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: 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