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: PLX3397 attenuates astrocytic scar formation and increases the inflammatory response following SCI. (A) Representative images of astrocytic scars in sagittal sections of the spinal cord at 14 days after spinal cord injury (SCI). In SCI mice, astrocytes surrounding the lesion area packed tightly together as an astrocytic scar barrier, and this astrocytic front was less prominent in mice with PLX3397 treatment. Scale bars: 50 μm. (B) In the SCI mice, the spread of CD68 + inflammatory cells was restricted by a dense glial scar, whereas the boundary of the PLX3397-treated mice was disrupted, and a significant infiltration of CD68 + cells was observed. Scale bars: 50 μm. (C) aCasp3/NeuN costaining in sagittal sections indicating apoptotic neurons. Scale bars: 50 μm. (D) PLX3397 treatment increased the lesion size (calculated as GFAP-negative staining area). Microglia elimination increased the concentration of the proinflammatory cytokines (E) TNF-α, (F) IL-6, and (G) IL-1β at 14 days post-injury (dpi). (H) The proportion of NeuN + /aCasp3 + cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test for E–G; unpaired t -test for D and H). Data are expressed as the mean ± SD. The experiments were repeated six times. aCasp3: Activated caspase-3; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; IL: interleukin; NeuN: neuronal nuclei; PLX3397: colony-stimulating factor 1 receptor inhibitor; SCI: spinal cord injury; TNF-α: tumor necrosis factor-α.

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: Negative Staining, Concentration Assay

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: Effect of Argon preconditioning on caspase-3 cleavage. (A) Evaluation of caspase-3 cleavage by western blotting. Densitometric analysis of western blots of caspase-3 cleavage, normalized against uncleaved caspase-3. 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, ** *P < 0.001). (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 caspase-3 cleavage.

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: 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 has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; 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: Western Blot, Staining

Journal: Oncology Reports

Article Title: Andrographolide suppresses aerobic glycolysis and induces apoptotic cell death by inhibiting pyruvate dehydrogenase kinase 1 expression

doi: 10.3892/or.2023.8509

Figure Lengend Snippet: List of antibodies used in the present study.

Article Snippet: Cleaved Caspase-3 , Cell Signaling Technology, Inc. , 9661 , 1:1,000 in 2% skim milk.

Techniques:

Journal: Oncology Reports

Article Title: Andrographolide suppresses aerobic glycolysis and induces apoptotic cell death by inhibiting pyruvate dehydrogenase kinase 1 expression

doi: 10.3892/or.2023.8509

Figure Lengend Snippet: AG induces more apoptotic cell death in H292 cells than A549 cells. (A and B) A549 and H292 cells were treated with various concentrations of AG (20, 50 and 75 µM) for 6 h, labeled with Annexin V-fluorescein isothiocyanate and propidium iodide, and analyzed via flow cytometry. (C) Histogram showing the percentage of apoptotic cells. Apoptotic cells were quantified by combining Q2 (late-stage of apoptotic cells, PI + /Annexin V + ) and Q3 (early-stage of apoptotic cells, PI − /Annexin V + ) regions after FACS analysis. AG significantly increased the apoptotic cell death in H292 cells, but not in A549 cells, in a dose-dependent manner. (D) To determine the protein levels of PARP, cleaved caspase-3, and cleaved caspase-9 via western blot analysis, A549 and H292 cells were treated with AG at the indicated dose for 24 h. Levels of all apoptotic marker proteins were significantly increased by AG treatment in H292 cells and only slightly increased in A549 cells. Data are represented as the mean ± SD compared with the control from three independent experiments. ***P<0.001 vs. control. AG, andrographolide; PARP, cleaved poly (ADP ribose) polymerase.

Article Snippet: Cleaved Caspase-3 , Cell Signaling Technology, Inc. , 9661 , 1:1,000 in 2% skim milk.

Techniques: Labeling, Flow Cytometry, Western Blot, Marker

Journal: Oncology Reports

Article Title: Andrographolide suppresses aerobic glycolysis and induces apoptotic cell death by inhibiting pyruvate dehydrogenase kinase 1 expression

doi: 10.3892/or.2023.8509

Figure Lengend Snippet: AG-induced cancer cell death is alleviated in PDK1-overexpressing H292 cells. (A) Overexpression of PDK1 protein in H292 cells was determined via western blotting. PDK1 expression vector was FLAG tagged to detect PDK1-overexpressing cells using an anti-FLAG antibody. (B) mRNA levels of PDK1 in H292 cells were determined via RT-qPCR. (C) After H292 cells were treated with AG, cell death was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. AG-induced cell death was alleviated in PDK1-overexpressing H292 cells. (D) Morphological alterations revealed that PDK1-overexpressing H292 cells showed reduced apoptosis compared with the control cells. (E) Numbers of dead cells (combination of the Q1 + Q2) after AG treatment was measured via FACS analysis. A histogram showing the percentage of dead cells is produced by the analysis of 10,000 cells after AG treatment in PDK1-overexpressing and empty vector cells. (F) Activation of apoptotic signals by PARP and caspase-3 cleavage was examined via western blotting. The cleavage of PARP and caspase-3 was slightly weak in PDK1-overexpressing H292 cells. GAPDH was used as the internal control. (G) Mechanism of AG-induced cancer cell death via PDK1 inhibition. Data are represented as the mean ± SD compared with the control from three independent experiments. **P<0.01 and ***P<0.001 compared with each group. AG, andrographolide; PDK, pyruvate dehydrogenase kinase; PARP, cleaved poly (ADP ribose) polymerase; EV, empty vector; OE, overexpressing.

Article Snippet: Cleaved Caspase-3 , Cell Signaling Technology, Inc. , 9661 , 1:1,000 in 2% skim milk.

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR, MTT Assay, Produced, Activation Assay, Inhibition

Journal: Gut Microbes

Article Title: Epithelial talin-1 protects mice from citrobacter rodentium -induced colitis by restricting bacterial crypt intrusion and enhancing t cell immunity

doi: 10.1080/19490976.2023.2192623

Figure Lengend Snippet: Loss of epithelial-specific talin-1 suppresses pathogen-induced epithelial apoptosis. (a) Representative images of colon tissues immunoperoxidase-stained for cleaved caspase-3. (b) the proportion of cleaved caspase-3-positive mucosa determined by measuring the total height of the mucosa and the height of the region with positive staining. Each dot represents measurements from a high-powered field; n = 12–15 fields from 3 different mice per group. All values reported with the median depicted as a thick line and the upper and lower quartiles as thin lines. (c) Expression of the gene encoding TNF-α analyzed by RT-qPCR. n = 4 uninfected mice and n = 6–7 infected mice per genotype. Each symbol is a different mouse. Values are reported as mean ± SEM. * P < 0.05 and ** P < 0.01 determined by (b) 1-way ANOVA and Tukey test and (c) 1-way ANOVA with Kruskal-Wallis test, followed by a Mann-Whitney U test. Scale bars represent 100 μm.

Article Snippet: Tissues were then incubated overnight at 4°C using the following antibodies: prediluted rabbit polyclonal anti-Ki-67 (Biocare, PRM325AA), prediluted rabbit monoclonal anti-MPO (Biocare, PP023AA), rabbit polyclonal anti-CD3 (Abcam, ab5690; 1:150), or rabbit monoclonal anti-cleaved caspase-3 (Cell Signaling, 9664; 1:400).

Techniques: Staining, Expressing, Quantitative RT-PCR, Infection, MANN-WHITNEY