Journal: Cell Death & Disease

Article Title: Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

doi: 10.1038/s41419-022-05431-2

Figure Lengend Snippet: Ratios of mean 50% growth-inhibitory concentrations (GI 50 ) for 28 top hit compounds between cancer cell lines carrying other TP53 mutations and cancer cell lines carrying nonsense TP53 mutations shown on the y-axis (Mut/Nonsense), and between cancer cell lines carrying WT TP53 and cancer cell lines carrying nonsense TP53 mutations shown on the x-axis (WT/Nonsense) in colon cancer cell lines (A) and renal cancer cell lines (B) . Each dot represents one compound. C , Full-length p53 induction by 5-FU in H1299-R213X cells is dose-dependent after 24, 48 or 72 h treatment, according to Western blotting with p53 antibody DO-1. GAPDH was used as loading control and DMSO (-) was used as negative control. Full membrane was blotted with DO-1 antibody, washed and blotted with GAPDH antibody. D , qRT-PCR analysis showing induction of p53 mRNA levels after 50 or 100 µM 5-FU treatment for 72 h in H1299-R213X cells, N = 3. DMSO (-) was used as negative control. E , IC 50 (µM) values of 5-FU treatment for 72 h in H1299-R213X and H1299-EV cells, N = 6. F , mRNA levels of p53 target genes p21, Zmat3, Puma, Noxa, Fas and Bax after 50 or 100 µM 5-FU treatment for 72 h measured by qRT-PCR in H1299-R213X cells and G , H1299-EV cells, N = 3. In D , F and G , expression of some genes was examined simultaneously in each cell line with a single GAPDH control; thus, same GAPDH value was used as control for several genes. Gene expression values were normalized to GAPDH expression and to the DMSO-treated sample as negative control. Statistical analyses for each gene were performed comparing each treatment to DMSO control treatment using repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.01) or Friedman test followed by Dunn’s multiple comparisons test ( # p ≤ 0.05) in genes with data not fitting normal distribution. In D , E , F and G , data are represented as mean ± SEM. Each dot represents an independent experiment.

Article Snippet: TaqMan probes used were TP53 (Hs00153340_m1), ZMAT3 (Hs00536976_m1), CDKN1A (Hs00355782_m1), FAS (Hs00531110_m1), BAX (Hs00414514_m1), PMAIP1 (Hs00560402), BBC3 (Hs00248075_m1) and GAPDH (Hs99999905_m1) (Applied Biosystems, USA), corresponding to p53, Zmat3 (Wig-1), p21, Fas, Bax, Noxa, Puma and GAPDH, respectively.

Techniques: Western Blot, Control, Negative Control, Membrane, Quantitative RT-PCR, Expressing, Gene Expression

