Journal: Molecular Cancer Research

Article Title: Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

doi: 10.1158/1541-7786.mcr-09-0373

Figure Lengend Snippet: FIGURE 1. PARP cleavage and upregulation in FA cells. A. Immunoblot showing PARP cleavage in FANCD2-deficient (PD20), FANCD2-proficient (PD20cor), or nonmonoubiquitinable mutant (K561R) cells. B. Immunoblot showing PARP processing in PD20 and PD20-corrected cells treated (+) or untreated (−) with PARP inhibitor 3AB. C. PARP cleavage is increased in primary fibroblasts transfected with FANCD2 siRNA for the corresponding time intervals. Arrows, mobility shift representing PARP phosphorylation. Note the reduction of PARP signal in the sample treated with λ-phosphatase or in the siRNA FANCD2–depleted cells pretreated with PKC inhibitor. D. Immunoblot of the above FANCD2 knockdown samples immunoprecipitated with PARP antibody followed by probing with phosphor-PARP or total PARP antibodies. E. Immunoblots showing PARP cleavage in different FA subgroups treated or untreated with the PARP inhibitor, 3AB.

Article Snippet: In addition to the antibodies used for Western blotting, cleaved PARP (Asp214) antibody (human specific) #9541 (Cell Signaling, Inc.) has been used.

Techniques: Western Blot, Mutagenesis, Transfection, Mobility Shift, Phospho-proteomics, Knockdown, Immunoprecipitation

Journal: Human reproduction (Oxford, England)

Article Title: Autotransplantation of the ovarian cortex after in-vitro activation for infertility treatment: a shortened procedure.

doi: 10.1093/humrep/deab143

Figure Lengend Snippet: Figure 2. Effects of in-vitro activation (IVA) stimulators on cultured human ovarian cortex. Human ovarian cortex was cut into large strips (1 1 cm, 1–2 mm thickness) or tiny pieces (1 1 mm, 1–2 mm thickness) and cultured with or without IVA stimulators, PA and 740Y-P for 1 h (IVA-N). (A) TUNEL (TdT-mediated dUTP Nick-End Labeling) assay to compare apoptosis between large strips and tiny pieces treated with (IVA-N) or without (control; C) stimulators. Left panel, TUNEL staining showed apoptosis signals in each group. Dotted line, apoptotic areas. Right panel, TUNEL positive cell counting in each group. Five areas were randomly chosen in each section. (B) Western blots of apoptotic proteins p-PARP (Poly(ADP-Ribose) Polymerase), cleaved Caspase3 in large ovarian strips and tiny ovarian pieces treated with or without stimulators (left panel). Right panel, densitometry of western blot was quantified and shown as p-PARP to tubulin and cleaved Caspase3 to tubulin ratios. The ratios of p-PARP/tubulin and cleaved Caspase3/tubulin in control large ovarian strips were designed as 1. Immunostaining (C) and western blot (D) of p-rpS6 (S235/236) in large ovarian strips after IVA treatment. Densitometry of western blot was quantified and shown in the right panel of (D) as p-rpS6/rpS6 ratio. The ratios in control were designed as 1. (E) Immunostaining of Foxo3a in large ovarian strips after IVA treatment. The activation of primordial follicles was quantified by counting cells with the translocation of Foxo3a from nucleus to cytoplasm (lower panel). Insets in (C) and (E), a representative dormant primordial follicle and an activated primordial follicle in control and treated larger ovarian cortex, respectively. Red arrows, primordial follicles shown in insets. All bars ¼ 50mm. All data were shown as means § SD. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies against Akt (2920), p-Akt (Ser473) (4060), rpS6 (Ribosomal Protein S6) (2217), p-rpS6 (S235/6) (4858), p-PARP (Poly(ADP-Ribose) Polymerase) (12061), PARP (12061), cleaved Caspase3 (9661) and b-actin (2128) were all rabbit antibodies and were purchased from Cell Signaling Technology (CST, Shanghai, China).

