lengend Snippet: Representative immunohistochemical staining of colon cancer sections Representative photomicrographs of colon cancer sections with ( A ) SREBF2 score = 0, ( B ) SREBF2 score = 1, ( C ) SREBF2 score = 2, and ( D ) SREBF2 score = 3. Representative photomicrographs of colon cancer sections with ( E ) DHCR7 score = 0, ( F ) DHCR7 score = 1, ( G ) DHCR7 score = 2, and ( H ) DHCR7 score = 3. Immunohistochemical staining of ( I ) SREBF2 and ( J ) DHCR7 in normal tissues. Scale bar, 200 μm.

Article Snippet: Immunohistochemical staining was performed using an anti-SREBP2 polyclonal antibody (1:1000, R&D Systems, MAB7119) or an anti-DHCR7 antibody (1:1200, Abmart, PHY2844) at 4 °C overnight, followed by incubation with polyclonal peroxidase-conjugated anti-rabbit IgG (Zhongshanjinqiao, Beijing, China) at room temperature for 20 min, according to the manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 1. Loss of SREBP2 inhibits the transcription of pro-inflammatory chemokines and upregulates expression of type I interferon signaling in TNFα-treated ECs. A: immunoblot of ALOD4, SREBP2-P (precursor), and SREBP2-C (cleaved) protein levels in HUVEC treated with SREBF2 siRNA (siSRE2) and with or without TNFα (10 ng/ml). Data are normalized to respective HSP90 levels and then to untreated cells (n = 3). B: volcano plot of RNA-seq analysis of differentially expressed genes of HUVEC treated with TNFα (10 ng/ ml) for 16 h and with or without siRNA targeting CTRL or SREBF2. Red lines indicate cutoffs used for pathway analysis (−1.5

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Expressing, Western Blot, RNA Sequencing, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 2. SREBP2 inhibition significantly attenuates IL6 and IL8 mRNA levels and protein expression. A: expression of chemokines that are altered with SREBF2 knockdown as well as several classical NF-κB genes that are unaffected by loss of SREBF2 from previous RNA-seq experiment. (n = 3). B: qRT-PCR analysis of RNA from HUVEC treated with 25-hydroxycholesterol (25HC) (10 μM) or siSCAP and with or without TNFα (10 ng/ml). HUVEC were treated with siRNA for 48 h prior to 16 h treatment with 25HC in full serum medium. Data are normalized to respective ACTB and then to untreated cells (n = 3). C: IL6 and IL8 ELISA from media collected from HUVEC treated with siRNA against SREBF2 or SCAP and with or without TNFα (10 ng/ml). (n = 3). D: qPCR analysis of mRNA from HUVEC treated with similar conditions as in (C). HMGCS1 is a key downstream target of SREBP2 and serves as a surrogate readout of SREBP2 activity. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Inhibition, Expressing, Knockdown, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig.3. SCAP and SREBF2 knockdown significantly increase type I interferon response genes and protein surface expression. A: heatmap of representative genes from Fig. 1B showing three independent donors. B: HLA-A, MX1, and IRF1 expression from RNA-seq experiment. C: flow cytometry analysis of surface ICAM1, VCAM1, or HLA-A,B,C levels in HUVEC treated with siSCAP or siSREBF2 at the indicated dose of TNFα. Expression was quantified as the mean fluorescence intensity of the respective fluorophore per cell (10,000 events/replicate, n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by either one-way ANOVA with Tukey’s multiple comparison test (A) or two-way ANOVA with Sidak’s multiple comparisons test (B).

