Fig 1: A Genome-wide CRISPR-Cas9 Screen Identifies Components Required for CYP51A1TM Degradation(A) Workflow of the CRISPR-Cas9 genome-wide screen.(B) Significance score of the genes analyzed in the screen calculated by the MAGeCK algorithm. The x axis represents the genes in alphabetical order. The y axis shows the -log(aRRA) significance value. The -log(aRRA) cutoff was arbitrarily set at 9 (dashed line). Significantly enriched genes are annotated.(C) Overview of the enriched genes identified and their proposed function.(D) Validation of several screen hits using independent sgRNAs. Levels of CYP51A1TM were analyzed by flow cytometry (based on GFP fluorescence).(E) Degradation of CYP51A1TM depends on RNF185, MBRL, SPP, and UBE2K. CYP51A1TM levels were assessed by flow cytometry (based on GFP fluorescence) in parental control cells and clonal RNF185, MBRL, SPP, UBE2K, and HRD1 KO cells expressing cDNAs encoding either WT, a CI mutant, or an EV.See also Figure S2.
Fig 2: Reducing nuclear HO-1 by SPP knockdown exacerbated endothelial senescence.A Screening of SPP interference sequences by Western blot. Sequence-3 was used for the following experiments. *P < 0.05 vs. NC-siRNA. n = 5. B Silencing of SPP decreased the expression of nuclear HO-1 rather than cytoplasmic HO-1. *P < 0.05 vs. NC-siRNA. n = 5. C Silencing of SPP reduced fluorescence signal of nuclear HO-1. n = 3. D Silencing of SPP increased the expression of p53 or p21. *P < 0.05 vs. NC-siRNA. n = 5. E Silencing of SPP increased the percentage cells at G0/G1 phase. *P < 0.05 vs. NC-siRNA. n = 5. F Silencing of SPP increased the proportion of SA-ß-gal staining cells. *P < 0.05 vs. NC-siRNA. n = 5. G Silencing of SPP decreased the ratio of EdU-positive cells. *P < 0.05 vs. NC-siRNA. n = 5.
Fig 3: Nuclear HO-1 was up-regulated in prematurely senescent endothelial cells with SPP activation.A Upregulation of total, cytoplasmic and nuclear HO-1 expression in HUVECs induced by Ang II, D-gal, ox-LDL and H2O2. *P <0.05 vs. Control. n = 5. B Immunofluorescence experiment showed that HO-1 was enriched in the nucleus of senescent endothelial cell induced by H2O2. n = 3. C Hemin increased the expression of total, cytoplasmic and nuclear HO-1. *P < 0.05 vs. Control. n = 5. D HO-1 accumulation occurred in the cytoplasm and nucleus induced by Hemin. n = 3. E The level of HO-1 increased in LCA with ligation, *P < 0.05 vs. RCA without ligation. n = 4. F Upregulation of SPP mRNA and protein level in HUVECs induced by H2O2. *P < 0.05 vs. Control. n = 5. G Hemin increased the mRNA and protein level of SPP. *P < 0.05 vs. Control. n = 5.
Fig 4: MARCH6 and TRC8 are required for the degradation of selected ER proteins following SPP-mediated intramembrane proteolysis Volcano plot showing TMT mass spectrometry analysis of whole-cell proteome of mCherry-CL1 cells compared to combined MARCH6/TRC8 null cells. Significance is shown in the y-axis (log(2) q-value) and log(2)-fold change on the x-axis. Proteins that were highly enriched in the combined MARCH6/TRC8 null cells and not in the single MARCH6 or TRC8 KO clones (detailed in Dataset EV2) are shown in purple.Schematic of membrane topology of the tail-anchored protein, HO-1, that fails to insert correctly, or before and after intramembrane cleavage by SPP, in comparison with the amphipathic mCherry-CL1 degron.[35S]methionine/cysteine-radiolabelling of HO-1 in HeLa mCherry-CL1 cells (WT) or the combined MARCH6/TRC8 null cells. Cells were pulse-radiolabelled for 10 min and immunoprecipitated for HO-1 from detergent lysates at the times indicated. Cells were treated with or without the SPP inhibitor (SPP inb), 10 µM (Z-LL)2 ketone for 24 h. Full-length (black) and SPP-cleaved form of HO-1 (blue) are indicated. The asterisk represents non-specific band observed in the HeLa mCherry-CL1 cells (see also Appendix Fig S5).[35S]methionine/cysteine-radiolabelling of HO-1 in wild-type HeLa cells, in HeLa TRC8 KO cells (see Appendix Fig S5C and D) or in the TRC8 KO clone depleted for MARCH6 by sgRNA (M6 KO), as described. Full-length and SPP-cleaved form of HO-1 are indicated.[35S]methionine/cysteine-radiolabelling of HO-1 in HeLa cells (WT) or combined MARCH6/TRC8 null cells, with or without overexpressed SPP. The SPP-cleaved form of HO-1 is indicated.Schematic for mechanism of HO-1 quality control at the ER membrane, requiring both MARCH6 and TRC8 to ubiquitinate misinserted or cleaved HO-1, targeting HO-1 for proteasome-mediated degradation.
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