Journal: bioRxiv

Article Title: Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

doi: 10.1101/2023.01.13.523943

Figure Lengend Snippet: Collagen biosynthesis positively regulates cell-based collagen clearance and MRC2 expression. (A) Heatmap of overall phenotype scores from original screens as indicated for top genes from ‘Collagen Biosynthetic Process’ GO gene-set. (B) Flow-cytometry based collagen-uptake assay in U937 cells after shRNA treatment as indicated vs. Scramble control. MFI = Mean Fluorescence Intensity. N=4 per group. (C) Q-RT-PCR in U937 cells after shRNA treatment as indicated vs. Scramble control. N=4 per group. (D) Western blot of U937 cells after shRNA treatment as indicated vs. Scramble control, representative of N=4 independent experiments. (E) Q-RT-PCR in U937 cells after shRNA treatment as indicated vs. Scramble control. N=4 per group. (F) Western blot of MRC5 fibroblasts after shRNA treatment as indicated vs. Scramble control, representative of N=3 independent experiments. (G) Representative confocal immunofluorescence images of MRC5 fibroblasts after shRNA treatment as indicated vs. Scramble control. (H) Flow-cytometry based collagen-uptake assay in MRC5 cells after shRNA treatment as indicated vs. Scramble control. N=4 per group. (I) Flow-cytometry based collagen-uptake assay in U937 cells after shRNA treatment as indicated vs. Scramble control. N=4-6 per group. (J-K) Q-RT-PCR in U937 cells after shRNA treatment as indicated vs. Scramble control. N=4-5 per group. (L-M) Flow-cytometry based collagen-uptake assay in U937 (L) or MRC5 (M) cells after drug treatment as indicated vs. vehicle control. N=4 per group. Western blot of corresponding cells and conditions shown below, each representative of N=3-4 independent experiments. (N-P) Q-RT-PCR in MRC5 cells after drug treatment as indicated vs. vehicle control. N=3-5 per group. Western blot of corresponding cells and conditions shown below, each representative of N=3 independent experiments. Statistics: (B-C, E, H-K) ANOVA with post-hoc Bonferroni testing; (L-P) Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: For MRC2 overexpression, pCI-Mrc2 (murine Mrc2 cloned into the pCI-Neo expression vector, from Promega) was used as we previously described .

Techniques: Expressing, Flow Cytometry, shRNA, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

doi: 10.1101/2023.01.13.523943

Figure Lengend Snippet: Sel1L positively regulates collagen uptake and Mrc2 expression. (A) Schematic of domain structure of SEL1L protein. FN2 = Fibronectin-2. (B) Flow-cytometry based collagen-uptake assay in MEF cells (mouse embryonic fibroblasts) after treatment with 4-hydroxytamoxifen (OHT) as indicated vs. vehicle control. WT = Sel1L f/f; ERcre+ , iKO = Sel1L f/f ;ERcre- . N=3-4 per group. (C) Western blot of MEFs after OHT treatment as indicated vs. control, representative of N=4 independent experiments. (D) Q-RT-PCR in MEFs after OHT treatment for genes as indicated. N=3 per group. (E) Flow-cytometry based measurement of MRC2 surface expression in MEFs after OHT treatment. N=4 per group. (F) Representative wide-field immunofluorescence images of MEFs after OHT treatment. (G) Q-RT-PCR of MRC5 cells transfected with overexpression vectors containing genes as indicated vs. vehicle control (lipofectamine only). N=4 per group. (H) Western blot of MRC5 cells transfected with overexpression vectors containing genes as indicated vs. vehicle control (lipofectamine only). N=2. Statistics: (B, G) ANOVA with post-hoc Bonferroni testing; (D-E) Student’s t -test. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Article Snippet: For MRC2 overexpression, pCI-Mrc2 (murine Mrc2 cloned into the pCI-Neo expression vector, from Promega) was used as we previously described .

Techniques: Expressing, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transfection, Over Expression

Journal: bioRxiv

Article Title: Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

doi: 10.1101/2023.01.13.523943

Figure Lengend Snippet: SEL1L-mediated regulation of MRC2 is ERAD-independent. (A) Flow-cytometry based collagen-uptake assay in 293T of different genotypes (compared with baseline control without fluorescent collagen). N=4-5 per group. (B) Western blot of 293T cells of different genotypes, representative of N=4 independent experiments. (C) Firefly luciferase assay of MRC2 promoter activity, normalized to constitutive Renilla luciferase in 293T cells of different genotypes. N=3 per group. (D) Western blot of HEK293T cells of different genotypes transfected with MRC2 overexpression vector vs. vehicle control, representative of N=2 independent experiments. (E) Representative wide-field immunofluorescence images of 293T cells of different genotypes transfected with MRC2 overexpression vector vs. vehicle control. (F) Flow-cytometry based collagen-uptake assay in HEK293T of different genotypes transfected with MRC2 overexpression vector (compared with baseline control without fluorescent collagen; note different scale compared with panel A). N=3-4 per group. Statistics: (A, C, F) ANOVA with post-hoc Bonferroni testing. * p < 0.05 ** p < 0.01, ns = not significant.

