Journal: Scientific Reports

Article Title: Identification and functional interpretation of miRNAs affected by rare CNVs in CAKUT

doi: 10.1038/s41598-022-22749-1

Figure Lengend Snippet: The difference in the relative expression of target genes mRNA between MIR484 +/− KO and HEK293 WT. Relative mRNA levels were standardized against GAPDH endogenous control and presented as a scatter plot of 2 −dCt values with standard errors of the mean from three independent replicates. ( A ) Relative levels of MDM2 mRNA , Student's t-test, P = 0.005. ( B ) Relative levels of APAF1 mRNA, Student's t-test, P < 0.0001 ( C ) Relative levels of NOTCH3 mRNA, Student's t-test, P = 0.014. ( D ) Relative levels of FIS1 mRNA, Student's t-test, P = 0.081. ( E ) Relative levels of PKD1 mRNA, Student's t-test, P = 0.742. * denotes a significant difference at P < 0.05; ** denotes a significant difference at P < 0.01; ns denotes a nonsignificant difference in relative target gene mRNA levels.

Article Snippet: Expression levels of the selected hsa-miR-484 target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan ® gene expression assay: Hs00947377_m1 (for PKD1 (polycystin-1)), Hs00540450_s1 (for MDM2 (Mouse double minute 2 homolog)), Hs00211420_m1 (for FIS1 (Mitochondrial Fission 1 Protein)), Hs01128537_m1 (for NOTCH3 (Neurogenic locus notch homolog)), Hs00559441_m1 (for APAF1 (Apoptosis protease-activating factor-1)), Hs02786624_g1 (for GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense

doi: 10.3389/fimmu.2022.933329

Figure Lengend Snippet: Bystander apoptosis is characterized by the HMGB1/p53 complex–mediated apoptosis. (A) p53 uptake by the EpCAM+/ABCG2+ versus EpCAM+/ABCG2- CSCs with or without anti-HMGB1 pre-treated BCG-CM. (B) Relative uptake of p53 by the EpCAM+/ABCG2+ and EpCAM+/ ABCG2- CSCs with or without neutralizing anti-HMGB1 pre-treated BCG-CM. (C) The induction of p53/MDM2 oscillation in EpCAM+/ABCG2+ CSCs following 12 h of BCG-CM treatment. (D) Significant induction of p53-related pro-apoptotic genes and HMGB1 gene in EpCAM+/ABCG2+ CSCs following 28 h of BCG-CM treatment. The real-time PCR data were compared with untreated EpCAM+/ABCG2+ CSCs to obtain fold change. (E) Significant increase in cleaved caspase-3 protein level in EpCAM+/ABCG2+ CSCs treated with BCG-CM with or without pifithrin alpha (2 µM in DMSO for 48 h) or anti-HMGB1 (10 µg/ml for 48 h; isotype control of same dose). Data represent means ± SEM (A–E) . N = 3 independent experiments (A–E) . *p < 0.05, **p < 0.01, and ***p < 0.001 ( A, B, E : one-way ANOVA with Dunnet post hoc test; C, D : Student’s t-test).

Article Snippet: The following TaqMan gene expression primers were used: human: ABCG2 (Hs00184979_m1), TLR2 (Hs02621280_s1), TLR4 (Hs00152939_m1), TLR7 (Hs01933259_s1), TLR9 (Hs00370913_s1), p53 (Hs01034249_m1), p21 (Hs00355782_m1), PUMA (Hs00248075_m1), Bax (Hs00180269_m1), GAPDH (Hs00266705_g1), MDM2 (Hs01066930_m1), and HMGB1 (Hs01923466_g1).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense

doi: 10.3389/fimmu.2022.933329

Figure Lengend Snippet: Tumor stemness defense (TSD) phenotype can amplify the pathogen-induced bystander apoptosis (PIBA). (A) The p53 uptake assay in the culture supernatant was measured from 0 to 16 h of BCG-CM treatment in the cells. The SCC-25 SP cells were obtained as described in Figure 1 Data represent mean +/- SEM, n= 3 independent experiments. ***p < 0.0001, student t test. (B) Potential mechanism of TSD phenotype–mediated niche defense of CSCs against BCG infection. In the infected CSCs, as part of the Altruistic stemness–based niche defense mechanism ( , , ) HMGB1 form a complex with cytoplasmic p53 to make an, “altruistic death signal”. TLR4 internalizes the altruistic death signal, leading to induction of p53/MDM2 oscillation and activation of p53-induced pro-apoptotic genes. The EpCAM+/ABCG2+ CSCs undergoing bystander apoptosis further release the HMGB1/p53 death complex into the TME, amplifying PIBA.