Journal: Cell Death & Disease

Article Title: Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

doi: 10.1038/s41419-022-05431-2

Figure Lengend Snippet: A , RNA-seq to Ribo-seq log2 fold-change/fold-change (FC/FC) plots showing differentially transcribed (x axis) and translated (y axis) genes for G418 (top) and FUr (bottom) treatments. Each dot on each graph represents a protein-coding gene. Curated TP53 target genes from Andrysik et al. are coloured red and curated TP53 target genes from Fischer are in blue. Genes found in both lists are coloured purple. Selected TP53 target genes validated by qRT-PCR in ( C and D ) are labelled by gene symbol, CDKN1A (p21), ZMAT3 (Zmat3), BBC3 (Puma), PMAIP1 (Noxa), FAS (Fas) and BAX (Bax). B , Heatmap showing gene expression of TP53 target gene lists as obtained from Andrysik et al. and from Fischer , organized per treatment condition in H1299-R213X cells, as measured by RNA-seq (green, left) and ribo-seq (blue, right). Statistical analyses for each treatment were performed using DESeq2 (ref. ), i.e., a Wald test with Benjamin-Hochberg multiple test correction (adjusted p -value ≤ 0.05, log2 fold change ≥ 1) to test for significant genes in Ribo-seq data, colours indicate differentially expressed genes. Normalised counts were transformed to Z-scores for visualisation. C , mRNA levels of p53 target genes genes p21, Zmat3, Puma, Noxa, Fas and Bax after 3 or 5 µM FUr or FdUr, or 50 or 100 µM G418 treatment for 72 h measured by qRT-PCR in H1299-R213X cells and D , in H1299-EV cells. N = 3-4. E , mRNA levels of p53 target genes p21, Zmat3, Puma, Noxa, Fas and Bax after 50 µM FUr or FdUr, or 100 or 200 µM G418 treatment for 72 h measured by qRT-PCR in HDQ-P1 cells. N = 3. In C , D and E , data are represented as mean ± SEM. Values were normalized to GAPDH expression and to the non-treated sample (NT). Statistical analyses for each cell line and each gene were performed comparing each treatment to only each control non-treated (NT) sample using repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) or Friedman test followed by Dunn’s multiple comparisons test ( # p ≤ 0.05, ## p ≤ 0.01) in genes with data not fitting normal distribution. In C , D, E , Figure and Figure , expression of some genes was examined simultaneously in each cell line with a single GAPDH control; thus, same GAPDH value was used as control for several genes.

Article Snippet: TaqMan probes used were TP53 (Hs00153340_m1), ZMAT3 (Hs00536976_m1), CDKN1A (Hs00355782_m1), FAS (Hs00531110_m1), BAX (Hs00414514_m1), PMAIP1 (Hs00560402), BBC3 (Hs00248075_m1) and GAPDH (Hs99999905_m1) (Applied Biosystems, USA), corresponding to p53, Zmat3 (Wig-1), p21, Fas, Bax, Noxa, Puma and GAPDH, respectively.

Techniques: RNA Sequencing, Quantitative RT-PCR, Gene Expression, Transformation Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: Consequences of Zmat3 loss in c-MYC - and mutant KRAS -driven tumorigenesis

doi: 10.1038/s41419-020-03066-9

Figure Lengend Snippet: a Zmat3 mRNA expression in pre-leukemic Eµ-Myc B lymphoid cells 0, 6, 12 and 24 h following 5 Gy γ-irradiation (IR) or treatment with 10 µM Nutlin3a (Nut3a). Mean ± SEM. b Lymphoma-free survival of Eµ-Myc ( n = 23), Eµ-Myc ; Zmat3 +/ − ( n = 21) and Eµ-Myc ; Zmat3 −/ − ( n = 17) mice. Median lymphoma-free survival: Eµ-Myc : 125 days; Eµ-Myc ; Zmat3 +/− : 94 days; Eµ-Myc ; Zmat3 −/ − : 93 days. Eµ-Myc vs Eµ-Myc ; Zmat3 +/ − p = 0.0718; Eµ-Myc vs Eµ-Myc ; Zmat3 −/ − p = 0.3231. c The proportions of Ig + B cell lymphomas and pro-B/pre-B cell lymphomas in sick Eµ - Myc ; Zmat3 −/− ( n = 4) and sick Eµ - Myc ( n = 3) mice. No significant differences were observed; p = 0.5 calculated by two-tailed t -test. d Spleen, thymus and lymph node weight (grams) in sick Eµ-Myc ( n = 7), Eµ-Myc ; Zmat3 +/ − ( n = 15) and Eµ-Myc ; Zmat3 −/ − ( n = 9) mice at ethical endpoint. Mean ± SEM.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Techniques: Expressing, Irradiation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Consequences of Zmat3 loss in c-MYC - and mutant KRAS -driven tumorigenesis

