Fig 2: RPS3 overexpression directly in SGC7901S cells increases the DDP resistance of SGC7901S cells. (A) RT-qPCR was performed to observe the effect of RPS3 knockdown and overexpression on the mRNA expression of RPS3 in SGC7901S cells. (B) Western blotting was performed to observe the effect of RPS3 knockdown and overexpression on the protein expression of RPS3 in SGC7901S cells. (C) CCK-8 assay was performed to observe the effect of cisplatin (DDP) on the proliferation of indicated cells: SGC7901S, SGC7901S-shRNA/NC, SGC7901S + SGC7901S-shRNA/RPS3, SGC7901S-pcDNA3.1, and SGC7901S-pcDNA3. 1-RPS3. The concentrations of DDP used for the drug dose-response curve analysis of indicated cells were 0, 125, 250, 500, 1,000, 2,000, 4,000, 8,000, and 16,000 μg/L. Each experiment was performed in triplicate. (D) ICP-MS analysis was applied to investigate the effect of RPS3 knockdown and overexpression on the DDP concentration in the cellular of SGC7901S cells. (E,F) Western blotting and quantitative analysis were performed to observe the effect of RPS3 knockdown and overexpression on the P-gp protein expression in SGC7901S cells. β-Actin was used as cellular control. (G) Flow cytometry and quantitative assay were performed to observe the shift of cell cycles in SGC7901S cells treated with RPS3-overexpressing or -silencing, or their respective controls. (H) Flow cytometry and quantitative assay were performed to observe the change of cell apoptosis in SGC7901S cells treated with RPS3-overexpressing or -silencing, or their respective controls. (I,J) Western blotting and quantitative assays of p-PI3K, p-AKT, PP1, PP2A, p-cofilin-1, and cofilin-1 proteins in the SGC7901S cells treated with RPS3-overexpressing or -silencing, or their respective controls. The level of each protein was normalized against those of β-actin (total proteins) or COXIV (mitochondrial proteins). Each experiment was performed at least in triplicate, and all the data are shown as mean ± SD. Student’s t-test was used to analyze the data. ∗P < 0.05, ∗∗P < 0.01.

Fig 3: RNase I digests small ribosomal subunit. U2OS cells were digested with MN and RNase I as for ribosome profiling and protein levels were analysed by mass spectrometry after ribosome separation using sucrose cushion centrifugation. (a) Correlation of protein abundances (log2 of emPAI scores) between the two digestions. Ribosomal proteins and Pearson correlation value (R2) are indicated. (b) Average protein abundances (normalized emPAI scores) for small subunit (RpS), large subunit (RpL) and mitochondrial (MitoRp) ribosomal proteins with MN and RNase I digestions. Relative enrichments of RpS compared to RpL proteins with RNase I and MN are indicated, suggesting differences in ribosome degradation. (c) Fraction of ribosome profiling sequencing reads mapping to 18S rRNA. S2* indicates Drosophila S2 cell sample from (8). (d) Visualization of the relative changes in RpS proteins based on the mass spec data. Upper left structure shows log2 relative abundance of RpS proteins (MN/RNase I) mapped on human 40S ribosome (PDB accessions 3J3D (18S rRNA) and 3J3A (RpS proteins)). Grey proteins were not identified by mass spectrometry. Structure on the right, shows the most MN-enriched proteins (log2 fold change > 1.5) colour coded individually. Other proteins are in light brown and rRNA is in grey. The views are from the solvent side of the 40S ribosome. Bar chart on the bottom left shows the relative enrichment of individual RpS proteins. (e) Sucrose gradient analysis of MN (left) and RNase I (right) digested HeLa cells. Undigested lysate is shown as a control. Equal amount of cell lysate was used for each sample. (f) Western blots of U2OS cell lysates digested with indicated amounts of nucleases and centrifuged through sucrose cushion. Some ribosomal proteins, like RpS3, are dissociated from ribosomes more with RNase I than with MN. See also panel (d). Nuclease digestions were carried out under same conditions as for the ribosome profiling samples.

Fig 4: USP10 and USP13 silencing has little effect on the UPR and cell viability under GS- or 2DG-stressed conditions. (a) Effects of USP10 and USP13 silencing on GRP78 in HT1080 cells were determined by western blotting. RPS3 was used as a loading control. The blot membranes were cut prior to hybridization with antibodies, according to Full range rainbow molecular weight markers. Original blots were presented in Supplementary Fig. S5. (b) Effects of USP10 and USP13 silencing on ATF4 and XBP1s induction in vehicle- or spautin-1-treated HT1080 cells under 2DG-stressed conditions were visualized using the Operetta CLS. (c,d) Mean intensities of nuclear (c) ATF4 and (d) XBP1s in (b) were determined using Harmony high-content analysis software. Data are shown as mean ± SD (n = 3). (e) Effects of USP10 and USP13 silencing on cell viability under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD (n = 3). (f) Effects of USP10 and USP13 silencing on preferential cytotoxicity of spautin-1 under GS were determined by the CellTiter-Glo luminescent cell viability assay. Data are shown as mean ± SD (n = 3).

Fig 5: Rad6 co-sediments with elongating ribosomes(A) Immunoblot of co-immunoprecipitation (coIP) of Rad6-FLAG with ribosomal proteins uS3 and uL5.(B) Rad6 presence on the ribosome pellet is independent of cellular lysate incubation with 10 mM DTT, 1 M NaCl, 1 mM H2O2, and DNAse (0.1 U/µg protein) and RNAse I (100 U/µg protein). 1% SDS was used to disrupt molecular interactions. Anti-uS3 and -uL5 were used as loading control for proteins from the 40S and 60S ribosome subunits, respectively.(C) Sucrose density sedimentation analysis and anti-HA immunoblot of lysates from cells expressing Rad6-HA, GFP-HA, or GFP-Rad6-HA constructs. Anti-uS3 and -uL5 were used as markers for the 40S and 60S ribosome subunits, respectively.(D) Purified Rad6, but not GFP, relocalizes to the polysomal fraction after incubation with ribosomes for 10 min at room temperature followed by sucrose density-sedimentation separation. Ribosomes were isolated from rad6?bre1? strain. Anti-uL5 was used as a marker for the 60S ribosome subunit. *Anti-Rad6 antibody unspecific band.(E) 3D classification of ribosomes incubated with purified Rad6 (RiboRad6mix) and ribosomes co-immunoprecipitated with FLAG-tagged Rad6 (RiboRad6IP). Ribosomes were observed in the pre-translocation (PRE), post-translocation (POST), and transition (PRE-to-POST) states.(F) Structural detail of RiboRad6IP at the pre-translocation stage with tRNAs in hybrid A/P and P/E positions. Rigid fit of the tRNAs was performed using coordinates from the 80S ribosome (PDB: 6GZ5) (Flis et al., 2018).(G) Focused classification of RiboRad6IP shows two classes with distinct conformations for the 40S beak and ribosomal proteins eS12 and eS31. The extended beak rRNA model was built using COOT (Emsley and Cowtan, 2004).