Techniques: In Vitro, Activation Assay, Cell Culture, TUNEL Assay, End Labeling, Control, Staining, Cell Counting, Western Blot, Immunostaining, Translocation Assay

Journal: Chemical Science

Article Title: Selective chemical reagents to investigate the role of caspase 6 in apoptosis in acute leukemia T cells †

doi: 10.1039/d2sc05827h

Figure Lengend Snippet: Caspase 6 activation in STS-treated Jurkat T cells. (A) Graphical representation of the intrinsic apoptosis pathway upon staurosporine treatment. The pale green background represents our working model to study hierarchical activation of executioner caspases and real-time imaging of caspase 6. (B) Activation of pro-caspase 3 (p32 → p17/p19) and pro-caspase 6 (p35) and processing of PARP (p116 → p89) and lamin A (p74 → p28) substrates demonstrate hierarchical activation of executioner caspases. (C and D) Detection of caspase 3/7 (DEVD), caspase 3/6/7 (VEID) and caspase 6 (EVEID) activity in STS-treated Jurkat T cells with AIE substrates. Cells were incubated with the appropriate AIE substrate for 2 hours, washed with PBS, stimulated with STS for 8 hours, and subjected to fluorescence readout (C) or confocal microscopy imaging (D). (E) Real-time imaging of caspase 3 activation in Jurkat T cells using Ac-DEVD-KTPE fluorogen. (F) Real-time imaging of caspase 6 activation in Jurkat T cells using Ac-EVEID-KTPE fluorogen. Panels E and F demonstrate hierarchical activation of caspase 3 followed by activation of caspase 6. An unpaired two-tailed T -test ( P < 0.05) was performed to demonstrate the statistical significance of the results (C).

Article Snippet: Other metal-tagged antibodies, anti-cleaved caspase 3 ( 172 Yb, clone 5 A1E, # 3172023) and anti-cleaved PARP ( 143 Nd, clone F21-852, # 3143011), were purchased from Fluidigm.

Techniques: Activation Assay, Imaging, Activity Assay, Incubation, Fluorescence, Confocal Microscopy, Two Tailed Test

Journal: Chemical Science

Article Title: Selective chemical reagents to investigate the role of caspase 6 in apoptosis in acute leukemia T cells †

doi: 10.1039/d2sc05827h

Figure Lengend Snippet: Analysis of caspase 6 activity by covalent inhibitors and mass cytometry. To assess the selectivity and potency of the designed inhibitors, we performed a set of cell-based studies using western blotting and single-cell mass cytometry analysis. (A) The kinetic parameters ( k obs / I , M −1 s −1 ) of tetra- and pentapeptide AOMK-based inhibitors toward recombinant apoptotic caspases. Inhibitors bearing VEID, hFVEID and EVEhFD peptides are the most potent inhibitors of caspase 6, whereas Ac-EVEID-AOMK is the most caspase 6-selective inhibitor. The k obs / I value of Ac-DEVD-AOMK for caspase-3 is presented in green as it is well of the scale ( k obs / I = 6 900 000 M −1 s −1 ). (B) The influence of small-molecule inhibitors on PARP and lamin A processing by caspases 3 and 6 in staurosporine-treated Jurkat T cells. The most potent caspase 6 inhibitors almost completely blocked lamin A cleavage, whereas most caspase 3 inhibitors failed to prevent PARP processing. (C) Schematic representation of the experimental setup to analyze caspase activation in mass cytometry format. Jurkat T cells were preincubated with inhibitors, treated with staurosporine and labeled with a cocktail of metal-tagged antibodies to dissect the processing of individual caspases and their substrates. We used four metal-tagged antibodies: anti-lamin A (142Nd), anti-cleaved PARP (143Nd), anti-caspase 6 (159Tb), and anti-cleaved caspase 3 (172Yb). (D) Mass cytometry diagram demonstrating PARP processing in Jurkat T cells (untreated, treated with staurosporine, and treated with staurosporine in the presence of caspase 3/7 Ac-DEVD-AOMK inhibitor). The results show that the DEVD inhibitor attenuates but does not fully prevent PARP processing. (E) Mass cytometry histograms demonstrating the processing of lamin A in the presence of caspase 3 (DEVD) and caspase 6 (hFVEID) inhibitors. The data demonstrate that the hFVEID inhibitor, but not the DEVD inhibitor, prevents lamin A processing, which is in line with western blotting analysis. (F) Mass cytometry histograms demonstrating the labeling of caspase substrates lamin A (upper) and cleaved PARP (lower) in staurosporine-treated Jurkat T cells in the presence of various inhibitors. The data demonstrate that VEID, hFVEID and IETD inhibitors, targeting mainly caspase 6, block lamin A processing, whereas the DEVD inhibitor attenuates but does not fully prevent PARP processing.

Article Snippet: Other metal-tagged antibodies, anti-cleaved caspase 3 ( 172 Yb, clone 5 A1E, # 3172023) and anti-cleaved PARP ( 143 Nd, clone F21-852, # 3143011), were purchased from Fluidigm.

