Journal: Cancer Cell International

Article Title: Caspase-2 is involved in cell death induction by taxanes in breast cancer cells

doi: 10.1186/1475-2867-13-42

Figure Lengend Snippet: Effect of paclitaxel on the level of (A) PIDD mRNA and (B) PIDD protein in SK-BR-3 and MCF-7 cells. After 0, 12, 24 and 36 h of incubation with paclitaxel (100 nM for SK-BR-3 cells and 300 nM for MCF-7 cells), the level of mRNA was determined using RT-PCR and relevant primers and the level of protein was determined using western blot analysis and relevant antibodies (see “Materials and Methods”). Each column of mRNA data represents the mean of 4 experimental values ± SEM. *P < 0.05, **P < 0.01 when comparing the effect with that at 0 h. Actin levels of protein data were used to confirm equal protein loading. The data shown were obtained in one representative experiment of three independent experiments.

Article Snippet: For western blot analysis, the following primary antibodies were used: mouse monoclonal antibody against caspase-3 (#9668), rabbit antibody against cleaved caspase-3 (#9661), rabbit antibody against cleaved caspase-7 (#9491), mouse monoclonal antibody against caspase-8 (#9746), and rabbit antibody against cleaved caspase-9 (#9505) from Cell Signaling Technology (Danvers, MA, USA), rat monoclonal antibody against caspase-2 (ALX-804-355-C100) and mouse monoclonal antibody against PIDD (ALX-804-837) from Enzo Life Science (Farmingdale, NY, USA), rabbit monoclonal antibody against caspase-2 (ab32021), rabbit polyclonal antibody against caspase-2 (ab 18737), goat polyclonal antibody against golgin-160 (anti-GOLGA3, ab40837), and rabbit monoclonal antibody against RAIDD (Ab52621) from Abcam (Cambridge, UK), rabbit polyclonal antibody against caspase-2 (H-145, sc-15379), mouse monoclonal antibody against p21 (F-5, sc-6246), goat polyclonal antibody against PIDD (S-17, sc-32161), and rabbit polyclonal antibody against PIDD (H-300, sc-2354) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and mouse monoclonal antibody against actin (AC-40, A3853) from Sigma-Aldrich.

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cancer Cell International

Article Title: Caspase-2 is involved in cell death induction by taxanes in breast cancer cells

doi: 10.1186/1475-2867-13-42

Figure Lengend Snippet: Effect of paclitaxel on coimmunoprecipitation of caspase-2 and PIDD with RAIDD in SK-BR-3 and MCF-7 cells. After 24 h of incubation with paclitaxel (100 nM for SK-BR-3 cells and 300 nM for MCF-7 cells), the level of precipitated RAIDD and coimmunoprecipitated caspase-2 as well as PIDD (detected by antibody against C-form) were determined using western blot analysis and relevant antibodies (see “Materials and Methods”). The heavy chain of the antibody used for RAIDD precipitation (Ab) is seen. The data shown were obtained in two independent experiments.

Article Snippet: For western blot analysis, the following primary antibodies were used: mouse monoclonal antibody against caspase-3 (#9668), rabbit antibody against cleaved caspase-3 (#9661), rabbit antibody against cleaved caspase-7 (#9491), mouse monoclonal antibody against caspase-8 (#9746), and rabbit antibody against cleaved caspase-9 (#9505) from Cell Signaling Technology (Danvers, MA, USA), rat monoclonal antibody against caspase-2 (ALX-804-355-C100) and mouse monoclonal antibody against PIDD (ALX-804-837) from Enzo Life Science (Farmingdale, NY, USA), rabbit monoclonal antibody against caspase-2 (ab32021), rabbit polyclonal antibody against caspase-2 (ab 18737), goat polyclonal antibody against golgin-160 (anti-GOLGA3, ab40837), and rabbit monoclonal antibody against RAIDD (Ab52621) from Abcam (Cambridge, UK), rabbit polyclonal antibody against caspase-2 (H-145, sc-15379), mouse monoclonal antibody against p21 (F-5, sc-6246), goat polyclonal antibody against PIDD (S-17, sc-32161), and rabbit polyclonal antibody against PIDD (H-300, sc-2354) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and mouse monoclonal antibody against actin (AC-40, A3853) from Sigma-Aldrich.

