cptc gmnn  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Isoform-Specific Activation of p53B and p53C in Response to Nucleolar Stress in Drosophila wing imaginal discs

doi: 10.1101/2025.08.07.669174

Figure Lengend Snippet: (A-C) Comet assay showing fluorescent images of nuclei stained with Hoechst (white) from cells of the wing imaginal discs from w 1118 third instar larvae (A), and from animals where NOC1 was temporarily reduced for 60 hours using the Act-Gal80 ts allele. (B), scale bar is 50 μ m. (C) Quantification of single-cell tail moment from comet assay performed on nuclei from the genotypes shown in A and B. Statistical analysis was calculated using the non-parametric Kruskal-Wallis as in . (D-E) Confocal images of wing imaginal discs from third instar w 1118 larvae (D), or from the same stage animals with reduced NOC1 expression in the wing pouch using the rotund ( rn ) promoter (E). Tissues were immunostained for the expression of p53 (green) and for Caspase3, as a marker of apoptosis (red). Nuclei were stained with Hoechst (blue). Scale bar is 100 μ m. (F) qPCRs from RNA extracted from wing imaginal discs from w 1118 animals or with reduced NOC1 using the rn promoter. p53 upregulation was analyzed using p53 pan isoform primers . In C, the asterisks represent the p-values from Student’s t -test in one out of three independent experiments performed, where the dots represent the numbers of nuclei analyzed. In F, the dots represent the number of independent biological replicates. * = p < 0.05 and **** = p < 0.0001, and the error bars indicate the standard deviations.

Article Snippet: After Ponceau staining, the membrane was blocked with 5% non-fat milk in TBS-T. Then the membranes were incubated with primary antibody mouse anti-p53 (1:200, Dmp53 H3, DSHB), and mouse anti-actin (1:1000; DSHB 224-236-1), followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Single Cell Gel Electrophoresis, Staining, Expressing, Marker

Journal: bioRxiv

Article Title: Isoform-Specific Activation of p53B and p53C in Response to Nucleolar Stress in Drosophila wing imaginal discs

doi: 10.1101/2025.08.07.669174

Figure Lengend Snippet: (A-B) Schematic representation of the p53 locus (gene ID 2768677) and of the p53 isoforms A, B, C , and E . (B-i), magnification of a common exon with the representation of the region where the primers that capture all p53 isoforms anneal . These primers were used in and will be referred to as “p53 pan”. (C) Schematic representation of the p53A A2 . 3 , p53B 41 . 5 , and p53 A5 . 1 . 4 null mutant alleles. p53A A2 . 3 null line was generated with a deletion of 23bp deletion, and a 7bp insertion spanning from the end of the first exon until the first base of the first intron (in red), generating multiple early STOP codons (Supplementary Figure 2). This deletion also affects the p53C isoform, leading to no production of p53C protein, according to Chakraborty et al. 2022. p53B 41 . 5 null line was generated with a 14bp deletion and a 1bp insertion in the second exon that causes a frameshift and introduces premature stop codons (Supplementary Figure 2). p53 A5 . 1 . 4 null line has a deletion of 3.3 kb, removing the common 3’ coding region (in red). (D) p53A isoform. (D-i and ii), magnification of exons 1 and 2 of p53A and p53A A2 . 3 null isoform, respectively, displaying where the primers designed in this study anneal (in green) and compared to previous primers (in pink). As shown in D-ii, the new primers can also be used to identify p53A in the p53A A2 . 3 null mutant, as the forward primer does not anneal within the deleted region. Conversely, 1 bp of the previous reverse primer (pink) mismatch in the p53A null line. Note that this new set of primers can also capture p53C isoform; therefore, they will be referred to as “p53A/C”. (E) p53B and p53C isoforms. (E- i, ii, and iii), magnification of exons 2 and 3, which are common to both isoforms, showing where the primers designed in this study anneal (in green). Note that the new pair can distinctly capture the p53B isoform, while the old primers (in pink) also capture p53C . Moreover, the new pair is also suitable for studying the p53B null line since the forward primer does not anneal in the deleted region marked in red (E-ii). This pair of primers will be referred to as “p53B”. (F) p53C isoform. (F-i), magnification of the region in exon 2 showing the sequence of 78 bp, from 440 to 518 nucleotides from the beginning of the p53C transcript (in orange), present only in this isoform. Optimized primers for the detection of this isoform are represented in green. This new set of primers will be referred to as “p53C”. (G) Photo of a 2% agarose gel showing qPCR amplified products from RNA extracted from wing imaginal discs from w 1118 third instar larvae using the indicated primers and actin as a control. A single band is visible for each primer pair, indicating the specificity of the new optimized primers.

Article Snippet: After Ponceau staining, the membrane was blocked with 5% non-fat milk in TBS-T. Then the membranes were incubated with primary antibody mouse anti-p53 (1:200, Dmp53 H3, DSHB), and mouse anti-actin (1:1000; DSHB 224-236-1), followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Generated, Sequencing, Agarose Gel Electrophoresis, Amplification, Control

Journal: bioRxiv

Article Title: Isoform-Specific Activation of p53B and p53C in Response to Nucleolar Stress in Drosophila wing imaginal discs

doi: 10.1101/2025.08.07.669174

Figure Lengend Snippet: qPCRs showing specific mRNA changes in RNAs extracted from whole third instar larvae of the annotated p53 mutant lines. (A) p53 pan recognizes all p53 isoforms. (B) p53A/C recognizes specifically only p53A and p53C isoforms. (C) p53B recognizes only the relative isoform. (D) p53C recognizes only the relative isoform. Expression levels were normalized to actin-5C and compared to the levels in control w 1118 animals. RNA was extracted from five whole larvae of the respective genotype. The asterisks represent the p- values from One-way analysis of variance (ANOVA) with Tukey multiple comparison: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001, and the error bars indicate the standard deviations. (E) Western blot analysis from larval extracts showing the relative amount of p53 protein from w 1118 animals or different p53 mutant isoforms. Actin was used as the loading control. The band visible between 60 and 75 kDa is a non- specific signal present in all lanes.

Article Snippet: After Ponceau staining, the membrane was blocked with 5% non-fat milk in TBS-T. Then the membranes were incubated with primary antibody mouse anti-p53 (1:200, Dmp53 H3, DSHB), and mouse anti-actin (1:1000; DSHB 224-236-1), followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Expressing, Control, Comparison, Western Blot