Techniques: Activity Assay, Mass Cytometry, Western Blot, Recombinant, Activation Assay, Labeling, Blocking Assay

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Notoginsenoside R1 alleviates high glucose-evoked damage in RSC96 cells through down-regulation of miR-503.

doi: 10.1080/21691401.2019.1671434

Figure Lengend Snippet: Figure 1. RSC96 cells were injured by high glucose (HG). RSC96 cells were cultivated in HG condition for 48 h. Cells in normal glucose (NG) were considered as con- trol. (A) Cell viability, (B) apoptosis rate, (C,D) cleavage of caspase-3 and PARP, (E) ROS generation, and (F,G) expression of neurotrophic proteins were examined by CCK-8 assay, flow cytometry, Western blot and DCFH-DA probe. Data were presented as mean ± SD (n ¼ 3). p < .05 (Student’s t-test).

Article Snippet: Following primary antibodies against pro-caspase-3 (orb35276; dilution 1:1000), cleaved-caspase-3 (orb106556; dilution 1:1000), pro-PARP (orb88930; dilution 1:1000), cleaved-PARP (orb323122; dilution 1:2000), NGF (orb379540; dilution 1:1000), BDNF (orb519283; dilution 1:500), PI3K (orb395443; dilution 1:1000), p-PI3K (orb338965; dilution 1:500), AKT (orb29949; dilution 1:500), p-AKT (orb344403; dilution 1:1000), b-catenin (orb500798; dilution 1:1000) and b-actin (orb181785; dilution 1:500, Biorbyt, San Francisco, CA) were utilized in immunoblotting.

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Notoginsenoside R1 alleviates high glucose-evoked damage in RSC96 cells through down-regulation of miR-503.

doi: 10.1080/21691401.2019.1671434

Figure Lengend Snippet: Figure 2. Notoginsenoside R1 (NGR1) pre-conditioning ameliorated high glucose (HG)-evoked injury in RSC96 cells. (A) NGR1 with various dosages ranged from 10 to 100 lM was utilized to treat RSC96 cells. (B) RSC96 cells were pre-treated with NGR1 and then stimulated by HG. Cell viability was examined by CCK-8 kit. 50 lM was applied as optimum dosage for use in following experiments. (C) Apoptosis rate, (D,E) cleavage of caspase-3 and PARP, (F) ROS generation and (G,H) expression of neurotrophic proteins were examined by flow cytometry, Western blot and DCFH-DA probe. Data were presented as mean ± SD (n ¼ 3). p < .05 (ANOVA).

Article Snippet: Following primary antibodies against pro-caspase-3 (orb35276; dilution 1:1000), cleaved-caspase-3 (orb106556; dilution 1:1000), pro-PARP (orb88930; dilution 1:1000), cleaved-PARP (orb323122; dilution 1:2000), NGF (orb379540; dilution 1:1000), BDNF (orb519283; dilution 1:500), PI3K (orb395443; dilution 1:1000), p-PI3K (orb338965; dilution 1:500), AKT (orb29949; dilution 1:500), p-AKT (orb344403; dilution 1:1000), b-catenin (orb500798; dilution 1:1000) and b-actin (orb181785; dilution 1:500, Biorbyt, San Francisco, CA) were utilized in immunoblotting.

Techniques: CCK-8 Assay, Expressing, Flow Cytometry, Western Blot

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Notoginsenoside R1 alleviates high glucose-evoked damage in RSC96 cells through down-regulation of miR-503.

doi: 10.1080/21691401.2019.1671434

Figure Lengend Snippet: Figure 4. Notoginsenoside R1 (NGR1) pre-conditioning ameliorated high glucose (HG)-evoked injury in RSC96 cells through miR-503. (A) miR-503 mimic and its respective control (NC mimic) were transfected into RSC96 cells. Transfection efficiency was examined by qRT-PCR. The transfected or non-transfected cells were then treated by NGR1, HG or both. (B) Cell viability, (C) apoptosis rate, (D,E) cleavage of caspase-3 and PARP, (F) ROS generation, and (G,H) expression of neuro- trophic proteins were examined by CCK-8 assay, flow cytometry, Western blot and DCFH-DA probe. Data were presented as mean ± SD (n ¼ 3). p < .05 (ANOVA).