Techniques: Incubation, Western Blot

Journal: bioRxiv

Article Title: PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

doi: 10.1101/2020.05.13.092486

Figure Lengend Snippet: (A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2 and RAIDD. HSP90 and PonceauS protein staining served as loading controls. (B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. Data are represented as median, statistical significance was defined as * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Article Snippet: Proteins of interest were detected using antibodies recognizing caspase-2 (rat monoclonal anti-Casp2 (11B4), Enzo Life Science, Farmingdale, NY, Cat# ALX-804-356; 1:1000), RAIDD (rabbit polyclonal anti-RAIDD, Proteintech, Rosemont, IL, Cat# 10401-1-AP, 1:1000), PIDD1 (mouse monoclonal anti-PIDD1 (Anto-1), Enzo Life Science, Cat# ALX-804-837, 1:500) and HSP90 (mouse monoclonal anti Hsp90, Santa Cruz, Dallas, TX, Cat# sc-13119, 1:10000).

Techniques: Western Blot, Staining, Flow Cytometry

Journal: Cellular signalling

Article Title: ATR signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death.

doi: 10.1016/j.cellsig.2020.109602

Figure Lengend Snippet: Fig. 1. Prolonged ATR kinase inhibition causes cell death in normal cells: Asynchronous culture of NHDFs were treated with specific chemical inhibitor (5 h) for ATRi, LatB, Chk1i, ATMi and DNAPKci, followed by cell death assays (Annexin V FITC PI assay and Trypan blue dye exclusion test). (a) Representative images of Annexin V FITC PI staining of the inhibitor treated samples (upper panel at 20× magnification; lower panel at 63× magnification). (b) Quantitation of samples described in panel-A (NT: “No Treatment”) N = 3, n = 100. Error bars represent the s.e.m. (c) Asynchronous culture of NHDFs were treated with Control siRNA and siRNA for ATR and Chk1 for 54 h followed by cell death assays (Annexin V FITC PI assay and Trypan blue dye exclusion test). Representative images of Annexin V FITC PI staining of the inhibitor treated samples (upper panel at 20× magnification; lower panel at 63× magnification). (d) Quantitation of samples described in panel-A (NT: “No Treatment”) N = 3, n = 100. Error bars represent the s.e.m. (e) HEK293 cells were treated with specific chemical inhibitor (5 h) for ATRi, Chk1i and Cisplatin, and immunoblotted for γH2AX, p53 and pChk2 using actin as a loading control. Cells from specific time-points of ATRi inhibition were immunoblotted for p53.

Article Snippet: Primary antibodies used were rabbit anti-pNPM (1:1000, Cell Signalling Technologies), rabbit anti-PIDD (1:1000, Biorad), rabbit anti-Caspase 2 (1:1000, Abcam), mouse anti-RAIDD (1:200, Santa Cruz Biotechnology), rabbit antipChk1 (Ser 345) (1:1000, Cell Signalling Technologies) mouse anti-p53 (1:200, Santa Cruz Biotechnology), rabbit anti-γH2AX (1:1000, Abcam), rabbit anti-pChk2 (1:1000, Abcam), mouse anti-NPM (1:5000, Abcam).

Techniques: Inhibition, Staining, Quantitation Assay, Control

Journal: Cellular signalling

Article Title: ATR signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death.

doi: 10.1016/j.cellsig.2020.109602

Figure Lengend Snippet: Fig. 2. ATR kinase inhibition neither hampers ATM kinase activity nor leads to its activation: (a) NHDFs from specific time-points of ATRi inhibition were fixed, nuclei counterstained with DAPI, or stained for pATM or p53. Chk1i and Cisplatin treated (5 h) samples were similarly processed. Nuclei Images were captured in ZEISS 510 microscope at 100× magnification. (b) Quantitation of percentage of pATM positive nuclei in the samples. At least 100 nuclei/sample were analysed and scored for pATM positivity and was averaged for three independent biological replicates. (c) NHDFs from specific time-points of ATRi inhibition were fixed, nuclei counterstained with DAPI, or stained for γH2AX and pChk2. Cisplatin treated (5 h) samples were similarly processed. Nuclei Images were captured in ZEISS 510 microscope at 100× magnification. (d) Quantitation of percentage of γH2AX/pChk2 positive nuclei in the samples. At least 100 nuclei/sample were analysed and scored for γH2AX/pChk2 positivity and was averaged for three independent biological replicates. (e) HEK293 from specific time-points of ATRi, Chk1i, ATRi + Cisplatin and Chk1i + Cisplatin treatment were fixed, nuclei counterstained with DAPI, or stained for γH2AX and pChk2. Nuclei Images were captured in ZEISS 510 microscope at 100× magnification. (f) Quantitation of percentage of γH2AX/pChk2 positive nuclei in the samples. At least 100 nuclei/sample were analysed and scored for γH2AX/pChk2 positivity and was averaged for three independent biological replicates. Statistical analysis was performed by Student's t-test where * represents P value ≤.05.

