Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: Microglial depletion inhibits phosphorylated STAT3 (pSTAT3) in astrocytes in spinal cord tissue. (A) Western blotting bands of STAT3 and pSTAT3 at 7 post-injury (dpi). (B) Quantification of pSTAT3 (one-way analysis of variance with Tukey’s post hoc test, n = 3). Data are expressed as the mean ± SD. (C) Double immunofluorescence staining of pSTAT3 with various markers at 7 dpi. Arrowheads indicate double-stained cells. Scale bars: 50 μm. (D) Colony-stimulating factor 1 receptor inhibitor PLX3397 treatment significantly decreased the proportion of pSTAT3 + astrocytes (unpaired t -test, n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001. Western blot experiments were repeated three times. Immunofluorescence staining was repeated five times. GFAP: Glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: The primary antibodies mouse anti-GFAP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-33673), rabbit anti-signal transducers and activators of transcription 3 (STAT3; 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9139), rabbit anti-phospho-STAT3 (p-STAT3; 1:2000, Cell Signaling Technology, Cat# 9145S), and rabbit anti-β-actin (Sigma, Cat# A5441) were incubated at 4°C overnight.

Techniques: Western Blot, Double Immunofluorescence Staining, Staining, Immunofluorescence, Binding Assay

Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: LPS-activated microglia promote astrocyte proliferation in vitro . (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU + /GFAP + astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: The primary antibodies mouse anti-GFAP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-33673), rabbit anti-signal transducers and activators of transcription 3 (STAT3; 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9139), rabbit anti-phospho-STAT3 (p-STAT3; 1:2000, Cell Signaling Technology, Cat# 9145S), and rabbit anti-β-actin (Sigma, Cat# A5441) were incubated at 4°C overnight.

Techniques: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: The number of mice in each experimental group

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: Western Blot, Staining, Concentration Assay

Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: Microglial depletion inhibits phosphorylated STAT3 (pSTAT3) in astrocytes in spinal cord tissue. (A) Western blotting bands of STAT3 and pSTAT3 at 7 post-injury (dpi). (B) Quantification of pSTAT3 (one-way analysis of variance with Tukey’s post hoc test, n = 3). Data are expressed as the mean ± SD. (C) Double immunofluorescence staining of pSTAT3 with various markers at 7 dpi. Arrowheads indicate double-stained cells. Scale bars: 50 μm. (D) Colony-stimulating factor 1 receptor inhibitor PLX3397 treatment significantly decreased the proportion of pSTAT3 + astrocytes (unpaired t -test, n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001. Western blot experiments were repeated three times. Immunofluorescence staining was repeated five times. GFAP: Glial fibrillary acidic protein; Iba1: ionized calcium binding adaptor molecule 1; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: Western Blot, Double Immunofluorescence Staining, Staining, Immunofluorescence, Binding Assay

Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: LPS-activated microglia promote astrocyte proliferation in vitro . (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU + /GFAP + astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

doi: 10.4103/1673-5374.355978

Figure Lengend Snippet: Argon preconditioning activates ERK1/2 and inhibits NF-κB phosphorylation – OxPAP partially reverses the argon effect part. Evaluation of ERK1/2 and NF-κB phosphorylation by western blotting. (A) Densitometric analysis of western blots of ERK1/2 phosphorylation, normalized against total-ERK1/2. (B) Representative western blot image of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on ERK1/2 phosphorylation. (C) Densitometric analysis of western blots of NF-κB phosphorylation, normalized against total NF-κB. (D) Representative western blot image of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on NF-κB phosphorylation. Between-group comparisons were performed with a one-way analysis of variance using the post hoc Holm Sidak test ( n = 6; mean ± SD; * P < 0.05, ** P < 0.01). ERK1/2: Extracellular-signal regulated kinase; NF-κB: nuclear transcription factor-κB.

