Additional file 1 of The TP53-activated E3 ligase RNF144B is a tumour suppressor that prevents genomic instability
Creators: name: Abad, Etna
name: Sandoz, Jérémy
name: Romero, Gerard
name: Zadra, Ivan
name: Urgel-Solas, Julia
name: Borredat, Pablo
name: Kourtis, Savvas
name: Ortet, Laura
name: Martínez, Carlos M.
name: Weghorn, Donate
name: Sdelci, Sara
name: Janic, Ana
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Figure 1. Patient disease-survival by TP53 status and RNF144B expression. Probability of ten-year overall survival of cancer patients in human cancer samples with TP53 wild-type or TP53 mutant status and RNF144B low (below the median) or high expression (above the median). Cancer types: bladder (BLCA), breast (BRCA), colon (COAD), glioblastoma (GBM), head and neck (HNSC), kidney (KICH), liver (LICH), lung squamous (LUSC), pancreatic (PAAD), esophageal (ESCA), sarcoma (SARC), stomach (STAD), low grade glioma (LGG) and prostate (PRAD). Significance evaluated by log-rank test. Supplementary Figure 2. TP53 regulates the expression of RNF144B in diverse cellular contexts (A) qRT-PCR analysis of CDKN1A mRNA expression in TP53 proficient and TP53 deficient MEFs, A549, 3KT, HCT116 and mKLC upon 6h treatment with 10 μM Nutlin-3a relative to untreated cells of the same genotype. N=3-4 independent experiments for each cell line and cell variant, in duplicates or triplicates. Mean ± SEM, ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. p-values, two-tailed unpaired t-test. (B) Western blot analysis of mKLC cell lines and derivatives showing TP53 expression upon 0, 6 or 24 h treatment with doxorubicin (0.2 μg/ml), as well as of HCT116 cell lines and derivatives upon 6h treatment with 10 μM Nutlin-3a. Probing for β-ACTIN was used as a loading control. Note that for mouse mKLC we used three independent TRP53 WT cell lines and two TRP53 deficient cell lines. The Western blots shown are from 1 independent experiment. (C) UCSC genome view of TP53 occupancy in the Rnf144b loci in human TP53 wild-type fibroblasts from Doxorubicin (Dox, light blue tracks) and Input control data (INPUT, black tracks). TP53 consensus binding site is shown, where R = A, G; Y = C, T; W = A, T, and matching bases to the TP53 consensus binding sequence are in red. Spacer between the two binding sites can be from 0 to 13 nucleotides. Below, TP53 binding sequence identified as p53BS1 (D) UCSC Genome Browser views of TRP53 ChIP-Seq peaks in Trp53WT control (mock, gray tracks), Trp53WT irradiated (IR, light blue tracks) or irradiated Trp53MUT (IR, dark blue) mouse B cells. Y-axis represents normalized read counts at each position. (E) Rnf144b and control Cdkn1a (p21) TP53-dependent regulation in mouse and human cells. Data accessed from www.targetgenereg.org. TP53-dependent significant gene upregulation (light blue), downregulation (dark blue) and non-significant regulation (gray). Supplementary Figure 3. Cells deficient for Rnf144b have growth advantage in vivo. (A) qRT-PCR analysis of Rnf144b mRNA expression in MEFs transduced with indicated shRNAs and treated with 10μM Nutlin-3a for 6h. The mRNA levels were standardised to Hmbs. Expression is relative to the untreated control shRNA targeting Renilla Luciferase (shCTRL). N=3 independent MEF cell lines. **P ≤ 0.01; *P ≤ 0.05, two-tailed unpaired t-tests. (B) MEF 1.01 transduced with indicated shRNAs were injected subcutaneously into nude mice and tumor volume was measured over 20 days. (Left) Tumour volume (mm3) of the same genotype. (Right) Tumour weight at ethical endpoint. N=4-10 tumours/shRNA from one MEFs cell line (1.01). Data are presented as Mean ± SEM. ****P ≤ 0.0001, ***P ≤ 0.001; **P ≤ 0.01, two-way ANOVA or two-tailed unpaired t-test, respectively. (C) Competition in vivo assay using MEFs 1.01 cell line. (Top) shCTRLMEF expressing mCherry were mixed 1:1 with shRNF144BMEF or shTRP53MEF expressing GFP, injected subcutaneously into nude mice and grown for 20 days. Tumours were analyzed by FACS. (Bottom) Representative FACS plots show input at day 0 (INPUT - cell population before inoculation) and tumour populations at day 20 with either mCherry/shCTRL, GFP/shRNF144B or GFP/shTRP53 labeled cells. Bar plots show the mean percentages ± SEM of cells expressing shRNF144B (green) or shTRP53 (blue) relative to all labeled cells (GFP+mCherry) in both input and tumour populations. N=6 tumours/shRNA. (D) qRT-PCR analysis of Rnf144b mRNA expression in A549 and 3KT transduced with indicated sgRNAs and treated with 10μM Nutlin-3a for 6h. The mRNA levels were standardised to Hmbs. Expression is relative to the untreated control sgRNA (sgNT). N=2-4 independent experiments for each cell line and cell variant, in triplicates. **P ≤ 0.01; *P ≤ 0.05, two-tailed unpaired t-tests. (E) Next generation DNA sequencing results of A549 (left) and 3KT (right) Rnf144b gene targeted cell lines derivatives showing alterations (INDELs) in the Rnf144b gene CRISPR targeted region. sgRNF144BA549 isogenic clone shows a 94,4% mutant genotype. sgRNF144B3KT cells show a 98.6% mutant genotype. (F) Circular plots showing frequency of lesions observed in lungs of mice 5 months after intravenous injection with 