DOI: 10.6084/m9.figshare.27132687
title: Additional file 1 of UBASH3B-mediated MRPL12 Y60 dephosphorylation inhibits LUAD development by driving mitochondrial metabolism reprogramming

description: Supplementary Material 1: Figure S1. MRPL12 is highly expressed in LUAD organoid, tissues, and cells and associated with poor survival. A Analysis of MRPL12 expression in KRAS mutant versus non-mutant LUAD tissues using TCGA databases. B Analysis of the correlation between MRPL12 and TP53 expression in LUAD tissues using TCGA databases. C Organoid morphology. Scale bar: 50 μm. D MRPL12 mRNA levels in LUAD. E MRPL12 mRNA levels in deceased and living LUAD patients. F Analysis of MRPL12 expression levels based on T, N, M clinical stage, and pathologic stage. G Overall survival analysis based on MRPL12 mRNA levels in LUAD. H Kaplan-Meier survival curves depicting the correlation between MRPL12 mRNA levels and the overall survival time of patients with lung squamous cell carcinoma. I-J Western blot and RT-PCR were employed to assess the efficiency of MRPL12 knockdown or overexpression in the indicated cells (I) and PDOs (J). **, p&lt;0.01;***, p&lt;0.001; ****, p&lt;0.0001. Figure S2. MRPL12 facilitates LUAD tumorigenesis in vitro. A-B Soft agar (A) and EDU (B) assays were conducted to evaluate cell proliferation and colony formation capabilities in A549 and H1299 cells with stable MRPL12 overexpression or knockdown. C EDU assays were performed to assess cell proliferation in A549 and H1299 cells with MRPL12 knockout or re-expression. D Transwell assays were conducted to assess cell migration and invasion ability in A549 and H1299 cells with stable MRPL12 overexpression or knockdown. E Transwell assays were conducted to assess cell migration and invasion ability in A549 and H1299 cells with MRPL12 knockout or re-expression. F Quantification of A549 and H1299 cells in trans-endothelial migration assays (referenced in Figure 3H). G-H Ingenuity Pathway Analysis (IPA) of the differentially expressed genes in A549 cells with MRPL12 knockdown. *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001; ****, p&lt;0.0001. Figure S3. Co‐expression analysis combined with KEGG enrichment analysis. A Co‐expression analysis of MRPL12. B Western blotting was used to analyze the expression of the indicated genes following MRPL12 knockdown or overexpression in A549 and H1299 cells. *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001, ****, p&lt;0.0001; ns, p&gt;0.05. Figure S4. MRPL12 promotes lung tumorigenesis via promoting mitochondrial oxidative phosphorylation. A Analysis of mtDNA copy number in H1299 cells subjected to MRPL12 knockdown or overexpression. B-C Evaluation of mRNA (B) and protein (C) levels of OXPHOS complexes in H1299 cells following MRPL12 knockdown or overexpression. D Measurement of OCR in H1299 cells with MRPL12 knockdown or overexpression using Seahorse XFe96. E Analysis of mitochondrial OXPHOS, including basal respiration, maximal respiration, ATP production, and spare respiratory capacity in H1299 cells with MRPL12 knockdown or overexpression. F Mitochondrial mass was measured using MitoTracker Red CMXRos. G-H Examination of the proliferation and migration capabilities of A549 and H1299 cells overexpressing MRPL12 under treatment with an OXPHOS inhibitor. *, p&lt;0.05; **, p&lt;0.01;***, p&lt;0.001; ****, p&lt;0.0001; ns, p&gt;0.05. Figure S5. UBASH3B interacts with MRPL12. A Analysis of UBASH3B expression across various cancer types using pan-cancer samples from the TIMER2.0 database. B Examination of UBASH3B expression in LUAD and corresponding normal tissues within the TCGA databases. C Assessment of UBASH3B expression in LUAD and adjacent normal tissues through analysis of the TCGA databases. D Comparative analysis of UBASH3B expression in LUAD and normal tissues utilizing the GEPIA2 website. E-F Kaplan-Meier survival curves illustrating the impact of UBASH3B expression on the overall survival of LUAD patients. G-H Evaluation of the migration (G) and proliferation (H) capabilities of A549 and H1299 cells with UBASH3B overexpression or knockdown. I-J Investigation of the proliferation and migration properties of A549 and H1299 cells overexpressing MRPL12, along with UBASH3B H391A or UBASH3B WT overexpression, assessed by EDU and Transwell assays. *, p&lt;0.05;**, p&lt;0.01; ***, p&lt;0.001; ****, p&lt;0.0001. Figure S6. The expression and role of UBASH3B in LUAD. A UBASH3B expression was assessed in LUAD tissues and PDO. B-C Analysis of the correlation between UBASH3B expression levels and TNM stages or pathological stage using TCGA datasets. D Transwell assays were conducted to assess cell migration and invasion ability in A549 and H1299 cells with UBASH3B knockdown, combined with MRPL12 WT, MRPL12 Y60A or MRPL12 Y60E overexpression. E EDU assays were conducted to assess cell proliferation in A549 and H1299 cells with UBASH3B overexpression, combined with MRPL12 WT, MRPL12 Y60E or MRPL12 Y60A overexpression. *,p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001; ****, p&lt;0.0001. Figure S7. UBASH3B dephosphorylates MRPL12 at residue Y60 and inhibits lung tumorigenesis. A-B Assessment of protein (A) and mRNA (B) levels of OXPHOS complexes in H1299 cells overexpressing the MRPL12 Y60A mutation or MRPL12 WT. C Analysis of mtDNA copy number in H1299 cells with overexpression of MRPL12 WT or the MRPL12 Y60A mutant. D Examination of oxygen consumption rate (OCR) in H1299 cells overexpressing MRPL12 Y60A or MRPL12 WT, performed using Seahorse XFe96. E Quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity in H1299 cells overexpressing MRPL12 Y60A or MRPL12 WT. F Co-ip assays conducted in A549 and H1299 cells, wherein cells were lysed and immunoprecipitated with an MRPL12 antibody, followed by western blot analysis with POLRMT. G-H Analysis of proliferation and migration characteristics in A549 and H1299 cells with overexpression of MRPL12 WT, MRPL12 Y60A, or Y152A mutations. *, p&lt;0.05; **, p&lt;0.01; ***,p&lt;0.001; ****, p&lt;0.0001; ns, p&gt;0.05.