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: For normalization, blots were re-probed with p44/42 MAPK (mitogen-activated protein kinase, ERK1/2) antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 9102, RRID: AB_330744), NF-κB p65 (D14E12) (rabbit, 1:1000, Cell Signaling Technology, Cat# 8242, RRID: AB_10859369), Akt antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 9272, RRID: AB_329827), caspase-3 antibody (rabbit, 1:4000, Cell Signaling Technology, Cat# 9662, RRID: AB_331439), β-actin antibody (rabbit, 1:1000, Cell Signaling Technology, Cat# 4967, RRID: 330288), and NFE2L2 (rabbit, 1:1000, Abcam, Cat# ab62352, RRID: AB_944418).

Techniques: Western Blot, Inhibition

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: PLOS ONE

Article Title: Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo

doi: 10.1371/journal.pone.0281063

Figure Lengend Snippet: A, heat map of results of RPPA analyses done to detect factors that regulate CRC cell proliferation and apoptosis in different KRAS-mutated CRC cell lines following treatment with trametinib (T), dasatinib (D), or both (T+D) for 48 hours. C, control. B , Western blots performed to detect the apoptosis markers cleaved PARP, total PARP, cleaved caspase 3, and total caspase 3 in multiple KRAS-mutated CRC cell lines. Vinculin was used as a loading control. The numbers below the blots denote the cleaved protein levels relative to total protein levels in each sample (Control cells standardized to 1).

Article Snippet: Antibodies against pERK1/2 (cat. #9101; RRID:AB_331646), ERK1/2 (cat. #4695; RRID:AB_390779), pAKT (cat. #9271; RRID:AB_329825), AKT (cat. #9272; RRID:AB_329827), pSRC (cat. #6943; RRID:AB_10013641), SRC (cat. #2123; RRID:AB_2106047), pFAK (cat. #3284; RRID:AB_10831810), FAK (cat. #3285; RRID:AB_2269034), pMEK (cat. #9154; RRID:AB_2138017), MEK (cat. #9126; RRID:AB_331778), PARP (cat. #9542; RRID:AB_2160739), cleaved PARP (cat. #9541; RRID:AB_331426), caspase 3 (cat. #9665; RRID:AB_2069872), and cleaved caspase 3 (cat. #9661; RRID:AB_2341188) were obtained from Cell Signaling Technology.

Techniques: Western Blot

Journal: The American Journal of Pathology

Article Title: Inhibition of a Microbiota-derived Peptide Ameliorates Established Acute Lung Injury

doi: 10.1016/j.ajpath.2023.03.003

Figure Lengend Snippet: Inhibition of apoptosis in mice with established acute lung injury treated with anticorisin monoclonal antibody. Mice received intratracheal instillation of lipopolysaccharide (LPS; 100 μg) or saline (SAL) and then treatment with anticorisin monoclonal antibody or control IgG. Mice receiving intratracheal saline (SAL) and treated with control IgG were the control mice. A, B, β-actin, noncleaved caspase-3 (casp-3) and the activation of caspase-3 were evaluated using specific antibodies by Western blotting. Number of mice in each group: n=5 in SAL/SAL group, n=5 in control LPS/control IgG group, and n=5 in LPS/anticorisin group. C, D, DNA fragmentation was evaluated by TUNEL method as described under materials and methods. Scale bars indicate 50 μm. Black arrows indicate TUNEL (+) cells. indicate Number of mice in each group: n=3 in SAL/SAL group, n=7 in LPS/control IgG group, and n=5 in LPS/anticorisin group. Data are expressed as the mean ± S.D. Statistical analysis by ANOVA with Neuman-Keuls test. *p<0.05; **p<0.01

Article Snippet: Nitrocellulose membranes, anti-cleaved caspase-3, anti-noncleaved caspase-3, or anti-βactin antibodies from Cell Signaling (Danvers, MA) were used to perform Western blotting.

Techniques: Inhibition, Activation Assay, Western Blot, TUNEL Assay