3KT cells transduced with indicated sgRNAs. Representative histological pictures below. Scale bar = 100 μm (G) Western blot showing overexpression of His-tagged RNF144B in TP53 deficient A549 and 3KT (sgTP53A549 and sgTP533KT) cells after 72h of transfection with pcDNA 3.1 construct carrying empty (OE-CTRL) or Rnf144b cDNA (OE-RNF144B). The Western blots shown are 1 independent blot from independent experiments. (H) Percentage of viable (AnnexinV- DAPI-) MEFs, A549 and 3KT cells after treatment with Doxorubicin (0.05 μg/ml or 0.2 μg/ml) or serum starvation (0% FBS) for 24h or 72 h, respectively. Data are presented as Mean ± SEM, relative to untreated cells. N=3-4 independent experiments for each cell line and cell variant. P-value **P ≤ 0.01, One-way ANOVA. Supplementary Figure 4. Identifying RNF144B regulated proteins. (A) Heatmaps of differentially abundant proteins in shRNF144BMEF vs shCTRLMEF classified in the four highly enriched pathways: cell cycle control (N=30 proteins), microtubule organization (N=14 proteins), DNA damage response (N=20 proteins) and chromatin remodeling (N=17 proteins). N=3 biological replicas/shRNA. (B) Western blot analysis from extracts from RNF144B-deficient and proficient MEF 1.04, A549 and 3KT cell lines probed for TP53 or β-ACTIN (loading control). (Left) Western blots shown are representative of 3-8 independent blots from independent experiments. (Right) Quantification of TP53 protein levels relative to respective controls. The protein levels were standardised to β-ACTIN. P-value *P ≤ 0.05, two-tailed t.student. Supplementary Figure 5. RNF144B deficiency leads to aneuploidy in oncogene expressing MEFs. DNA quantification (Edu incorporation plus propidium iodide FACS analysis) for profiling of more than 4N (aneuploid analysis) cells in MEFs transduced with indicated shRNAs. (Left) Representative histogram profiles. (Right) DNA content quantification for profiling more than 4N (aneuploid) cells (&gt;G2). N=2-3 independent biological replicates per MEF cell line/shRNA. Data is represented by Mean ± SEM.****P ≤ 0.0001; ***P ≤ 0.001; *P ≤ 0.05, one-way ANOVA. Supplementary Figure 6. RNF144B deficiency triggers chromosomal instability and correlates with high aneuploidy in human tumors. (A) Centrosomes analysis. (Left) Representative pictures with staining for γ-Tubulin and DAPI in MEF 1.04 cell line. Scale bar: 10 μm (Right) Quantification of the centrosome number. Minimum of 300 cells/shRNA were analyzed in triplicates. **P ≤ 0.01, one-way ANOVA. (B) Mitotic pole analysis of MEF 1.01 cell line transduced with indicated shRNAs. (Left) Representative images of α-tubulin and DAPI staining. Scale bar: 10 μm (Right) Quantification of multipolar mitosis. N=200 mitoses/shRNA were analyzed in two independent experiments. Data presented as mean ± SEM, *P ≤ 0.05, One-way ANOVA. (C) Mitotic analysis. (Left) Representative anaphase images of α-tubulin and DAPI staining in MEF 1.01 cell line released after induced G2 arrest with 15 μm of RO-3306 transduced with indicated shRNAs. Arrows show lagging chromosomes. Scale bar: 10 μm. (Right) Quantification of aberrant mitosis containing lagging chromosomes or DNA bridges. Minimum of 200 mitoses (same proportion of the different phases of mitosis) per cell line were analyzed in two independent experiments. Data presented as mean ± SEM ****P ≤ 0.0001; ***P ≤ 0.001, calculated by one-way ANOVA. (D) Violin plots of the tumour aneuploidy scores in high versus low RNF144B mRNA expression in TCGA PANCAN cohort. ***P ≤ 0.001. two-tailed t-test. (E) GSEA analysis of proteins upregulated in shRNF144BMEF detected in the proteomics analysis (Fig 3) compared with the CIN70 aneuploidy signature list, described in S. Carter et al. [66]. GSEA shows enrichment of aneuploidy CIN70 signature in shRNF144BMEF protein expression signature. Top panel indicates the enrichment score, the bottom panel shows the ranking metrics of each gene. Supplementary Figure 7. RNF144B deficient lung cells are more resistant to several cytotoxic drugs. (A) In vitro growth of NLS-GFP tagged 3KT lung cells transduced with indicated sgRNAs treated for 48h with a panel of cytotoxic drugs: Palbociclib (1 μM, 3 μM), Abemaciclib (1 μM, 3 μM), Paclitaxel (5 nM, 10 nM), Docetaxel (5 nM, 20 nM), Etoposide (10 μM, 20 μM), Doxorubicin (0,05μg/ml, 0,2 μg/ml), Carboplatin (50 μM, 100 μM), RO-3306 (5 μM) and Nutlin-3a ( 20 μM). Cell growth was measured by measuring nuclear GFP signal of images acquired with the Operetta High Content Screening System in confocal mode. Cell growth was normalized to day 0 of the same genotype/well. At least 1000 cells/well were analysed in 3-5 independent experiments, in duplicates. ****P ≤0.0001; **P ≤ 0.01; *P ≤ 0.05, paired t-test student. (B) Western blot analysis of TP53 and γH2AX proteins in A549 and 3KT cell lines transduced with indicated sgRNAs after 24h treatment with Palbociclib (3 μM), Abemaciclib (3 μM), RO-3306 (5 μM), Etoposide (20 μM), Doxorubicin (0.2μg/ml), Carboplatin (50 μM), Paclitaxel (10 nM) and Docetaxel (20nM). Probing for β-ACTIN was used as a loading control. The Western blots shown are from 1 independent blot from independent experiments.