doi: 10.1038/s41419-020-03066-9

Figure Lengend Snippet: a Representative hematoxylin and eosin (H&E) staining of lungs from Zmat3 −/ − , littermate Zmat3 +/+ (wt) and p53 −/ − control mice aged 16-18 weeks. Scale, 200 μm. b Flow cytometric analysis of the lymphoid and myeloid cells present in the lungs from Zmat3 −/− ( n = 4), p53 −/ − ( n = 2) and wt ( n = 4) mice. Immune cell populations are normalized to wt control mice. c Growth analysis of primary cell lines derived from the lung epithelium of wt ( n = 8) , Zmat3 −/ − ( n = 8) and p53 −/ − ( n = 4) mice. Mean ± SEM. d Accumulative cell number following 8 passages of primary wt ( n = 8) , p53 − / − ( n = 4) and Zmat3 −/− ( n = 8) cell lines. Mean ± SD, **** p < 0.0001. e Representative light microscope images of Zmat3 +/+ (wt) , p53 −/− and Zmat3 −/ − cell lines 8 passages following their derivation. Scale, 10 μm.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Techniques: Staining, Control, Derivative Assay, Light Microscopy

Journal: Cell Death & Disease

Article Title: Consequences of Zmat3 loss in c-MYC - and mutant KRAS -driven tumorigenesis

doi: 10.1038/s41419-020-03066-9

Figure Lengend Snippet: a Schematic of experimental design. Kras LSL-G12D/+ (K) mice were crossed with p53 −/ − mice (KPnull) or Zmat3 −/ − mice (KZ). b Kaplan–Meier tumor-free survival analysis of K ( n = 6), KPnull ( n = 6) and KZ ( n = 12) mice following i.n. infection of Ad5-CMV-Cre virus. c Representative H&E staining and immunohistochemistry of Ki67, Nkx2.1/TTF-1 and Hmga2 in end-stage lung tumors from Kras G12D , Kras G12D ; p53 −/ − and Kras G12D ; Zmat3 −/ − mice. Scale, 100 μm.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Techniques: Infection, Virus, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Consequences of Zmat3 loss in c-MYC - and mutant KRAS -driven tumorigenesis

doi: 10.1038/s41419-020-03066-9

Figure Lengend Snippet: a Representative low magnification H&E stained lungs of Kras G12D/+ (K), Kras G12D/+ ; p53 −/− (KPnull) and Kras G12D/+ ; Zmat3 −/− (KZ) mice 6- and 10-weeks following i.n. administration of Ad5-CMV-Cre. Scale, 1 mm. b Lesion classification in the lungs of K ( n = 4), KPnull ( n = 4) and KZ ( n = 5) mice 10 weeks following i.n. Ad5-CMV-Cre infection. c Wet weight (mg) of the left lung lobe of K ( n = 3), KPnull ( n = 3) and KZ ( n = 8) mice 6 weeks following i.n. Ad5-CMV-Cre infection. Violin plot indicates the median weight and distribution.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Techniques: Staining, Infection

Journal: Cell Death & Disease

Article Title: Consequences of Zmat3 loss in c-MYC - and mutant KRAS -driven tumorigenesis

doi: 10.1038/s41419-020-03066-9

Figure Lengend Snippet: a Growth analysis of primary cell lines derived from the lung epithelium of KPnull ( n = 8) and KZ ( n = 12) mice 10 weeks following i.n. Ad5-CMV-Cre infection. Mean ± SEM. b Accumulative cell number following 8 passages of primary KPnull ( n = 8) and KZ ( n = 12) cell lines. Mean ± SD, *** p < 0.0001. c Representative light microscope images of Kras G12D / p53 −/ − (KPnull) and Kras G12D / Zmat3 −/ − (KZ) cell lines 8 passages following their derivation. Scale, 10 μm. d Schematic representation of p53 target genes and their role in tumor suppression.

Article Snippet: Quantitative RT-PCR was performed using TaqMan probes (Thermo Fisher Scientific) for Zmat3 (Mm01292424_m1), Puma (Mm00519268_m1), p21 (Mm00432448_m1) and analyzed by the ΔΔCT method relative to HMBS (Mm01143545_m1).

Techniques: Derivative Assay, Infection, Light Microscopy