Journal: Journal of Biological Chemistry

Article Title: Mechanisms Underlying Neuronal Death Induced by Chromogranin A-activated Microglia

doi: 10.1074/jbc.m009711200

Figure Lengend Snippet: FIG. 3. Effect of conditioned medium from resting or chromogranin A-stimulated microglia on JNK and p38 MAPK in neurons. A, Western blotting showing the protein level of the total and phosphorylated forms of JNK and p38 MAP kinases in neurons incubated for 15 min with medium from microglia cultured without (2CGA) or with 10 nM CGA (1CGA). Triton X-100-soluble cytosolic (lane 1) and Triton X-100- insoluble cytoskeletal (lane 2) protein fractions from neurons were resolved by electrophoresis and transferred onto nitrocellulose, and blots were probed with antibodies against p46 JNK1 (JNK), phosphorylated JNK (pJNK), p38 MAP kinase (p38), and phosphorylated p38 MAP kinase (pp38). Immunodetection of actin (arrowhead) is shown as an internal standard for protein loading. The results shown are representative of four independent experiments. B, the activity of JNK or p38 MAPK was measured in immunoprecipitates from neurons exposed for the indicated period of time to CM from resting (2CGA) or CGA-stimulated (1CGA) microglia. To inhibit p38 MAPK, neurons were incubated for 1 h with 30 mM SB 203580 before the addition of CM. The radioactivity incorporated into the substrates was estimated after electrophoresis by optical scanning densitometry on the corresponding autoradiogram. Values are expressed relative to the kinase activities detected in neurons incubated for 15 min with CM from resting microglia. Results are representative of three independent experiments. Student’s t test was used for estimating significance: **, p , 0.001. C, neurons were incubated for 48 h with CM from resting (2CGA) or CGA-stimulated (1CGA) microglia in the presence of the indicated concentrations of SB 203580 added 1 h before treatment with CM. The number of apoptotic nuclei was assessed by Hoechst 33342 staining. Data are the mean percentage of apoptotic cells 6 S.E. and are representative of three independent experiments. Student’s t test results: * p , 0.01.

Article Snippet: The following antibodies to MAP kinases were used: anti-p38 MAP kinase, anti-phospho-specific p38 MAP kinase (Thr-180/Tyr-182), anti-c-Jun, anti-phospho-specific c-Jun (Ser-63) II (New England Biolabs, Beverley, MA), anti-p46 c-Jun N-terminal kinase 1 (JNK1) (C-17) (Santa Cruz, CA), and anti-active JNK pAb (Promega, Madison WI).

Techniques: Western Blot, Incubation, Cell Culture, Electrophoresis, Immunodetection, Activity Assay, Radioactivity, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Signaling pathways regulating VDAC1 overexpression associated with apoptosis, pyroptosis, and ferroptosis

doi: 10.1186/s12964-025-02647-5

Figure Lengend Snippet: Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA (si-NT) or si-c-Jun (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant

Article Snippet: The nucleotides in italic were 2′-O-methyl modified. si-c-JUN (CST-6203 S) and si-p38-MAPK (CST-6564 S) were purchased from Cell Signaling Technology (Danvers, MS).

Techniques: Over Expression, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Control, Transfection, Expressing