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Knockdown, Expressing, RNA Sequencing, Flow Cytometry, Fluorescence, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 4. Knockdown of key proteins involved in exogenous cholesterol uptake or endogenous cholesterol synthesis increase che- mokine expression in ECs under inflammatory stress. A: qRT-PCR analysis of RNA from HUVEC incubated in fetal bovine serum (FBS) for 24 h and treated with LDLR siRNA and with or without TNFα for an additional 16 h (10 ng/ml). Data are normalized to respective ACTB and then to untreated cells (n = 3). B: representative immunoblot of SREBP2-P (precursor), SREBP2-C (cleaved), and LDLR protein levels in HUVEC incubated in FBS for 24 h, treated with LDLR siRNA, and with or without TNFα for an additional 16 h (10 ng/ml). (n = 3). C: qRT-PCR analysis of RNA from HUVEC incubated in lipoprotein-depleted serum (LPDS) for 24 h and treated with HMGCR siRNA and with or without TNFα for an additional 16 h (10 ng/ml). Data are normalized to respective ACTB and then to untreated cells (n = 3). D: representative immunoblot of SREBP2-P (precursor), SREBP2-C (cleaved) , and LDLR protein levels in HUVEC incubated in LPDS for 16 h, treated with HMGCR siRNA, and with or without TNFα for an additional 16 h (10 ng/ml). (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Incubation, Western Blot, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 6. Endogenous SREBP2 ChIP-seq in ECs treated with TNFα. A: schematic of SREBP2 ChIP-seq protocol in HUVEC treated with or without TNFα (10 ng/ml) for 10 h in lipoprotein-depleted serum (LPDS) (n = 4). B: quantification of representative ChIP-seq tags for LDLR, HMGCS1, and HMGCR gene loci from (A). C: SREBP2 binding to BHLHE40 gene locus. D: SREBP2 binding to KLF6 gene locus. E: quantification of ChIP-seq tags for BHLHE40 and KLF6 gene loci from (A). F: BHLHE40 and KLF6 expression from previous RNA-seq experiments (15). Data for ChIP-seq analysis is available in GEO under accession code GSE223094 and RNA-seq analysis is available in GEO under accession code GSE201466. Data for (D) and (E) are scaled from 0 (bottom) to 15 (top). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test (F) or t test (B and E).

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: ChIP-sequencing, Binding Assay, Expressing, RNA Sequencing, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 5. Restriction of exogenous lipoproteins upregulates pro-inflammatory chemokine transcription in cytokine-treated ECs. A: representative immunoblot of ALOD4, SREBP2-P (precursor), SREBP2-C (cleaved), and LDLR protein levels in HUVEC incubated in fetal bovine serum (FBS) or lipoprotein depleted serum (LPDS) for 24 h and treated with TNFα (10 ng/ml) for indicated time. B: qRT- PCR analysis of RNA from HUVEC incubated in FBS or LPDS for 24 h and treated with TNFα (10 ng/ml) for indicated time. Data are normalized to respective ACTB and then to untreated cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Western Blot, Incubation, Quantitative RT-PCR

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 7. Lentiviral expression of constitutively active N-terminal SREBP2 is sufficient to upregulate BHLHE40 and KLF6 levels. A: representative immunoblot of FLAG (N-SREBP2), endogenous SREBP2 (endog), and LDLR protein levels in HUVEC expressing lentiviral-driven FLAG-N-SREBP2 (FLAG-N-SRE2) and treated with or without TNFα (10 ng/ml). Negative controls were treated with empty vector lentivirus. B: qRT-PCR analysis of classical SREBP2-dependent gene expression in HUVEC expressing lentiviral- driven FLAG-N-SREBP2 (S2) and treated with or without TNFα (10 ng/ml). Data are normalized to respective ACTB and then to untreated cells (n = 3). C: qRT-PCR analysis of BHLHE40 and KLF6 expression in HUVEC expressing lentiviral-driven FLAG-N- SREBP2 (S2) and treated with or without TNFα (10 ng/ml). Data are normalized to respective ACTB and then to untreated cells (n = 3). D: qRT-PCR analysis of KLF6, BHLHE40, HMGCS1, and ICAM1 expression in HUVEC treated with or without TNFα (10 ng/ml) after overnight incubation in lipoprotein-depleted serum (LPDS) or LPDS+LDL (50 μg/ml). (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test (B) and (C) or two-way ANOVA with Sidak’s multiple compar- isons test (D).

