Fig 2: SYVN1 affects cell proliferation in serum‐deficient conditions. (A) Cell proliferation under serum‐sufficient conditions was detected in LM3 and HUH7 cells at different time points using a CCK‐8 kit. Error bars represent the standard error of the mean from two or three biological replicates with eight technical replicates each. (B‐D) Stable shNC‐ and shSYVN1‐transfected LM3 and HUH7 cells were injected subcutaneously into nude mice, and tumor lesions, volume, and weight were measured. Error bars represent the standard deviation. (E) Lysates from subcutaneous tumors formed by stable shNC‐ and shSYVN1‐transfected LM3 cells were subjected to SDS‐PAGE and Western blotting to detect the expression of SYVN1. (F) Cell proliferation under serum‐free conditions was detected at different time points using a CCK‐8 kit. Error bars represent the standard error of the mean from two or three biological replicates with eight technical replicates each. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: NC, negative control

Fig 3: Erg11TM and CYP51A1TM Engage Distinct ERAD Branches(A) Schematic representation of the Erg11TM and CYP51A1TM constructs. Erg11TM and CYP51A1TM were C-terminally tagged with sfGFP and a 3xHA-tag. The Erg11TM and CYP51A1TM amphipathic helix (AH) and transmembrane (TMD) domain are aligned and highlighted in red.(B) Subcellular fractionation of Erg11TM- and CYP51A1TM-expressing cells. Postnuclear supernatants (W) were fractionated into a cytosolic soluble fraction (C) and a crude membrane pellet (P1). Membranes in P1 were salt washed and carbonate treated (P2) to remove all peripherally associated proteins. Fractions were subjected to SDS-PAGE and western blotting analysis. The partitioning of the HA-tagged substrates was compared to endogenous soluble (p97 and GAPDH) and integral membrane (BAP31) proteins.(C and D) Erg11TM and CYP51A1TM levels are stabilized upon inhibition of p97 and the proteasome but are unaffected by blocking protein delivery to lysosomes. Flow cytometry (C) and immunoblotting (D) analysis of tetracycline-induced expression of Erg11TM and CYP51A1TM in Flp-In T-REx 293 cells. Analysis was performed 24 h post-induction in cells left untreated, incubated 4 h with inhibitors to p97 (CB-5083; 2.5 µM [CB]) or to the proteasome (Bortezomib; 500 nM [Btz]), or incubated 24 h with bafilomycin A (250 nM [Baf]) that inhibits lysosomal delivery. Flp-In T-REx 293 cells without any GFP-tagged substrate were used as control (no substrate). In (D), Erg11TM and CYP51A1TM were detected with anti-HA antibodies. LC3 was analyzed to confirm effectiveness of bafilomycin A treatment and was detected with an anti-LC3 antibody. Tubulin was used as a loading control and was detected with an anti-tubulin antibody.(E) Degradation of Erg11TM and CYP51A1TM was analyzed after inhibition of protein synthesis by cycloheximide (CHX) in the absence or presence of the p97 inhibitor CB-5083 (4 h; 2.5 µM). Cell extracts were analyzed by SDS-PAGE and immunoblotting. Erg11TM and CYP51A1TM were detected with anti-HA antibodies. GAPDH was used as a loading control and detected with an anti-GAPDH antibody.(F) Erg11TM and CYP51A1TM follow distinct ERAD pathways. Cells expressing Erg11TM and CYP51A1TM as well as plasmids encoding sgRNAs targeting the indicated genes were analyzed by flow cytometry (based on GFP fluorescence).(G) Degradation of Erg11TM depends on TEB4, UBE2G2, and UBE2J2. Erg11TM levels were analyzed by flow cytometry (based on GFP fluorescence) in parental control cells and in clonal TEB4, UBE2G2, UBE2J2, and HRD1 KO cells expressing cDNAs encoding either the corresponding wild type (WT), a catalytically inactive (CI) mutant, or an empty vector (EV).See also Figure S1.

Fig 4: HERPUD1 affects the ubiquitination of GSS by acting on MDM2.A Half-life of GSS in Bel-7402 or Bel-7404 cells with or without HERPUD1 knocked out was detected by CHX chase experiments. The relative protein levels of GSS were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. B Total ubiquitination of GSS was detected by co-IP experiments and measured by an anti-total ubiquitin antibody. C Bel-7402 and Bel-7404 cells were seeded at the indicated density in 10 cm petri dish and cultured for 24 h, then treated with: 10 μM erastin or 5 μM RSL3; 10 μM CA; pretreatment with 10 μM CA for 1 h followed by 10 μM erastin or 5 μM RSL3, in complete medium for 24 h. The drug-treated protein was then collected, co-IP was performed with anti-GSS antibody according to the previous steps, and measured by anti-total ubiquitin antibody. D UbiBrowser online software and the literature was used to identify GSS-related ubiquitinating enzymes. E Reciprocal IP experiments of GSS, MDM2 and SYVN1 in wild-type Bel-7402 and Bel-7404 cells. F Reciprocal IP experiments for detecting GSS, MDM2 and SYVN1 in Bel-7402 and Bel-7404 cells which HERPUD1 were overexpressed. G Endogenous MDM2 was immunoprecipitated by anti-MDM2 antibody and ubiquitination of MDM2 were measured by anti-ubiquitin antibody in Bel-7404 and Bel-7402 cells with or without HERPUD1 knocked out. Representative images of IB are shown in the figure. H Half-life of MDM2 was detected by CHX chase experiments in Bel-7402 and Bel-7404 cells with or without HERPUD1 knocked out. The relative protein levels of MDM2 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. Data were expressed as mean ± SD. Images of all the immunoblots are representative of three independent experiments.

Fig 5: 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.