Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 1 The effects of DIL on histological and immunohistochemical analyses of cardiac muscular tissue in rats with MI/R injury. (A) Hematoxylin and eosin (HE) staining of cardiac muscular tissue in the three groups. Original magnification: ×100, scale bar = 100 μm (upper row); ×200, scale bar = 50 μm (lower row). (B) Representative images of immunohistochemical (IHC) staining of DUSP1, JNK, and BNIP3L/NIX in the three groups. (C–E) Comparison of the average IOD values of DUSP1, JNK, and BNIP3L/ NIX expression in myocardial tissue among the three groups determined by IHC analysis. **P < 0.01 relative to the sham group, ##P < 0.01 relative to the MI/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Comparison, Expressing, Paraffin-embedded Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 5 RT‒qPCR analysis of the mRNA levels of Beclin 1, DUSP1, JNK, and BNIP3L/NIX in the control, OGD/R, and OGD/R + DIL groups. Compared to those in the control group, the expression levels of Beclin 1 (A), JNK (C) and BNIP3L/NIX (D) were significantly greater in the OGD/R group, but only DUSP1 expression (B) was significantly lower (all P < 0.01). Remarkably, this effect could be reversed by DIL intervention (all P < 0.01). **P < 0.01 relative to the control group; ##P < 0.01 relative to the OGD/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Control, Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 6 Western blot analyses of the Beclin 1, LC3B, DUSP1, JNK, and BNIP3L/NIX proteins in the control, OGD/R, and OGD/R + DIL groups. (A) The electrophoresis results of mitophagy-related proteins from the Western blot. The analyses indicated significantly increased protein expression of Beclin 1 (B), LC3B (C), JNK (E), and BNIP3L/NIX (F) and decreased DUSP1 protein expression (D) in the OGD/R group compared to the control group. DIL intervention reversed these changes (all P < 0.05). *P < 0.05, **P < 0.01 relative to the control group; #P < 0.05, ##P < 0.01 relative to the OGD/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Western Blot, Control, Electrophoresis, Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 7 RT‒qPCR analysis of the mRNA levels of the DUSP1, JNK, and BNIP3L/NIX genes after treatment with DUSP1, JNK, and BNIP3L/NIX inhibitors or activators in the different groups. (A) The DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (B) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (C) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/ NIX expression in the OGD/R+DIL or OGD/R+BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R +MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 8 Western blot analyses of DUSP1, JNK, and BNIP3L/NIX after treatment with DUSP1, JNK and BNIP3L/NIX inhibitors or activators in the different groups. (A and B) Compared with that in the OGD/R group, the DUSP1 gene expression level was significantly upregulated in the OGD/R+DIL and OGD/R+CTB (an activator of DUSP1) groups (P < 0.01). Moreover, the DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (C and D) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (E and F) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/NIX expression in the OGD/R+DIL or OGD/R +BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R+MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Western Blot, Gene Expression, Expressing

Journal: Journal of Neuroscience

Article Title: Breakdown of Axonal Synaptic Vesicle Precursor Transport by Microglial Nitric Oxide

doi: 10.1523/jneurosci.3887-04.2005

Figure Lengend Snippet: Figure 4. Immunohistochemistry of phosphorylated JNK in cultured hippocampal neurons. a, Neurons were identified by antibodies directed against the axonal marker protein tau (green). Only rare punctate staining of phosphorylated JNK (red) was detected in untreated neurons. Scale bar, 10 m. b, Phosphorylated JNK (red) was observed in most tau (green)-positive axons after treatment of the cultures with 300 M DEA/NONOate for 5 min. Area selected for inset as indicated. Scale bar, 10 m. c, Percentageofaxonsidentifiedbyimmunolabelingwithantibodiesdirectedagainsttauthatshowdouble-labelingforphosphor- ylatedJNK.Neuronsareeitheruntreatedortreatedwith300MDEA/NONOatefor5,10,or15min.Dataarepresentedasmean SEM. **p 0.005.

Article Snippet: Hippocampal neurons cultured for 1 week were fixed with 4% paraformaldehyde, incubated with a mouse monoclonal antibody specific for phosphorylated JNK (2 g/ml; Santa Cruz Biotechnology, Santa Cruz, CA), followed by secondary fluorochrome Cy3-conjugated goat antibody directed against mouse IgG (2 g/ml; Dianova).

Techniques: Immunohistochemistry, Cell Culture, Marker, Staining