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Expressing, Western Blot, Plasmid Preparation, Quantitative RT-PCR, Gene Expression, Incubation, Comparison

Journal: Journal of lipid research

Article Title: SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6.

doi: 10.1016/j.jlr.2023.100411

Figure Lengend Snippet: Fig. 8. RNAseq analysis of KLF6 knockdown in ECs reveals a significant inhibition chemokine gene expression that shares overlap with SREBF2 knockdown. A: volcano plot of RNA-seq analysis of differentially expressed genes of HUVEC treated with TNFα (10 ng/ ml) for 16 h and with or without siRNA targeting CTRL or KLF6. Red lines indicate cutoffs used for pathway analysis (−1.5

Article Snippet: A cocktail of antibodies targeting the N-terminal domain of SREBP2 were used for SREBP2 pull-down (Sigma #MABS1988, Novus #NBP1-54446, Abcam #ab30682).

Techniques: Knockdown, Inhibition, Gene Expression, RNA Sequencing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Functional Interaction Between PRDM16 and the SREBP Pathway Controls Lipid Metabolism

doi: 10.3390/ijms262110246

Figure Lengend Snippet: PRDM16 targets the nuclear forms of SREBP1a, SREBP1c, and SREBP2. ( A ) HepG2 cells were transfected with the HMG-CoA synthase promoter-luciferase construct (SYNSRE) together with GFP or PRDM16 (P16) cDNA in the absence (EV) or presence of cDNA encoding the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. ( B ) HepG2 cells were transfected with the HMG-CoA synthase promoter-luciferase construct (SYNSRE) together with non-targeted (C) or PRDM16 (P16) shRNA in the absence (EV) or presence of cDNA encoding the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. EV, empty vector. ( C ) HepG2 cells were transfected with a minimal promoter-luciferase construct containing a single binding site for the yeast transcription factor Gal4 (G1E1B-Luc) together with expression vectors for the DNA binding domain of Gal4 (Gal4), or the same DNA binding domain fused to the nuclear forms of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2), and either GFP or PRDM16 (P16). Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. ( D ). HepG2 cells were transfected with the same promoter-reporter construct as in ( A ) together with the DNA binding domain of Gal4 (Gal4), or the same DNA binding domain fused to the transactivation domain (TAD) of SREBP1a (nS1a), SREBP1c (nS1c), or SREBP2 (nS2), and either GFP or PRDM16 (P16) cDNA. Forty-eight hours after transfection, cells were lysed, and luciferase activity was measured. Cells were also transfected with the β-galactosidase gene as an internal control for transfection efficiency. Luciferase values (relative light units, RLU) were calculated by dividing the luciferase activity by the β-galactosidase activity. The data represent the average −/+ SEM of at least three independent experiments performed in duplicates. The RLU of WT promoter-reporter constructs transfected with either GFP or non-targeted shRNA were set to 1. p -values lower than 0.05 were considered statistically significant. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: Antibodies against HMG-CoA synthase (sc-271543), SREBP1 (sc-8984 and sc-13551), GST (sc-138), Myc (sc-40), and HA (sc-805) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and the SREBP2 antibody (AF7119) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transfection, Luciferase, Construct, Activity Assay, shRNA, Plasmid Preparation, Binding Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: The Functional Interaction Between PRDM16 and the SREBP Pathway Controls Lipid Metabolism

