Fig. 6: PPP1R15B promotes malignant phenotypes of multiple myeloma cells in vitro. | Nature Communications

Fig. 6: PPP1R15B promotes malignant phenotypes of multiple myeloma cells in vitro.

From: Super enhancer acquisition drives expression of oncogenic PPP1R15B that regulates protein homeostasis in multiple myeloma

Fig. 6

a, b Knockdown of PPP1R15B was confirmed by RT-qPCR (a) and western blot (b) in NCI-H929 transfected with scramble and PPP1R15B shRNAs. n = 3 biological replicates. For RT-qPCR, significant differences were determined using one-way ANOVA with Tukey’s post hoc test (p values in scramble vs shRNA-1 = 8.23 × 10-11; scramble vs shRNA-2 = 5.22 × 10-9). All western blots are representative of 3 biological replicates. β-actin was used as a loading control. c Cell cycle analysis of NCI-H929 after transfection with the indicated shRNAs. Significant differences were determined using two-way ANOVA with Tukey’s post hoc test (Detailed calculations of p values are provided in the Source Data file). Data were obtained from 3 independent experiments, with representative plots shown. d Apoptosis detection by Annexin-V/PI staining in NCI-H929 cells at day 4 post-transfection with scramble or PPP1R15B shRNAs. Bar chart showing the percentage of apoptotic cells (Annexin V + , right). n = 3 biological replicates. One-way ANOVA with Tukey’s post hoc test was performed (p values in scramble vs shRNA-1 = 0.00074; scramble vs shRNA-2 = 0.0012). e Protein synthesis analysis by flow cytometry in PPP1R15B knockdown or add-back NCI-H929 cells. Mean fluorescence intensity (MFI) for HPG incorporation (AF488) was measured and normalized to scramble control, with representative plots shown. n  =  3 biological replicates. One-way ANOVA with Tukey’s post hoc test was performed (p values in scramble vs shRNA-1 = 5.65×10-5; scramble vs add-back = 0.037). f Flow cytometric analysis of cytoplasmic immunoglobulin κ light chain expression upon PPP1R15B knockdown. n = 3 biological replicates. One-way ANOVA with Tukey’s post hoc test was performed (p values in scramble vs shRNA-1 = 9.05 × 10-7; scramble vs shRNA-2 = 1.79 × 10-6). g Secretion of M protein measured by ELISA in PPP1R15B knockdown cells in NCI-H929. Results are shown as the relative MFI compared to the control. n = 3 biological replicates. One-way ANOVA with Tukey’s post hoc test was performed (p values in scramble vs shRNA-1 = 2.70 × 10-6; scramble vs shRNA-2 = 3.23 × 10-6). Source data are provided as a Source Data file.

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