Article Snippet: After protein transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Schwalbach, Germany), the membranes were blocked with 5% skim milk in Tween20/PBS (TBST) or BSA (bovine serum albumin) and incubated in the recommended dilution of protein-specific antibody (p44/42 MAP kinase (phospho-ERK1/2 [extracellular-signal regulated kinase]) (Thr202/Tyr204), rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9101, RRID: AB_331646; phospho-NF-κB (Ser536) (93H1), rabbit, 1:1000, Cell Signaling Technology, Cat# 3033, RRID: AB_331284; phospho-Akt (protein kinase B) antibody (Ser473), rabbit, 1:1000, Cell Signaling Technology, Cat# 9271, RRID: AB_329825; Bax antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2772, RRID: AB_10695870; Bcl-2 antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2876, RRID: AB_2064177; and cleaved caspase-3 antibody (Asp175), rabbit, 1:500, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188; NF-E2-related factor 2 (Nrf2) antibody [nuclear factor erythroid 2-related factor 2, phosphor-S40], rabbit, 1:60000, Abcam, Cat# ab76026, RRID: AB_1524049).

Techniques: Western Blot, Inhibition

Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of thrombin/protease-activated receptor 1 on the mitogen-activated protein kinase (MAPK)/nuclear factor kappa-B (NFκB) pathway in astrocytes. (A) Western blot analysis of phosphorylated extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38 kinase (P38), and NFκB protein levels following astrocyte stimulation with 0, 0.5, 1, or 2 U/mL thrombin for 24 hours. (B–E) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. (F) Western blot analysis of phosphorylated ERK, JNK, P38, and NFκB protein levels following astrocyte stimulation with 1 U/mL thrombin in the presence of 0–3 μM SCH79797 for 24 hours. (G–J) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). SCH79797: Protease-activated receptor 1 inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of mitogen-activated protein kinase (MAPK) or nuclear factor kappa-B (NFκB) inhibitors on astrocyte cholesterol-25-hydroxylase (CH25H) expression in response to stimulation with thrombin. (A) Western blot analysis of NFκB expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (B) Quantification of NFκB protein expression, normalized to β-actin. (C) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (D) Quantification of CH25H expression, normalized to β-actin. (E) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence or absence of 10–100 μM of the NFκB inhibitor PDTC for 24 hours. (F) Quantification of CH25H expression, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). DMSO: Dimethyl sulfoxide; PD98059: extracellular regulated protein kinases inhibitor; SB203580: P38 inhibitor; SP600125: c-Jun N-terminal kinase inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Expressing, Western Blot

Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI attenuates inflammation through the p38–NF-κB signaling pathway. BMDMs were stimulated by LPS in the presence of DMSO or BCI, and the treated cells were used for qRT-PCR, immunostaining and western blotting. (A) qRT-PCR showing expression levels of LPS-induced Inos , Il1b , Il6 , Il12b and Cd14 mRNAs in BMDMs with DMSO or BCI ( n =3 per group). (B) qRT-PCR showing expression levels of LPS-induced Mcp1 , Ccl4 , Ccr2 and Cxcl9 mRNAs in BMDMs with DMSO or BCI ( n =3 per group). (C) Western blots and quantification of LPS-induced p-p65 and p65 in BMDMs with DMSO or BCI treatment ( n =3 per group). (D) Immunofluorescent staining and quantification of LPS-induced p-p65 (green) in BMDMs with DMSO or BCI treatment ( n =3 per group; scale bars: 20 μm). (E) Immunofluorescent staining and quantification of LPS-induced reactive oxygen species measured by dihydrorhodamine 123 (DHR123) (green) in BMDMs with DMSO or BCI treatment ( n =3 per group; scale bars: 20 μm). (F-H) Western blots and quantification of LPS-induced p-ERK (F), p-p38 (G) and p-JNK (H) in BMDMs with DMSO or BCI treatment ( n =3 per group). One-way ANOVA followed by Dunnett's multiple comparison test; mean±s.e.m.; * P <0.05, ** P <0.01, *** P <0.001; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Quantitative RT-PCR, Immunostaining, Western Blot, Expressing, Staining