Article Snippet: The following TaqMan gene expression primers were used: human: ABCG2 (Hs00184979_m1), TLR2 (Hs02621280_s1), TLR4 (Hs00152939_m1), TLR7 (Hs01933259_s1), TLR9 (Hs00370913_s1), p53 (Hs01034249_m1), p21 (Hs00355782_m1), PUMA (Hs00248075_m1), Bax (Hs00180269_m1), GAPDH (Hs00266705_g1), MDM2 (Hs01066930_m1), and HMGB1 (Hs01923466_g1).

Techniques: Infection, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways

doi: 10.1007/s00018-020-03552-5

Figure Lengend Snippet: Pathways containing H2Bub1-enriched genes in response to DNA damage

Article Snippet: Gene expression was analyzed in triplicate using TaqMan assays: TP53, Hs01034249_m1; CDKN1A, Hs00355782_m1; BBC3, Hs00248075_m1; MDM2, Hs00242813_m1; BAX, Hs00180269_m1; GADD45A , Hs00169255_m1; PLK2 , Hs00198320_m1 and HMBS, Hs00609297_m1 (Life Technologies, Mulgrave, VIC, Australia) and the iTaq Universal Probes Supermix (Bio-Rad Laboratories, Gladesville, NSW, Australia).

Techniques: Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways

doi: 10.1007/s00018-020-03552-5

Figure Lengend Snippet: Wild-type p53 cells treated with cisplatin show H2Bub1 enrichment in p53 target genes. a University of California Santa Cruz (UCSC) genome browser hg19 images of representative p53 target genes CDKN1A and b MDM2 (A2780 cells treated with saline or cisplatin). RNA-seq and ChIP-seq signals are shown, as is the layered image of the active transcription mark H3K27Ac of seven cell lines from ENCODE. Exonic regions are marked by boxes in RefSeq tracks. c UCSC genome browser hg19 image of TP53, comparing H2Bub1-enriched chromatin in response to DNA damage as above

Article Snippet: Gene expression was analyzed in triplicate using TaqMan assays: TP53, Hs01034249_m1; CDKN1A, Hs00355782_m1; BBC3, Hs00248075_m1; MDM2, Hs00242813_m1; BAX, Hs00180269_m1; GADD45A , Hs00169255_m1; PLK2 , Hs00198320_m1 and HMBS, Hs00609297_m1 (Life Technologies, Mulgrave, VIC, Australia) and the iTaq Universal Probes Supermix (Bio-Rad Laboratories, Gladesville, NSW, Australia).

Techniques: Saline, RNA Sequencing, ChIP-sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways

