Journal: Pathogens

Article Title: Hydrogen Sulfide (H2S) Mitigates Sepsis-Induced Adrenal Dysfunction via Inhibition of TNFα-Mediated Necroptosis

doi: 10.3390/pathogens14050439

Figure Lengend Snippet: Figure 1. The role of necroptosis in sepsis mice. (A) Hematoxylin and eosin (H&E) staining of tissue sections from the control and cecal ligation and puncture (CLP) groups. Scale bar: 50 µm. (B) Inflammatory factor expression levels in the control and CLP groups. Data are presented as mean ± standard deviation (SD). **** p < 0.0001 indicates statistically significant differences compared to the control group (n = 6 in each group). (C) Western blot analysis of RIPK1, P-RIPK1, MLKL, P-MLKL, and β-Actin in the wild-type (WT) and CLP groups (n = 6 in each group). (D) Western blot analysis of MLKL in the WT and conditional knockout (MLKL-KO) groups. Data are presented as mean ± standard deviation (SD). **** p < 0.0001 indicates statistically significant differences compared to the control group. (E) H&E staining of tissue sections from the control, CLP, and MLKL-KO + CLP groups. Scale bar: 50 µm. (F) Inflammatory factor expression levels in the control, CLP, and MLKL-KO + CLP groups. Data are presented as mean ± standard deviation (SD). ** p < 0.01 and **** p < 0.0001 indicate statistically significant differences compared to the CLP group (n = 6 in each group).

Article Snippet: After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h, the samples were incubated with antibodies against MLKL (Proteintech, Wuhan, China), P-MLKL (Proteintech, Wuhan, China), RIPK1 (Proteintech, Wuhan, China), P-RIPK1 (Proteintech, Wuhan, China), or β-Actin (Proteintech, Wuhan, China) overnight at 4 C at a dilution of 1:500 or 1:1000.

Techniques: Staining, Control, Ligation, Expressing, Standard Deviation, Western Blot, Knock-Out

Journal: Pathogens

Article Title: Hydrogen Sulfide (H2S) Mitigates Sepsis-Induced Adrenal Dysfunction via Inhibition of TNFα-Mediated Necroptosis

doi: 10.3390/pathogens14050439

Figure Lengend Snippet: Figure 2. The role of necroptosis in Y1 cells. (A) Bar graph showing the viability of Y1 cells treated with PBS or TNFα over time (0, 24, 48, and 72 h). Data are presented as percentage of control (untreated cells). ** p < 0.01 and **** p < 0.0001 indicate statistically significant differences compared to the control group (n = 3 in each group). (B) Western blot analysis of P-RIPK1, total RIPK1, P- MLKL, total MLKL, and β-Actin in Y1 cells treated with PBS or TNFα for 48 h (n = 3 in each group). (C) Bar graph showing the viability of Y1 cells treated with PBS, TNFα, or TNFα plus NSA over time (0, 24, 48, and 72 h). Data are presented as percentage of control (untreated cells). ** p < 0.01 and **** p < 0.0001 indicate statistically significant differences compared to the control group (n = 3 in each group).

Article Snippet: After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h, the samples were incubated with antibodies against MLKL (Proteintech, Wuhan, China), P-MLKL (Proteintech, Wuhan, China), RIPK1 (Proteintech, Wuhan, China), P-RIPK1 (Proteintech, Wuhan, China), or β-Actin (Proteintech, Wuhan, China) overnight at 4 C at a dilution of 1:500 or 1:1000.

Techniques: Control, Western Blot

Journal: Pathogens

Article Title: Hydrogen Sulfide (H2S) Mitigates Sepsis-Induced Adrenal Dysfunction via Inhibition of TNFα-Mediated Necroptosis

doi: 10.3390/pathogens14050439

Figure Lengend Snippet: Figure 3. The important role of H2S. (A) Western blot analysis of CBS and β-Actin protein lev- els in wild-type (WT) and CBS-KO samples. Data are presented as percentage of control (WT mice). **** p < 0.0001 indicates statistically significant differences compared to the control (n = 6 in each group). (B) H&E staining of tissue sections from the CLP and CKO + CLP groups. Scale bar: 50 µm. (C) Inflammatory factor expression levels in the control, CLP, CBS-KO + CLP, and CBS-KO + CLP + NsHS (10 mg/kg) groups. Data are presented as mean ± standard deviation (SD). *** p < 0.001 and **** p < 0.0001 indicate statistically significant differences compared to the CBS-KO + CLP group (n = 6 in each group). (D) Western blot showing the protein expression levels of P-RIPK1, RIPK1, P-MLKL, MLKL, and β-Actin in the WT, CBS-KO, CLP, CBS-KO + CLP, and CBS-KO + CLP + NaHS (1 mg/kg, 10 mg/kg) groups (n = 6 in each group).

