Journal: Physiological Genomics

Article Title: Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle

doi: 10.1152/physiolgenomics.00128.2016

Figure Lengend Snippet: Treatment with eplerenone or mifepristone alone caused gene expression changes in normal human myotubes. A: treatment with the selective MR antagonist eplerenone resulted in 42 gene expression changes and functional clustering of these genes revealed: 3 apoptotic, 5 immune or defense response, 3 transcriptional regulators, 4 ion binding, 3 cell adhesion, 3 alternative splicing, 12 muscle contraction, and 9 genes with unknown or specific functions. B: treatment with the GR antagonist mifepristone resulted in 27 gene expression changes and functional clustering of these genes revealed: 1 apoptotic, 4 immune or defense response, 9 regulators of transcription, 2 ion binding, 2 alternative splicing, 2 developmental, and 7 genes with unknown or specific functions.

Article Snippet: Cells were serum restricted for 5 days in high-glucose Dulbecco’s modified medium (DMEM, Invitrogen) and supplemented with 2% horse serum and 100 U/ml penicillin-streptomycin, followed by 48 h treatments with: aldosterone (10 µM; MR EC 50 : 1.3 nM; GR EC 50 : 80 nM), eplerenone (10 µM; MR IC 50 : 81 nM; Pfizer Compound Transfer Program), mifepristone (1 µM; GR IC 50 : 2.6 nM; Cayman Chemical), prednisolone (1 µM; GR EC 50 : 4.4 nM), spironolactone (10 µM; MR IC 50 : 1.6 nM; GR IC 50 : 2.9 µM), or vehicle (drugs were purchased from Sigma and dissolved in 100% DMSO unless specified otherwise).

Techniques: Expressing, Functional Assay, Binding Assay

Journal: Physiological Genomics

Article Title: Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle

doi: 10.1152/physiolgenomics.00128.2016

Figure Lengend Snippet: Gene expression changes with low-dose aldosterone compared with aldosterone plus antagonists in normal human myotubes. A: co-incubation with aldosterone plus eplerenone increased 8 genes and decreased 85 genes (93 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 4 apoptotic, 7 transcriptional regulators, 5 ion binding, 7 transmembrane, 6 cell adhesion, 11 cytoskeletal protein binding, 4 muscle contraction, 27 cell cycle, and 22 genes with unknown or specific functions. B: co-incubation with aldosterone plus spironolactone increased 24 genes and decreased 90 genes (114 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 9 apoptotic, 8 immune or defense response, 4 transcriptional regulators, 9 ion binding, 6 transmembrane, 13 cell adhesion, 8 structural constituents of muscle, 30 tissue development, and 27 genes with unknown or specific functions. C: co-incubation with aldosterone plus mifepristone increased 251 genes and decreased 370 genes (621 total) compared with myotubes treated with aldosterone alone. Functional clustering revealed: 33 apoptotic, 38 immune or defense response, 62 transcriptional regulators, 63 ion binding, 45 transmembrane, 18 cell adhesion, 63 ECM or cytoskeletal protein binding, 30 alternative splicing, 106 vasculature or muscle structure development, 25 oxidative stress responsive, 54 cell cycle, 7 oxidoreductase, 9 GTPase, and 68 genes with unknown or specific functions.

Article Snippet: Cells were serum restricted for 5 days in high-glucose Dulbecco’s modified medium (DMEM, Invitrogen) and supplemented with 2% horse serum and 100 U/ml penicillin-streptomycin, followed by 48 h treatments with: aldosterone (10 µM; MR EC 50 : 1.3 nM; GR EC 50 : 80 nM), eplerenone (10 µM; MR IC 50 : 81 nM; Pfizer Compound Transfer Program), mifepristone (1 µM; GR IC 50 : 2.6 nM; Cayman Chemical), prednisolone (1 µM; GR EC 50 : 4.4 nM), spironolactone (10 µM; MR IC 50 : 1.6 nM; GR IC 50 : 2.9 µM), or vehicle (drugs were purchased from Sigma and dissolved in 100% DMSO unless specified otherwise).

Techniques: Expressing, Incubation, Functional Assay, Binding Assay, Protein Binding

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A–E) Indicated proteins were transiently transfected into HEK293 cells. After 24 hr, cytosolic extracts (CE) and nuclear extracts (NE) were immunoblotted with the indicated antibodies. SREBP-1c cleavage by rhomboid protease family (A), SREBP-1a cleavage by RHBDL4 (B), SREBP-1c cleavage by RHBDL4 mutant S144A (C), SREBP-1c cleavage after coexpression of RHBDL4 and Insig1 (D), and SREBP-1c mutant 3M cleavage by RHBDL4 (E) are shown. HSV-SREBPs were immunoblotted with HSV antibodies. (−), mock; R1, RHBDL1; R2, RHBDL2; R3, RHBDL3; R4, RHBDL4; WT, wild type; S144A, the catalytically inactive mutant of RHBDL4; 3M, three known cleavage sites for CPP32, S2P, and S1P mutant of SREBP-1c, P, precursor; N, nuclear. (F) An in vitro cleavage assay was performed using recombinant HSV-SREBP-1c-HA and RHBDL4-V5 purified from a cell-free protein synthesis system using wheat germ. Indicated proteins were analyzed by immunoblotting. Arrowheads show precursor (P) and nuclear (N) SREBP-1c. (G) Eight-week-old male C57BL/6J mice were injected with green fluorescent protein (GFP) or RHBDL4 expressing adenovirus via the tail vein. Six days after injection, the mice (n = 3–4 per group) were subjected to fasting and refeeding. Immunoblot analysis of indicated proteins in whole cell lysates (WCL) and NE from mouse livers was performed. See also and .

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Transfection, Mutagenesis, In Vitro, Cleavage Assay, Recombinant, Purification, Western Blot, Injection, Expressing

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) Indicated proteins were expressed in HEK293 cells. After 24 hr, cytosolic extracts (CE) and nuclear extracts (NE) were immunoblotted with indicated antibodies. HSV-SREBPs were immunoblotted with HSV antibodies. P, precursor; N, nuclear. (B and C) Schematic overview of placental alkaline phosphatase (PLAP) assay (B). (C) PLAP-SREBP-1c (431-1123) and PLAP-SREBP-1c (490-1123) were prepared by fusing the catalytic domain of PLAP with the indicated amino acid residues of SREBP-1c. Indicated proteins were expressed in HEK293 cells. After 24 hr, PLAP activities in the medium were measured. Quantification was performed on six samples. (D) Diagram of SREBP-1c (451-550). Transmembrane domains are embedded in gray boxes. Numbers indicate the number of amino acid residues. S1P and S2P recognition and cleavage sites are indicated in bold. Rhomboid recognition motif and mutated amino acids are indicated in pink boxes and red, respectively. (E) Immunoblot analysis of SREBP-1c in CE and NE of HEK293A cells expressing RHBDL4 and either SREBP-1c WT or SREBP-1c harboring the indicated mutants. (F) HEK293A cells were transfected with Gal4-RE-Luc, pRL-SV40, and the indicated expression plasmids. After 24 hr, cell extracts were examined using luciferase assays. Quantification was performed on six samples. Data are represented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Western Blot, Expressing, Transfection, Luciferase

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: The RHBDL4 protease core is formed by six helical transmembrane domains (green) and contains a catalytic dyad of serine and histidine. The transmembrane domain of SREBP-1c as the substrate (cyan) interacts with the RHBDL4 active site harboring the catalytic dyad. The S2P cleavage site of SREBP-1c in sterol-regulated classical pathway is located near the catalytic dyad of RHBDL4.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques:

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) Cellular localization of RHBDL4 and SREBP-1c. HEK293A cells expressing the indicated YFP-RHBDL4 and DsRed-ER (top) or mCherry-SREBP-1c (bottom) constructs are shown. Nuclei were stained with DAPI. Scale bar, 20 μm. (B–E) Immunoprecipitation assay. HEK293A cells were transfected with the indicated expression plasmids. After 24 hr, cell extracts were immunoprecipitated (IP) with the indicated antibodies. Bound proteins were immunoblotted with the indicated antibodies. Interaction of SREBP-1c and RHBDL4 (B), SCAP and RHBDL4 (C), Insig1 and RHBDL4 (D), SCAP and SREBP-1c and Insig1 (E) are shown. (F and G) Translocation of SREBP-1c to the nucleus by RHBDL4. HeLa cells expressing YFP-SREBP-1c-CFP and mCherry-SCAP, mCherry-RHBDL4, or mCherry-RHBDL4 S144A are shown. Cells with nuclear fluorescence of YFP were counted and the percentages expressed as bar graphs (G). Scale bar represents 25 μm. (H) Luciferase assay for SREBP-1c activity. HEK293 cells were transfected with SRE-Luc, pRL-SV40, and the indicated expression plasmids. After 24 hr, cell extracts were examined using luciferase assays. Quantification was performed on four samples. Data are represented as means ± SEM. ** P < 0.01.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Expressing, Construct, Staining, Immunoprecipitation, Transfection, Translocation Assay, Fluorescence, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A–C) HepG2 cells (A and C) and HEK293 cells (B) were transfected with control siRNA or two independent RHBDL4 siRNAs (#1 and #2). Twenty-four hr after transfection, cells were incubated with 10% DLS containing 3 μM 25-hydroxycholesterol for 24 hr. (A and B) Immunoblot analysis of the indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE). P, precursor; N, nuclear. (C) qRT-PCR analysis of SREBP-1 and related genes. Data are represented as means ± SEM. Quantification was performed on six samples. * P < 0.05, ** P < 0.01. See also .