doi: 10.1007/s00018-020-03552-5

Figure Lengend Snippet: H2Bub1 enrichment in p53 target genes in the presence of wild-type or mutant p53. a Increased H2Bub1 enrichment in exons and introns of p53 target genes in response to cisplatin is observed in A2780 wild-type p53 cells treated with saline (s) or cisplatin (c; IC75 dose) for 24 h following ChIP with an anti-H2Bub1 antibody or IgG followed by qRT-PCR using either primers within (+) or downstream (−) of the coding regions of p53 target genes. Data within replicates was normalized to the experimental mean and presented as mean ± SEM (N = 3) analyzed using one-way ANOVA with Tukey’s post hoc test. b p53 target genes under the same conditions as above showed increased expression following treatment with cisplatin (N = 4; data expressed relative to saline treated cells (one-sample t test); GOI gene of interest. c OVCAR-3 p53 mutant cells treated with saline or cisplatin (IC80 dose; Suppl. Figure 1) for 24 h underwent ChIP and qRT-PCR as above (N = 3). d Expression of p53 target genes in OVCAR-3 cells in response to an IC80 dose of cisplatin as above (N = 3). (*p < 0.05, **p < 0.005). e Transfection of wt p53 into p53 null SKOV-3 cells leads to increased H2Bub1-enrichment at the coding region of p53 target genes relative to transfection with mutant p53, in parallel with increased gene expression. Transfected cells underwent ChIP with anti-H2Bub1 or IgG antibodies followed by qRT-PCR using either primers within (+) or downstream (−) of the coding regions of p53 target genes. Data within replicates was normalized to the experimental mean and presented as mean ± SEM (N = 4) analyzed using one-way ANOVA with Tukey’s post hoc test. f Expression of p53 target genes in transfected cells (N = 4). Data expressed relative to saline treated cells (one sample t test), (*p < 0.05, **p < 0.005). g Treatment of A2780 cells with the MDM2 inhibitor nutlin-3a leads to H2Bub1-enrichment at the coding region of p53 target genes. A2780 wt p53 cells treated with vehicle (DMSO) or nutlin-3a (21.3 µM, correlating with an IC75 dose) for 24 h underwent ChIP with an anti-H2Bub1 antibody or IgG followed by qRT-PCR using either primers within (+) or downstream (−) of the coding regions of p53 target genes. Data within replicates was normalized to the experimental mean and presented as mean ± SEM (N = 3) analyzed using one-way ANOVA with Tukey’s post hoc test. h Expression of p53 target genes in A2780 cells in response to an IC75 dose of nutlin-3a, or vehicle (DMSO) for 24 h (N = 4). Data expressed relative to DMSO treated cells (one sample t test), (*p < 0.05, **p ≤ 0.005)

Article Snippet: Gene expression was analyzed in triplicate using TaqMan assays: TP53, Hs01034249_m1; CDKN1A, Hs00355782_m1; BBC3, Hs00248075_m1; MDM2, Hs00242813_m1; BAX, Hs00180269_m1; GADD45A , Hs00169255_m1; PLK2 , Hs00198320_m1 and HMBS, Hs00609297_m1 (Life Technologies, Mulgrave, VIC, Australia) and the iTaq Universal Probes Supermix (Bio-Rad Laboratories, Gladesville, NSW, Australia).

Techniques: Mutagenesis, Saline, Quantitative RT-PCR, Expressing, Transfection, Gene Expression

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Schematic of the structure of the genetically modified Trp53 allele. The coding sequence for the triple-FLAG tag was inserted after the start codon of the Trp53 gene. ( B ) PCR analysis showing correct insertion of the coding sequences for the triple-FLAG tag into the genetically modified Trp53 allele. A band of 225 bp indicates the presence of a wt (unmodified) allele. A band of 291 bp indicates a FLAG-Trp53 allele. Each lane represents DNA taken from an individual mouse (#32, 38, 40, 44, 45) followed by DNA from a control wt mouse, control DNA from a FLAG-Trp53 KI/+ mouse, and a water-only control. ( C ) Table with observed and expected genotype distribution from inter-crosses of heterozygous FLAG-Trp53 KI/+ mice. ( D ) Table with observed and expected sex distribution from all matings that could give rise to FLAG-Trp53 KI/+ or FLAG-Trp53 KI/KI mice. Frequencies compared by Chi-square test p = 0.0262. ( E ) Kaplan–Meier survival curve showing overall survival of wt, FLAG-Trp53 KI/+ , and FLAG-Trp53 KI/KI mice. Differences in animal survival were compared using the Log-rank (Mantle–Cox) test, * p values ≤0.05. Mice were censored if harvested when healthy for use in experiments. ( F ) Thymocytes from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated for 48 h in vitro with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 1 µg/mL ionomycin or exposed to one dose of 1.25 Gy γ-radiation and then kept for 48 h in culture. Cell viability was measured at various time points by flow cytometry after staining with fluorochrome-conjugated annexin-V and DAPI. The percentages of live, i.e. annexin-V/DAPI double-negative cells, are plotted. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( G ) Mouse dermal fibroblasts (MDFs) were derived from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice and treated for 72 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide or 250 nM taxol. Cellular senescence was measured as a percentage of fluorescein-di-β- d -galactopyranoside (FDG) positive cells by flow cytometry. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( H ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice after they had been treated for 6 h with DMSO (vehicle control) or 10 µM nutlin-3a in vitro. qRT-PCR was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma , and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change of drug-treated compared to DMSO-treated cells. Data were presented as mean ± SD. n = 4 mice of each genotype. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests were non-significant (i.e. had a p value >0.05). .

