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: Development (Cambridge, England)

Article Title: Postnatal eye size in mice is controlled by SREBP2-mediated transcriptional repression of Lrp2 and Bmp2

doi: 10.1242/dev.200633

Figure Lengend Snippet: SREBP2 promotes mouse eye size growth during early postnatal development. (A) Schematic showing the experimental design. (B,C) Expression of the GFP reporter starts in the RPE at 1 day post AAV-CMV-GFP infection (1E9 vg/eye). Boxed area is enlarged and shown in C. Scale bars: 500 μm (B); 50 μm (C). (D-G) Representative images of uninjected eyes and eyes infected by AAV8-CMV-GFP/nSrebp2/flSrebp2 (1E9 vg/eye) at P0 and harvested at P14. Scale bars: 1 mm. uninj, uninjected; OE, overexpression. (H) Growth curve of mouse eye. AL, axial length; ED, equatorial diameter. P0 n =22; P7 n =19; P14 n =37; P30 n =58; P60 n =28; P90 n =6. (I,J) Time-course examination of the AL and ED increase induced by nSREBP2 overexpression. Data are represented as the ratio of injected right eye (R)/uninjected left eye (L). GFP: P0 n =10; P7 n =4; P14 n =4; P30 n =6; P60 n =6; nSREBP2: P0 n =11; P7 n =4; P14 n =7; P30 n =5; P60 n =6. Data are mean±s.e.m.; * P <0.05, ** P <0.01 (unpaired Student's t -test).

Article Snippet: The mouse Srebp2 coding sequence was cloned from a pLKO-puro FLAG Srebp2 plasmid (Addgene plasmid # 32018 ).

Techniques: Expressing, Infection, Over Expression, Injection

Journal: Development (Cambridge, England)

Article Title: Postnatal eye size in mice is controlled by SREBP2-mediated transcriptional repression of Lrp2 and Bmp2

doi: 10.1242/dev.200633

Figure Lengend Snippet: SREBP2 functions in the RPE to control eye growth. (A-C) Size comparison of eyes infected by different viruses. The diagrams on the left illustrate the cell types with the targeted gene expression (green) by the AAV8 virus with different promoters. The indicated viruses were injected at P0, and eyes were harvested at P14. All viruses were injected at a concentration of 1E9 vg/eye. Data are represented as the ratio of injected right eye (R)/uninjected left eye (L). Data are mean±s.e.m.; * P <0.05, ** P <0.01 (one-way ANOVA analysis with post-hoc Tukey test) (A,B) or unpaired Student's t -test (C). AC, amacrine cell; BP, bipolar cell; ns, no significant difference.

Article Snippet: The mouse Srebp2 coding sequence was cloned from a pLKO-puro FLAG Srebp2 plasmid (Addgene plasmid # 32018 ).

Techniques: Control, Comparison, Infection, Targeted Gene Expression, Virus, Injection, Concentration Assay

Journal: Development (Cambridge, England)

Article Title: Postnatal eye size in mice is controlled by SREBP2-mediated transcriptional repression of Lrp2 and Bmp2

doi: 10.1242/dev.200633

Figure Lengend Snippet: SREBP2 transcriptionally suppresses Lrp2 . (A,B) Representative eye images of control mice ( Lrp2 fl/fl without Cre) or Lrp2 conditional knockout (cko) mice. Lrp2 cko was induced by injecting AAV8-Best1-Cre (1E7 vg/eye) to Lrp2 fl/fl mouse eyes at P0. Scale bars: 1 mm. (C) Quantification of eye size. AAV8-Best1-Ctrl sh/ Lrp2 sh1/ Lrp2 shRNA2 ( sh2 ) viruses were injected at a concentration of 1E9 vg/eye, and AAV8-Best1-Cre was injected at a concentration of 1E7 vg/eye. (D,E) Expression levels of Srebp2 , Lrp2 , Hmgcr and Ldlr determined by qPCR when nSREBP2 was overexpressed (D) or knocked down (E) in the mouse RPE. The mouse eyes were injected by AAV8-Best1-GFP/nSrebp2 (D) or Ctrl sh/Srebp2 sh (E) (1E9 vg/eye) at P0 and harvested at P14. Expression levels were normalized to Gapdh mRNA and expressed relative to the GFP/Ctrl sh control. (F) Left: Schematic of the experimental design. Right: Expression levels of Lrp2 , Hmgcr and Ldlr determined by qPCR in RPE explant cultures with or without BF175 treatment. Expression levels were normalized to Gapdh mRNA and expressed relative to the vehicle-treated control. (G) Quantification of eye size. Eyes were injected with AAV8-Best1-Lrp2 sh1 alone, AAV8-Best1-Lrp2 sh1+AAV8-Best1-Ctrl sh or Srebp2 sh. For combined injection, viruses were mixed at a 1:1 ratio and injected at a total concentration of 2E9 vg/eye. (H) Quantification of eye size. Eyes were injected with AAV8-Best1-Lrp2 sh1 with vehicle or BF175. (I) Relative luciferase activity was determined in HEK293 cells. A luciferase reporter containing the human LDLR promoter (−335/+3) or LRP2 promoter (−505/−13) was co-transfected with pCAG-Cre (Ctrl) or pCAG-human n SREBP2 . Relative luciferase activity was normalized to Renilla luciferase activity. Schematic on the left shows the designs of the reporter constructs. (J) An illustration showing a working model, in which Srebp2 promotes mouse eye size by repressing Lrp2 , which is an inhibitor of eye overgrowth. All viruses were injected at P0, and eyes were harvested at P14 (C,G,H). Data are represented as the ratio of injected right eye (R)/uninjected left eye (L) (C,G,H). All data are shown as mean±s.e.m. * P <0.05, ** P <0.01 (one-way ANOVA analysis with post-hoc Tukey test for C,G or unpaired Student's t -test for D-F,H,I). ns, no significant difference

Article Snippet: The mouse Srebp2 coding sequence was cloned from a pLKO-puro FLAG Srebp2 plasmid (Addgene plasmid # 32018 ).