Article Snippet: For MRC2 overexpression, pCI-Mrc2 (murine Mrc2 cloned into the pCI-Neo expression vector, from Promega) was used as we previously described .

Techniques: Flow Cytometry, Western Blot, Luciferase, Activity Assay, Transfection, Over Expression, Plasmid Preparation, Immunofluorescence

Journal: bioRxiv

Article Title: Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

doi: 10.1101/2023.01.13.523943

Figure Lengend Snippet: SEL1L binds collagen as it is being synthesized via its FN2 domain and this interaction is necessary for regulation of MRC2 expression. (A) Representative confocal immunofluorescence images of MRC5 fibroblasts treated as indicated or vehicle control. (B) Western blot of MRC5 fibroblasts after immunoprecipitation with anti-SEL1L antibody (vs. controls as indicated), representative of N=2 independent experiments. (C) Western blot after immunoprecipitation with anti-procollagen type I antibody (with input controls as indicated) of HEK293T cells transfected with collagen and WT or mutant SEL1L constructs as indicated, representative of N=3 independent experiments. (D) Representative confocal immunofluorescence images of HEK293T cells transfected with collagen and WT or mutant SEL1L constructs as indicated. (E) Western blot of MEFs after treatment with WT or mutant SEL1L constructs as indicated, representative of N=3 independent experiments. (F) Flow-cytometry based measurement of MRC2 surface expression on MEFs after treatment with WT or mutant SEL1L constructs as indicated, vs. Empty Vector (EV). N=6 per group. (G) Q-RT-PCR of SEL1L-KO HEK293T cells after transfection with WT or mutant SEL1L as indicated vs. empty vehicle control. (H) Comparison of protein domain structures of Sel1L orthologs across phylogenetically diverse organisms; FN2 = Fibronectin-2 domain, tpr = Tetratricopeptide repeat domain. N=3 per group. Statistics: (F-G) ANOVA with post-hoc Bonferroni testing. * p < 0.05, ** p < 0.01, ns = not significant.

Article Snippet: For MRC2 overexpression, pCI-Mrc2 (murine Mrc2 cloned into the pCI-Neo expression vector, from Promega) was used as we previously described .

Techniques: Synthesized, Expressing, Immunofluorescence, Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Construct, Flow Cytometry, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

doi: 10.1101/2023.01.13.523943

Figure Lengend Snippet: Collagen biosynthesis and MRC2 expression are positively correlated in lung tissue, but this relationship is impaired in human fibrotic lung disease. (A) UMAP plot with cell type annotation of scRNAseq data from multiple available datasets of human lung control and IPF tissue (GSE136831, GSE135893, GSE121611, GSE128033, GSE132771). N=184,672 single cells. (B-C) Gene expression of individual genes as indicated in UMAP as generated in panel (A). (D) Comparison of gene expression in all cells from data as in panel (A) that had non-zero expression of COL1A1 or MRC2 . (E) Comparison of gene expression in whole human lung microarray data from GSE110147 analyzed via GEO2R in pulmonary fibrosis lungs vs. control specimens. N=11 vs. 37 per group. (F) Comparison of microarray gene expression in human lung bronchoalveolar lavage specimens from GSE70867 analyzed via GEO2R from Idiopathic Pulmonary Fibrosis (IPF) lungs vs. control specimens. N=20 vs. 112. (G) Comparison of microarray gene expression in peripheral blood mononuclear cell (PBMC) specimens from GSE37858 analyzed via GEO2R from Idiopathic Pulmonary Fibrosis (IPF) lungs vs. control specimens. N=35 vs. 70. (H) Comparison of gene expression in whole mouse lung microarray data from GSE40151 analyzed via GEO2R from Bleomycin-treated mice vs. Saline control mice. N=55 vs. 56. Statistics: ANCOVA, p-values indicated in figures. Pearson correlations are displayed in panel (D).

Article Snippet: For MRC2 overexpression, pCI-Mrc2 (murine Mrc2 cloned into the pCI-Neo expression vector, from Promega) was used as we previously described .

Techniques: Expressing, Generated, Microarray