Article Snippet: Mdm2 , Mm01233138_m1 , Thermo Fisher Scientific.

Techniques: Genetically Modified, Sequencing, FLAG-tag, Control, In Vitro, Flow Cytometry, Staining, Derivative Assay, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Next-generation sequencing results for the inserted sequences encoding the triple-FLAG tag in the F1 generation of FLAG-Trp53 KI/+ mice. Each line represents the reads from 1 independent F1 mouse. A black dot indicates a matching base in the sequencing reads compared to the reference sequence. ( B ) Representative histology from aged wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice at the time of harvest. No tumour samples showed TRP53 staining by immunohistochemistry, indicating that they did not have mutant TRP53 driving their malignancy. Scale bar = 100 um. ( C ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice that had been treated for 6 h with DMSO (vehicle control) or 1.25 Gy γ-radiation in vitro. qRT-PCR analysis was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change compared to DMSO-treated samples. Data were presented as mean ± SD. n = 4 mice of each genotype and treatment. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests showed that differences were not significant (had a p value >0.05).

Article Snippet: Mdm2 , Mm01233138_m1 , Thermo Fisher Scientific.

Techniques: Next-Generation Sequencing, FLAG-tag, Sequencing, Staining, Immunohistochemistry, Mutagenesis, Control, In Vitro, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Thymocytes from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated for 24 h with DMSO (vehicle control), 10 µM nutlin-3a or 5 Gy γ-radiation in the presence of the broad-spectrum caspase inhibitor QVD-oPH (to prevent degradation of protein due to apoptosis). Western blot analysis was performed on protein extracts, probing with antibodies against TRP53, FLAG and GAPDH (the latter used as a protein loading control). The asterisk indicates a non-specific band detected when probing for TRP53. Relative density for TRP53 and FLAG bands (normalised to GAPDH) are listed. ( B ) MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated for 24 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide or 250 nM taxol in the presence of QVD-oPH. Western blot analysis was performed on protein extracts, probing with antibodies against TRP53, FLAG and HSP70 (the latter used as a protein loading control). Relative density for TRP53 and FLAG bands (normalised to HSP70) are listed. ( C ) Thymocytes from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated with 10 µM nutlin-3a for 0, 6, 12 or 24 h in the presence of the broad-spectrum caspase inhibitor QVD-oPH. Western blot analysis was performed on protein extracts, probing with antibodies against TRP53, FLAG, HSP70 and β-ACTIN (the latter two used as protein loading controls). ( D ) Bar graph showing summary data of relative protein densities of TRP53, and TRP53-FLAG proteins quantitated from Western blots from three individual mice for each genotype normalised to β-ACTIN, and displayed as mean ± SD. ( E ) Heatmap from global analysis of CUT&RUN data showing the read coverage 2 kbp up- and downstream of enriched peaks found genome-wide in MDFs derived from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice, treated with either DMSO (vehicle) or 10 µM nutlin-3a for 6 h. Peaks were called with MACS2. ( F ) FLAG antibody CUT&RUN tracks for the known TRP53 target genes Mdm2, Pmaip1/Noxa, Bbc3/Puma and Cdkn1a/p21 . MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated in vitro for 6 h with either DMSO (vehicle control) or 10 µM nutlin-3a. Data shown are from MDFs from n = 3-4 mice of each genotype pooled in analysis, normalised to library size. .

Article Snippet: Mdm2 , Mm01233138_m1 , Thermo Fisher Scientific.

Techniques: Control, Western Blot, Genome Wide, Derivative Assay, In Vitro

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: TaqMan TM probes for qRT-PCR.

Article Snippet: Mdm2 , Mm01233138_m1 , Thermo Fisher Scientific.

Techniques: