Fig 2: TP induces ribosomal stress in A549 ×enografts. (A) Protein levels of p53, NCL and RPL23 in xenografts from control and TP-treated animals as determined via immunohistochemistry. Quantification of the levels of (B) p53, (C) NCL and (D) RPL23. Data are presented as the mean ± SD. Student's t-test was used to analyze the difference between two groups. *P<0.05. TP, triptolide; NCL, nucleolin; RPL23, ribosomal protein L23.

Fig 3: TP increases the binding of RPL23 with MDM2 and activates p53 and p53-regulated proteins. (A) Cell lysates of TP-treated cells (5×106) were immunoprecipitated with anti-MDM2 antibodies, followed by immunoblotting with anti-MDM2, p53 and RPL23 antibodies. For each lysate, 20% of the quantity used for IP was loaded as an input control. (B) Cell lysates from 5×106 A549 cells receiving various treatments for 48 h were subjected to western blot analysis with the indicated antibodies; GAPDH was used as an internal control. (C) Densitometric analysis of protein expression. Data are presented as the mean ± SD of at least three independent experiments. Data were analyzed using one-way ANOVA combined with Tukey's multiple comparisons test. ***P<0.001, ****P<0.0001 vs. control. TP, triptolide; MDM2, mouse double minute 2; RPL23, ribosomal protein L23; PUMA, p53-upregulated modulator of apoptosis, C-Cas, cleaved caspase; p, phosphorylated; IP, immunoprecipitation.

Fig 4: uL14 KD regulates antigen presentation. (A) Proteomics analysis of Mel624 cells reveals altered protein levels upon uL14 KD in untreated (Ctrl) cells and cells treated with IFNγ/TNFα (Treated). n = 3 independent experiments, and P-values were calculated using a two-tailed t-test. (B) uL14 KD decreases HLA I surface levels in Mel624 tumor cells. HLA surface levels were measured by flow cytometry using the MFI. n = 3 independent experiments each assessed in triplicates. Data are represented as mean ± SEM, and P-values were calculated using a two-tailed t-test. (C) uL14 KD results in low basal HLA I surface levels in Mel624 tumor cells treated with IFNγ/TNFα compared to the control. n = 3 independent experiments each assessed in triplicates. Data are represented as mean ± SEM, and P-values were calculated using a two-tailed t-test.

Fig 5: uL14 KD alters protein expression. (A) uL14 association with translating ribosomes (poly) is unaltered in Mel624 and M026 tumor cells treated with IFNγ/TNFα compared to untreated cells. uL30 was used as a control for data normalization. n = 1 experiment per cell line. uL14 and uL30 expression was additionally assessed in the total protein fraction (total) and the sub-polysomal fraction (sub). (B) Transduction efficiency (measured by % of cells with mCherry expression) of Mel624 cells was measured by flow cytometry gated on live cells. n = 3 independent experiments each assessed in triplicates represented as mean ± SEM. (C) RT-qPCR analysis confirming efficient uL14 KD in Mel624 tumor cells. ACTB was used as a reference. n = 3 technical replicates represented as mean ± SD. P-values were calculated using a two-tailed t-test. (D) Western blot analysis confirming efficient uL14 KD in Mel624 tumor cells. α-Tubulin was used as loading control. n = 1 experiment per cell line. (E) 35S-methionine incorporation assay showing decreased protein synthesis in shL14 Mel624 tumor cells compared to the control. Cycloheximide was used as a control to inhibit protein synthesis. n = 3 technical replicates represented as mean ± SD. P-values were calculated using a two-tailed t-test. (F) CellTrace violet cell proliferation assay showing decreased cell proliferation in shL14 Mel624 cells compared to the control. Proliferation was assessed by flow cytometry using the MFI. n = 3 biological replicates represented as mean ± SEM. P-values were calculated using a two-tailed t-test. (G) Western blot showing decreased HLA I protein levels in shL14 Mel624 tumor cells compared to the control. HSP90 was used as loading control. n = 1 independent experiment.