Article Snippet: After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h, the samples were incubated with antibodies against MLKL (Proteintech, Wuhan, China), P-MLKL (Proteintech, Wuhan, China), RIPK1 (Proteintech, Wuhan, China), P-RIPK1 (Proteintech, Wuhan, China), or β-Actin (Proteintech, Wuhan, China) overnight at 4 C at a dilution of 1:500 or 1:1000.

Techniques: Western Blot, Control, Staining, Expressing, Standard Deviation

Journal: Pathogens

Article Title: Hydrogen Sulfide (H2S) Mitigates Sepsis-Induced Adrenal Dysfunction via Inhibition of TNFα-Mediated Necroptosis

doi: 10.3390/pathogens14050439

Figure Lengend Snippet: Figure 4. The important role of H2S in Y1 cells. (A) Bar graphs showing the cell viability of Y1 cells treated with PBS, TNFα, and TNFα combined with NaHS (10 µM) over different time periods (0, 24, 48, and 72 h). Data are presented as percentage of control (untreated cells). * p < 0.05, *** p < 0.001, and **** p < 0.0001 indicate statistically significant differences compared to the control (n = 3 in each group). (B) Western blot analysis of protein expression levels of P-RIPK1, RIPK1, P-MLKL, MLKL, and β-Actin in Y1 cells treated with TNFα and NaHS (1 µM, 10 µM, and 100 µM). The blots show the changes in protein expression under different treatment conditions (n = 3 in each group). (C) Immunofluorescence images showing the localization of P-MLKL in Y1 cells treated with PBS, TNFα, and TNFα combined with NaHS. The images demonstrate the effect of these treatments on P-MLKL distribution within the cells. Scale bar: 200 µm. (D) Bar graphs show the cell viability of Y1 cells treated with PBS, TNFα, and TNFα combined with AOAA over different time periods (0, 24, 48, and 72 h). Data are presented as percentage of control (untreated cells). ** p < 0.01 and **** p < 0.0001 indicate statistically significant differences compared to the control (n = 3 in each group). (E) Western blot analysis of protein expression levels of P-RIPK1, RIPK1, P-MLKL, MLKL, and β-Actin in Y1 cells treated with TNFα, AOAA, and NaHS (10 µM). The blots show the changes in protein expression under different treatment conditions (n = 3 in each group).

Article Snippet: After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h, the samples were incubated with antibodies against MLKL (Proteintech, Wuhan, China), P-MLKL (Proteintech, Wuhan, China), RIPK1 (Proteintech, Wuhan, China), P-RIPK1 (Proteintech, Wuhan, China), or β-Actin (Proteintech, Wuhan, China) overnight at 4 C at a dilution of 1:500 or 1:1000.

Techniques: Control, Western Blot, Expressing, Immunofluorescence

Journal: Journal of Ophthalmology

Article Title: Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF- α

doi: 10.1155/2022/2285663

Figure Lengend Snippet: The DOR agonist TAN-67 suppresses TNF- α levels and the expression of necroptotic and apoptotic proteins in ARPE19 cells induced by OGD24/R36. (a) TNF- α levels in cell lysates from each group were measured by ELISA ( n = 5 per group). (b–c) The level of DOR expression was examined by Western blotting, and the density values were normalized to the levels of β -actin ( n = 3 per group). (d–k) Western blot showing the activation or total abundance of p-RIP1 (RIP1), p-RIP3 (RIP3), p-MLKL (MLKL), and cleaved Caspase-3 (Caspase-3) as indicated ( n = 3 per group). P-RIP1, phosphorylated RIP1; p-RIP3, phosphorylated RIP3; p-MLKL, phosphorylated MLKL. Data are presented as means ± SEM, and group differences were analyzed by one-way ANOVA with Dunnett's post hoc test. ## P < 0.01, ### P < 0.001 compared with the control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.01, compared with the OGD24/R36 group.

Article Snippet: The following primary antibodies were used in this study: RIP1 (1 : 2000, cell signaling, 45726, USA), p-RIP1 (1 : 2000, cell signaling, 53286, USA), RIP3 (1 : 2000, Abcam, ab226297, USA), p-RIP3 (1 : 2000, Abcam, ab195117, USA), MLKL (1 : 2000, Abcam, ab243142, USA), p-MLKL (1 : 2000, Abcam, ab187091, USA), Caspase3 (1 : 2000, cell signaling, 9662, USA), cleaved Caspase3 (1 : 2000, cell signaling, 9664, USA), DOR (1 : 2000, Millipore, AB1560, USA), and β -actin (1 : 2000, Sigma, A5441, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Control