Article Snippet: Primary antibodies used were rabbit anti-pNPM (1:1000, Cell Signalling Technologies), rabbit anti-PIDD (1:1000, Biorad), rabbit anti-Caspase 2 (1:1000, Abcam), mouse anti-RAIDD (1:200, Santa Cruz Biotechnology), rabbit antipChk1 (Ser 345) (1:1000, Cell Signalling Technologies) mouse anti-p53 (1:200, Santa Cruz Biotechnology), rabbit anti-γH2AX (1:1000, Abcam), rabbit anti-pChk2 (1:1000, Abcam), mouse anti-NPM (1:5000, Abcam).

Techniques: Inhibition, Activity Assay, Activation Assay, Staining, Microscopy, Quantitation Assay

Journal: bioRxiv

Article Title: PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

doi: 10.1101/2020.05.13.092486

Figure Lengend Snippet: (A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2 and RAIDD. HSP90 and PonceauS protein staining served as loading controls. (B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. Data are represented as median, statistical significance was defined as * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Article Snippet: Proteins of interest were detected using antibodies recognizing caspase-2 (rat monoclonal anti-Casp2 (11B4), Enzo Life Science, Farmingdale, NY, Cat# ALX-804-356; 1:1000), RAIDD (rabbit polyclonal anti-RAIDD, Proteintech, Rosemont, IL, Cat# 10401-1-AP, 1:1000), PIDD1 (mouse monoclonal anti-PIDD1 (Anto-1), Enzo Life Science, Cat# ALX-804-837, 1:500) and HSP90 (mouse monoclonal anti Hsp90, Santa Cruz, Dallas, TX, Cat# sc-13119, 1:10000).

Techniques: Western Blot, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

doi: 10.1101/2020.05.13.092486

Figure Lengend Snippet: (A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b . PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, “C” and “CC” indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. (B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Data are represented as median, statistical significance was defined as * p<0.05, ** p<0.01. (C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.

Article Snippet: Proteins of interest were detected using antibodies recognizing caspase-2 (rat monoclonal anti-Casp2 (11B4), Enzo Life Science, Farmingdale, NY, Cat# ALX-804-356; 1:1000), RAIDD (rabbit polyclonal anti-RAIDD, Proteintech, Rosemont, IL, Cat# 10401-1-AP, 1:1000), PIDD1 (mouse monoclonal anti-PIDD1 (Anto-1), Enzo Life Science, Cat# ALX-804-837, 1:500) and HSP90 (mouse monoclonal anti Hsp90, Santa Cruz, Dallas, TX, Cat# sc-13119, 1:10000).

Techniques: Western Blot, Staining, Control, Generated, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: PIDDosome-induced p53-activation for ploidy restriction facilitates hepatocarcinogenesis

doi: 10.1101/2020.05.13.092486

Figure Lengend Snippet: (A) Genes co-expressed with the PIDDosome components were examined with respect to the associated Hallmark pathways taking positive (pos) and negative (neg) correlation into account. (B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Pearson’s correlation coefficient. Data are represented as median. (C-E) Correlation (Pearson) of CASP2, RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.

Article Snippet: Proteins of interest were detected using antibodies recognizing caspase-2 (rat monoclonal anti-Casp2 (11B4), Enzo Life Science, Farmingdale, NY, Cat# ALX-804-356; 1:1000), RAIDD (rabbit polyclonal anti-RAIDD, Proteintech, Rosemont, IL, Cat# 10401-1-AP, 1:1000), PIDD1 (mouse monoclonal anti-PIDD1 (Anto-1), Enzo Life Science, Cat# ALX-804-837, 1:500) and HSP90 (mouse monoclonal anti Hsp90, Santa Cruz, Dallas, TX, Cat# sc-13119, 1:10000).

Techniques: Expressing