Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 1. Effect of the caspase inhibitor Q-VD-OPh on activated caspase-3. Representative blot demonstrating complete inhibition of caspase-3 protein expression by Q-VD-OPh at a concentration of 50 μM, compared with DMSO or saline in mouse RTECs sub- jected to CS/REW. β-actin is used as a protein loading control.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Inhibition, Expressing, Concentration Assay, Saline, Control

Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 3. Effect of the caspase inhibitor Q-VD-OPh on caspase-3 and caspase-1 activity. A, Q-VD-OPh significantly reduced caspase-3 ac- tivity in mouse RTECs exposed to CS/REW compared with saline or DMSO. (*P < 0.001 vs Q-VD-OPh, *P < 0.01 vs DMSO and #P < 0.001 vs Q-VD-OPh). B, RTECs exposed to CS/REW in presence of saline, DMSO or Q-VD-OPh had no significant change in caspase-1 activity. (P = not significant). Data were analyzed using 1-way ANOVA and post–multiple-comparison Newman-Keuls test. Values are means ± SEM. n = 4 in each group.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Activity Assay, Saline, Comparison

Journal: Transplantation

Article Title: Caspase Inhibition During Cold Storage Improves Graft Function and Histology in a Murine Kidney Transplant Model

doi: 10.1097/tp.0000000000002218

Figure Lengend Snippet: FIGURE 2. Effect of the caspase inhibitor Q-VD-OPh on tubular cell death. A, Representative flow cytometry images of mouse RTECs ex- posed to CS/REW in presence of saline, DMSO or Q-VD-OPh. B, Cell death percentage was calculated after detection of annexin V–positive and/or PI-positive cells. Cell death was significantly decreased by Q-VD-OPh vs saline or DMSO in RTECs subjected to CS/REW (*P < 0.001 vs Q-VD-OPh; *P < 0.05 vs DMSO and **P < 0.001 vs Q-VD-OPh). Data were analyzed using 1-way ANOVA and post–multiple-comparison Newman-Keuls test. Values are means ± SEM. n = 4 in each group.

Article Snippet: M-1 (ATCC CRL-2038) RTECs were subjected to CS in cold saline for 24 hours at 4°C and rewarmed (REW) in normal media at 37°C for 24 hours as previously described.16-18 Renal tubular epithelial cells were incubated with either dimethyl sulfoxide (DMSO) or a caspase inhibitor Q-VD-OPh (R&D systems, OPH001) at a final concentration of 50 μM during CS/REW.

Techniques: Flow Cytometry, Saline, Comparison

Journal: Frontiers in Immunology

Article Title: Dynamics of necroptosis in kidney ischemia-reperfusion injury

doi: 10.3389/fimmu.2023.1251452

Figure Lengend Snippet: Comparison of cleaved Caspase-3 staining in WT and Mlkl -ko mice at 24 hrs post-reperfusion. (A) : WT; (B) : Mlkl -ko; (C) : sham; (D) : semi-quantitative analysis. Mann Whitney U test with * p<0.05 compared to sham procedure and # p<0.05 compared to WT mice. Data presented as mean ± SEM. Image magnification x 200.

Article Snippet: Sections were then incubated with rabbit monoclonal anti-phospho-MLKL (pSer345) (ab196436, 1:100, Abcam, Melbourne, Australia) or rabbit monoclonal anti-human/mouse cleaved Caspase-3 (cC3) (Asp175) (IC835G, 1:200, RD Systems, Noble Park, Australia) in 2% swine serum at 4°C overnight.

Techniques: Comparison, Staining, MANN-WHITNEY

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 1. TNF or LPS activates IKK. (A) DPSCs were treated with TNF (10 ng/mL) and P. gingivalis LPS (1 g/mL) for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against IB (1:1000) or monoclonal antibodies against phospho-specific antibodies (1:1000). For loading control, the blots were stripped and re-examined with monoclonal antibodies against - tubulin (1:15, 000). (B) E. coli LPS activates IKK. DPSCs were treated with E. coli LPS (200 ng/mL) or TNF for the indicated time points. Western blot analysis was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Bioprocessing, Control, Western Blot

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 2. TNF or LPS induces the nuclear translocation of NF-B. (A) DPSCs were treated with TNF (10 ng/mL) for the indicated time. Five- g aliquots of nuclear proteins were incubated with 32P-labeled NF-B oligonucleotide probes. For the supershift assay, nuclear proteins were pre-incubated with polyclonal antibodies against p65 for 10 min and then probed with32P-labeled NF-B oligonucleotide probes. (B) DPSCs were treated with P. gingivalis LPS (1 g/mL) for the indicated times. EMSA was performed as described in (A).

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Translocation Assay, Incubation, Labeling

Journal: Journal of dental research

Article Title: NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

doi: 10.1177/154405910508401105

Figure Lengend Snippet: Figure 3. LPS induces IL-8 expression in DPSCs. (A) DPSCs were treated with TNF or P. gingivalis LPS for the indicated times. Fifty-g aliquots of protein extracts were probed with polyclonal antibodies against phospho-specific p65. For loading control, the membrane was stripped and re-probed with polyclonal antibodies against p65. (B) DPSCs were treated with TNF for the indicated times. Ten-g aliquots of total RNAs were hybridized with 32P-labeled IL-8 cDNA probes. For loading control, the blot was stripped and re-probed with 32P-labeled glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA probes. (C) DPSCs were treated with P. gingivalis LPS for the indicated times. Northern blot analysis was performed as described in (B). (D) DPSCs were treated with TNF for the indicated times. Total RNAs were probed with 32P-labeled IL- 6 cDNA probes. (E) DPSCs were treated with E. coli LPS for the indicated times. Total RNAs were probed with 32P-labeded IL-6 cDNA probes.

Article Snippet: For the super-shift assay to confirm NF- B-binding specificity, we added 1 L of rabbit polyclonal antibodies against p65 subunit (Rockland) of NF- B to nuclear extracts for 15 min prior to the addition of poly(dI-dC)poly(dI-dC) and a 32P-labeled oligonucleotide probe, and then separated them on 5% polyacrylamide gels (Wang et al., 1999a,b).

Techniques: Expressing, Control, Membrane, Labeling, Northern Blot