Fig 2: 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 3: Schematic diagram of the mechanism of TP-induced lung cancer cell inhibition via ribosomal stress initiation. TP induces nucleolus degradation and inhibits 45S rRNA synthesis by reducing the binding of Pol I and UBF to the rDNA promoter. The 18S rRNA degraded by TP disrupts ribosomal synthesis, resulting in the liberation of RPL23. The combination of RPL23 with MDM2 activates p53, which drives apoptosis, growth inhibition and cell cycle arrest. TP, triptolide; Pol I, RNA polymerase I; UBF, upstream binding factor; RPL23, ribosomal protein L23; MDM2, mouse double minute 2; NCL, nucleolin; B23, nucleophosmin; PUMA, p53-upregulated modulator of apoptosis; C, cleaved; p, phosphorylated; r, ribosomal.

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 the presented peptide pool. (A) Immunopeptidomics analysis reveals a decrease in the number of uniquely presented peptides identified in uL14 KD Mel624 tumor cells compared to the shScr control. n = 2 independent experiments, and data are represented as mean ± SEM. (B) uL14 KD in Mel624 cells results in high numbers of Down peptides (downregulated compared to the shScr control + peptides exclusively unique to the shScr control) (Down) compared to Up peptides (upregulated compared to the shScr control + peptides exclusively unique to uL14 KD) (Up). Significant peptides were determined using a two-tailed t-test without imputation of missing peptides. FDR threshold was set at 0.01 with a dispersion correction factor of 0.1. (C) uL14 KD in Mel624 cells results in a significant increase in net positive charge of Up peptides (upregulated + peptides exclusively unique to uL14 KD). Each violin-boxplot represents the distribution of theoretical net charge of presented Down peptides (downregulated + peptides exclusively unique to the shScr control) and Up peptides (upregulated + peptides exclusively unique to uL14 KD) using the Lehninger pKa scale at a pH of 7.0. P-values were calculated using a two-tailed Wilcoxon test (****P < .0001). (D) Peptide N-terminus and anchor residues (position 2 and C-terminus) are altered between Down and Up peptides in shL14 Mel624 cells compared to shScr. Logo plots represent the conservation of amino acid residues at each position and across peptides identified in each group of Down and Up peptides. (E) uL14 KD in Mel624 cells results in significantly lower predicted binding affinity to Mel624-specific HLA alleles in Down peptides compared to Up peptides. Each violin-bar plot represents the distribution of predicted log-transformed affinity (nM) of peptides to HLA alleles A02:01, A03:01, B07:02, B14:01, C07:02, and C08:02. P-values were calculated using a two-tailed Wilcoxon test (***P < .001). (F) uL14 KD in Mel624 cells results in significantly lower predicted peptide–MHC (pMHC) complex stability in Down peptides compared to Up peptides. Each violin-bar plot represents the distribution of predicted log-transformed pMHC half-life against Mel624-specific HLA alleles (A02:01, A03:01, B07:02, B14:01, C07:02, and C08:02). P-values were calculated using a two-tailed Wilcoxon test (****P < .0001).