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Transfection, Control, Incubation, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) qRT-PCR analysis of SREBP related genes. HepG2 cells were transfected with control siRNA or two independent RHBDL4 siRNAs (#1 and #2). Twenty-four hr after transfection, cells were incubated with 10% DLS containing 3 μM 25-hydroxycholesterol for 24 hr. qRT-PCR analysis of SREBP-1 related genes was performed. Data represented as means ± SEM. Quantification was performed on six samples. ** P < 0.01. (B and C) Immunoblot analysis of the cleavage of SREBP-1 by RHBDL4. HepG2 cells (B) and HEK293 cells (C) were transfected with control siRNA or two independent RHBDL4 siRNAs (#1 and #2). Twenty-four hr after transfection, cells were incubated with 10% DLS for 24 hr. Immunoblot analysis of indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE) was performed. P, precursor; N, nuclear.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Quantitative RT-PCR, Transfection, Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: Eight-week-old male wild type (WT) and RHBDL4 knockout (KO) mice were fed with normal chow or a Western diet (WD) for 14 days (A, B and D), or a high-sucrose diet (HS) for 7 days (C). (A and B) Body weight and liver weight (A), white adipose tissue (WAT) weight (B) of mice. Data are represented as means ± SEM. Quantification was performed on eight samples (A) or four samples (B). * P < 0.05, *** P < 0.001. (C) Representative immunoblot analysis of the indicated proteins in the membrane fraction and nuclear extracts (NE) of mouse livers. P, precursor; N, nuclear. (D) Functional annotations associated with KO upregulated (left) and downregulated (right) genes compared to WT mice fed a normal chow diet.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Knock-Out, Western Blot, Membrane, Functional Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: Eight-week-old male wild type (WT) and RHBDL4 knockout (KO) mice were fed on normal chow or a Western diet (WD) for 14 days. (A) Representative immunoblot analysis of the indicated proteins in the membrane fraction and nuclear extracts (NE) of mouse livers. P, precursor; N, nuclear. (B) qRT-PCR analysis of lipogenic genes from mouse livers. (C and D) RNA-seq analysis of livers of WT and KO mice fed on normal chow or a WD for 14 days (FDR < 0.05, n = 4). (C) Venn diagram of overlap between upregulated genes from mice fed on a WD (WD induced genes > 1.5-fold versus normal chow, top) and downregulated genes in KO (KO reduced genes > 1.5-fold versus WT mice fed on a WD, bottom). (D) Functional annotations associated with KO downregulated genes compared to WT mice fed on a WD and overlapped genes with WD upregulated genes compared to normal chow. (E and F) qRT-PCR analysis of lipoprotein assembly and remodeling genes (E) and PUFA producing genes (F) in mouse livers. Data are represented as means ± SEM. Quantification was performed on eight samples. * P < 0.05, ** P < 0.01 and *** P < 0.001. See also .

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Knock-Out, Western Blot, Membrane, Quantitative RT-PCR, RNA Sequencing, Functional Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: Eight-week-old male wild type (WT) and RHBDL4 knockout (KO) mice were fed normal chow or a Western diet (WD) for 14 days. (A) Plasma triglyceride (TG), plasma cholesterol (TC) levels, liver TG, and liver TC contents of mice. Data are represented as means ± SEM. Quantification was performed on eight samples. * P < 0.05, *** P < 0.001. (B) Total fatty acids (left) and fatty acid composition (right) of liver tissue in WT and KO fed a WD for 14 days. Data are represented as means ± SEM. Quantification was performed on four samples. * P < 0.05, ** P < 0.01. (C and D) Representative hematoxylin and eosin (H&E) stained section (C) and Oil red O (ORO) stained section (D) of mouse livers. Scale bar represents 100 μm. See also .