Techniques: Control, Knock-Out, Injection, Concentration Assay, Expressing, Luciferase, Activity Assay, Transfection, Construct

Journal: Development (Cambridge, England)

Article Title: Postnatal eye size in mice is controlled by SREBP2-mediated transcriptional repression of Lrp2 and Bmp2

doi: 10.1242/dev.200633

Figure Lengend Snippet: BMP2 is the downstream effector of Srebp2 and Lrp2 . (A) Schematic showing the experimental design. (B) Volcano plots illustrating genes that were differentially expressed between the enlarged eye groups and controls. Genes significantly upregulated and downregulated (BH-adjusted P <0.05, |log2FC|>1) are shown in red and green, respectively. Values are presented as −log10 (BH-adjusted P -value). (C) GSEA suggests five significantly enriched canonical pathways shared by the three enlarged eye groups. The number of significantly enriched ( P <0.05) pathways in each group is also indicated in the circle. (D) Heatmap of the gene expression levels of BMP pathway components. Genes were clustered based on hierarchical clustering on z -normalized expression levels (red: high; blue: low). (E) Left: Schematic showing the experimental design. All viruses were injected at a concentration of 1E9 vg/eye. Right: Quantification of AL and ED. Data are represented as the ratio of injected right eye (R)/uninjected left eye (L). Ctrl sh n =12; Bmp2 sh1 n =11; Bmp2 sh2 n =3; Bmp4 sh1 n =5; Bmp4 sh2 n =3; Bmp6 sh1 n =7; Bmp6 sh2 n =3; Bmp7/11 sh1/2 n =3. Data are mean±s.e.m. * P <0.05, ** P <0.01 (one-way ANOVA with post-hoc Tukey test). ns, no significant difference

Article Snippet: The mouse Srebp2 coding sequence was cloned from a pLKO-puro FLAG Srebp2 plasmid (Addgene plasmid # 32018 ).

Techniques: Gene Expression, Expressing, Injection, Concentration Assay

Journal: Development (Cambridge, England)

Article Title: Postnatal eye size in mice is controlled by SREBP2-mediated transcriptional repression of Lrp2 and Bmp2

doi: 10.1242/dev.200633

Figure Lengend Snippet: The SREBP2-LRP2-BMP2 signaling axis regulates postnatal eye growth. (A) Bmp2 expression levels determined by qPCR in mouse RPE with nSREBP2 overexpression or with Srebp2 knockdown. Mouse eyes were injected with AAV8-Best1-GFP/nSrebp2 or Ctrl sh/Srebp2 sh (1E9 vg/eye) at P0 and harvested at P14. Expression levels were normalized to Gapdh mRNA and expressed relative to the GFP/Ctrl sh control. GFP/n Srebp2 n =3; Ctrl sh n =4; Srebp2 sh n =3. (B) Top: Illustration showing the two E-box motifs in intron 1 of the human BMP2 gene and the ChIP-qPCR primer positions. P1, primer set 1; P2, primer set 2; P3, primer set 3; TSS, transcription start site. Bottom: ChIP-qPCR showed SREBP2 protein enrichment at the promoter as well as at the first E-box motif of the human BMP2 gene in ARPE19 cells. (C) Relative luciferase activity of reporters containing the human LDLR promoter (−335/+3), BMP2 promoter (−500/−1) or BMP2 intron I (+1271/+1778) fused with a minimal promoter (minP). (D) Bmp2 expression levels determined by qPCR in mouse RPE with Lrp2 knockdown. Ctrl sh n =4; Lrp2 sh1 n =3; Lrp2 sh2 n =6. (E-G) Relative mRNA expression and western blotting of Lrp2 , Bmp2 and Srebp2 in the RPE of wild-type mice at three different ages. P0 n =5; P14 n =7; P30 n =7. Expression levels were normalized to Gapdh mRNA and expressed relative to P0. All data are shown as mean±s.e.m. * P <0.05, ** P <0.01 (unpaired Student's t -test for A-C and one-way ANOVA with post-hoc Tukey test for D,E). ns, no significant difference. (H) Schematic illustrating a working model based on our data. High Srebp2 and low Lrp2 / Bmp2 promote the rapid eye size increase in neonatal mice, whereas low Srebp2 and high Lrp2 / Bmp2 ensure that eye growth stops at the proper size in adult mice.

Article Snippet: The mouse Srebp2 coding sequence was cloned from a pLKO-puro FLAG Srebp2 plasmid (Addgene plasmid # 32018 ).

Techniques: Expressing, Over Expression, Knockdown, Injection, Control, ChIP-qPCR, Protein Enrichment, Luciferase, Activity Assay, Western Blot

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