doi: 10.3390/ijms262110246

Figure Lengend Snippet: PRDM16 interacts with nuclear SREBP1/2 through its zinc finger domains. ( A ) HEK 293 cells were transfected with cDNA encoding the nuclear forms of either SREBP1c or SREBP2 in the absence (−) or presence (+) of HA-tagged PRDM16. Forty-eight hours after transfection, cells were lysed, precleared, and PRDM16 was immunoprecipitated with anti-HA antibodies. The immunoprecipitated proteins were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes. The amount of nuclear SREBP1c and SREBP2, and PRDM16 in the immunoprecipitated material was determined by Western blotting (left). The levels of nuclear SREBP1c and SREBP2, PRDM16, and α-tubulin (loading control) in the whole cell lysates (Input) was determined by Western blotting (right). ( B ) A schematic structure of PRDM16 (top) with the numbering corresponding to the fragments used for GST pulldown assays, and a schematic structure of nuclear SREBP1a and the different deletion mutants used in the GST pulldown assays (bottom). ( C ) HEK293 cells were transfected with expression vectors for the nuclear forms of SREBP1a, SREBP1c, or SREBP2, and whole-cell lysates were used in GST pulldown experiments with the indicated PRDM16 fragments. The captured proteins were separated on SDS-PAGE gels, and the amount of the individual nuclear SREBP (nSREBP) proteins were analyzed by Western blotting. GST alone was used as a negative control in the pulldown experiments and was run out on the same gel together with the whole-cell lysates (Input). The Coomassie staining of the PRDM16 GST fusion proteins is shown in . ( D ) HEK293 cells were transfected with expression vectors encoding full-length nuclear SREBP1a (nSREBP1a), or the mutants illustrated in ( B ). These mutants contained deletions of the N-terminal transactivation domain (ΔTAD), the C-terminal regulatory domain (ΔC), or both domains (ΔTADΔC). Whole-cell lysates were used in GST pulldown assays using GST-ZF2 as bait. The pull-down material (top) and whole-cell lysates (Input, bottom) were analyzed by Western blotting. The Coomassie staining of the GST-ZF1 and GST-ZF2 proteins is shown in . ( E ) Nuclear extracts were prepared from MCF7 cells grown in lipoprotein-deficient media to fully activate SREBP1/2. The nuclear extracts were used in GST pulldown assays using GST-ZF1 and ZF2 of PRDM16, with GST alone as negative control. The captured material was separated on SDS-PAGE gels together with an aliquot of the nuclear extracts used in the pulldown assays (Input) and analyzed by Western blotting. The Coomassie staining of GST-ZF1 and ZF2 is shown in .

Article Snippet: Antibodies against HMG-CoA synthase (sc-271543), SREBP1 (sc-8984 and sc-13551), GST (sc-138), Myc (sc-40), and HA (sc-805) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and the SREBP2 antibody (AF7119) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Control, Expressing, Negative Control, Staining

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage

doi: 10.1002/art.39337

Figure Lengend Snippet: Pharmacologic cholesterol inhibition attenuates ADAMTS5 expression in human osteoarthritic (OA) cartilage. A, Results of real‐time polymerase chain reaction (PCR) analyses in human OA cartilage explants, showing a reduction in expression of the OA markers MMP13 and ADAMTS5 in samples treated with statin (shaded bars) as compared to vehicle control (solid bars) (n = 4 per group). Results are the mean fold change (with 95% confidence intervals) relative to controls (set at 1.0). ∗ = P < 0.05 versus controls. B, Luciferase activity from the ADAMTS5 promoter construct after transfection into primary human OA chondrocytes with or without statin treatment. Results, measured in triplicate, are the mean ± SEM relative light units (RLU). C, Enrichment of ADAMTS5 gene expression in ATDC5 cells. Chromatin immunoprecipitation was performed in ATDC5 cells with antibodies to SREBF2 (red bars) or IgG as a control (black bars). ADAMTS5 expression was assessed by real‐time PCR with primers designed in the proximal promoter region (Proximal) or a control region (Distal). Results are the fold change in ADAMTS5 expression with anti‐SREBF2 relative to IgG (set at 1.0), D, Schematic diagram of the ADAMTS5 promoter, showing a sterol regulatory element (SRE) consensus binding site (red) that is conserved in both mice and humans (results obtained from Mulan analysis; see http://mulan.dcode.org ). UTR = untranslated region.

Article Snippet: A SREBF2 antibody (catalog no. AF7119‐SP; R&D Systems) and a negative control IgG antibody (Active Motif) were used to immunoprecipitate the DNA–protein complexes.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control, Luciferase, Activity Assay, Construct, Transfection, Gene Expression, Chromatin Immunoprecipitation, Binding Assay