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Knock-Out, Western Blot, Clinical Proteomics, Staining

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A and B) Eight-week-old male wild type (WT) and RHBDL4 knockout (KO) mice were fed a Western diet (WD) for 14 days, and treated with 5 % EPA-E once the last day. (A) Representative immunoblot analysis of SREBP-1/2 and RHBDL4 in the membrane fraction and nuclear extracts (NE) of mouse livers. P, precursor; N, nuclear. (B) qRT-PCR analysis of lipogenic genes in mouse livers. Data represented as means ± SEM. Quantification was performed on 9-10 samples. * P < 0.05, ** P < 0.01, *** P < 0.001. (C-E) HEK293 cells were transfected with HSV-SREBP-1c and RHBDL4 expression plasmids. After 4 hr, cells were incubated with 10% DLS containing the indicated concentration of each fatty acid for 20 hr. Immunoblot analysis of the indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE) was performed. SREBP-1c cleavage by AA, EPA, and DHA (C), SREBP-1a cleavage by PA (D), SREBP-1c cleavage by 100 μM PA and 10 μM EPA (E) were shown. P, precursor; N, nuclear. See also .

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Knock-Out, Western Blot, Membrane, Quantitative RT-PCR, Transfection, Expressing, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) HEK293 cells were transfected with HSV-SREBP-1c and RHBDL4 expression plasmids. After 4 hr, cells were incubated with 10% DLS containing the indicated concentration of each fatty acid for 20 hr. Immunoblot analysis of the indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE) was performed. SREBP-1c cleavage by various fatty acids were shown. P, precursor; N, nuclear. (B) The quantification of the relative levels of SREBP-1c nuclear form to the vehicle treatment of panel (A). Data are represented as means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with vehicle group. (C) The effect of EPA on the interaction of RHBDL4 and SREBP-1c. HEK293A cells were transfected with HSV-SREBP-1c and RHBDL4-V5 expression plasmids. After 4 hr, cells were incubated with 10% DLS containing with 3 μM EPA for 20 hr. Cell extracts were immunoprecipitated (IP) with anti-HSV. Bound proteins were immunoblotted with anti-HSV and anti-V5 antibodies. (D and E) The suppressive effect of EPA on the cleavage of pTα and MPZ L170R induced by RHBDL4. HEK293 cells were transfected with RHBDL4 and HA-tagged pTα (D) or MPZ L170R (E) expression plasmids. After 4 hr, cells were incubated with 10% DLS containing with 50 μM EPA for 20 hr. Immunoblot analysis of the indicated proteins in cell extracts was performed.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Transfection, Expressing, Incubation, Concentration Assay, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

doi: 10.1101/2021.08.24.457590

Figure Lengend Snippet: (A) qRT-PCR analysis of SREBP-1 related genes. HepG2 cells were transfected with the indicated siRNA. 24 hr after transfection, cells were incubated with 10% DLS containing 3 μM 25-hydroxycholesterol for 24 hr. Data are represented as means ± SEM. Quantification was performed on six samples. *P < 0.05, ** P < 0.01. (B) Luciferase assay of SREBP-1c regulated by p97. HEK293A cells were transfected with Gal4-RE-Luc, pRL-SV40, and the indicated expression plasmids. After 24 hr, cell extracts were examined using luciferase assays. Data are represented as means ± SEM. Quantification was performed on six samples. *P < 0.05, ** P < 0.01. (C) The RHBDL4 with mutation of ubiquitin interaction motif (UIM) show weaker cleavage activity for SREBP-1c. HEK293 cells were transfected with HSV-SREBP-1c and either RHBDL4 WT or mutant RHBDL4 (ΔUIM) plasmid. The indicated proteins in cytosolic extracts (CE) and nuclear extracts (NE) were immunoblotted. HSV-SREBPs were immunoblotted with HSV antibodies. P, precursor; N, nuclear.

Article Snippet: The membranes were incubated with the antibodies as follows: antibodies for HSV tag (Millipore, 69171), SREBP-1 (Santa Cruz Biotechnology, sc-8984 or LifeSpan Biosciences, LS-C179707 or Novus, NB 600-582), RHBDL1 (Abnova, H00009028-A01), RHBDL2 (Abcam, ab179726), RHBDL3 (Sigma-Aldrich, SAB1104094), RHBDL4 (Proteintech, 20869-1-AP), PARL (Abnova, PAB13061), SCAP (Santa Cruz Biotechnology, sc-13553), p97/VCP (Cell Signaling Technology, #2648), UFD1L (Cell Signaling Technology, #13789), NPLOC4 (Cell Signaling Technology, #13489), Insig1 (Abcam, ab112248), V5 tag (BioLegend, #903801), HA tag (Roche, #12013819001 or Cell Signaling Technology, #3724), Myc tag (Santa Cruz Biotechnology, sc-789, sc-40, or Cell Signaling Technology, #2272), SREBP2 (Cayman chemical, #10007663), respectively.

Techniques: Quantitative RT-PCR, Transfection, Incubation, Luciferase, Expressing, Mutagenesis, Ubiquitin Proteomics, Activity Assay, Plasmid Preparation