Article Snippet: Following primary antibodies against pro-caspase-3 (orb35276; dilution 1:1000), cleaved-caspase-3 (orb106556; dilution 1:1000), pro-PARP (orb88930; dilution 1:1000), cleaved-PARP (orb323122; dilution 1:2000), NGF (orb379540; dilution 1:1000), BDNF (orb519283; dilution 1:500), PI3K (orb395443; dilution 1:1000), p-PI3K (orb338965; dilution 1:500), AKT (orb29949; dilution 1:500), p-AKT (orb344403; dilution 1:1000), b-catenin (orb500798; dilution 1:1000) and b-actin (orb181785; dilution 1:500, Biorbyt, San Francisco, CA) were utilized in immunoblotting.

Techniques: Control, Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Indian Journal of Experimental Biology

Article Title: Apoptotic and antiproliferative effects of Urtica dioica L. extract on K562 chronic myeloid leukemia cell line

doi: 10.56042/ijeb.v61i11.1574

Figure Lengend Snippet: Fig. 5 — (A) Representative immunoblot image; and (B) densitometric analysis showing cleaved PARP protein expression in K562 cells. The Western Blot analyse was carried out using primary antibody for (89 kD)-cleaved-PARP fragment after treatment with Urtica dioica extract at the end of 24 h incubation. β-actin protein was used as the loading control.

Article Snippet: Primary antibodies used in the study were β-actin (Novus Biologicals, NB600-501), Bax (SPM336, NBP2-32809), Bcl-2 (100/D5, NBP2-15200) and cleaved PARP (194C1439, NBP2-27335) monoclonal antibodies produced in mouse (Novus Biologicals).

Techniques: Western Blot, Expressing, Incubation, Control

Journal: Cells

Article Title: New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection

doi: 10.3390/cells10030599

Figure Lengend Snippet: Response to 16 h LPS treatment of bovine PBMC. ( A ) Representative histograms overlay of PAR content in control (black line) and LPS treated PBMC (green line). ( B ) Histogram showing the decrease in MFI PAR (Geometric mean) in PBMC in response to 16h LPS treatment ( p = 0.048). ( C ) Histogram overlay showing the amount of active caspase-3 positive PBMC; ( D ) histogram overlay showing the amount of cleaved PARP-1 positive PBMC. ( E ) Percentage of active Caspase-3 positive cells in control and LPS treated PBMC. ( F ) Percentage of cleaved-PARP-1 positive cells in control and LPS treated PBMC. Results are presented as mean ± SD of n = 3 cows. * Significant differences with p < 0.05, paired Student’s t -test.

Article Snippet: In order to verify apoptotic inactivation of PARP after prolonged LPS stimulation, we evaluated the activation of Caspase-3 and the cleavage of PARP-1 by using the PE- conjugated anti-active Caspase-3 (clone C92-605—Becton Dickinson) and the FITC- conjugated anti-cleaved PARP-1 (Asp214) (clone F21-852, BD—Becton Dickinson) monoclonal antibodies, respectively.

Techniques:

Journal: Cells

Article Title: New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection

doi: 10.3390/cells10030599

Figure Lengend Snippet: Flow cytometric evaluation of PAR in bovine milk leukocytes and PARP-1 activation in milk cells during infection. At the top, representative flow cytometry dot plots showing the gating strategy for the identification of leukocyte subpopulations: ( A ) Identification of single cells on FSC-H vs. FSC-A dot-plot, ( B ) identification of milk leukocytes (CD45 + ) in SSC vs. PE dot-plot, ( C ) identification of lymphocytes (CD14 − ) neutrophils (CD14 low/− ) and macrophages (CD14 + ) in SSC vs. Per-CP dot-plot. In the middle, histograms overlay showing PAR levels of the three different cell populations: ( D ) Lymphocytes, ( E ) macrophages, and ( F ) neutrophils. Below, box-whisker plots (median, 25th–75th percentiles, maximum and minimum values) showing the PAR MFI differences of the leukocyte subpopulations between not infected (NI, n = 9) and infected (I, n = 10) milk samples ( G ), lymphocytes; ( H ), macrophages, ( I ), neutrophils). A significant increase in PAR levels was observed in lymphocytes ( p = 0.02) and macrophages ( p < 0.01) in infected conditions after the unpaired Student’s t -test. * Significant differences with p < 0.05.

Article Snippet: In order to verify apoptotic inactivation of PARP after prolonged LPS stimulation, we evaluated the activation of Caspase-3 and the cleavage of PARP-1 by using the PE- conjugated anti-active Caspase-3 (clone C92-605—Becton Dickinson) and the FITC- conjugated anti-cleaved PARP-1 (Asp214) (clone F21-852, BD—Becton Dickinson) monoclonal antibodies, respectively.

Techniques: Activation Assay, Infection, Flow Cytometry, Whisker Assay