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: Scientific Reports

Article Title: Mycophenolic acid directly protects podocytes by preserving the actin cytoskeleton and increasing cell survival

doi: 10.1038/s41598-023-31326-z

Figure Lengend Snippet: MPA increases cell viability during TNF-α and CHX induced cell death. ( A ) Cells were treated with either Vehicle or MPA (M) for 48 h, or Emricasan (E) for 24 h. All cells received Tumor Necrosis Factor-α (T) and Cycloheximide (C) for 24 h. An additional control group received only MPA for 48 h (M). This resulted in 5 treatment groups (Control, TC, TCM, TCE and M), which were analyzed with a cell viability assay. ( B ) Quantification of cell viability after treatment with combinations of Tumor Necrosis Factor-α (TNF-α; T), Cycloheximide (CHX; C), Mycophenolic Acid (MPA; M) and Emricasan (E). Control cells received only vehicles. Cell viability of each sample was normalized against the control of their respective passage. TC-treatment resulted in a drastic decrease of cell viability. Additional MPA treatment significantly increases cell viability compared to TC only (36.8 vs. 62.9%). Emricasan was able to prevent cell death in a very high degree (91%). Treatment with only MPA showed no signs for proliferative activity of the drug. ** p < 0.01 ( C ) Western Blot Analysis of cleaved Caspase-3 levels. Lane 1, Control. Lane 2, TC. Lane 3, TCM. Treatment with TC results in a steep increase of cleaved Caspase-3 levels. TCM treated cells display a reduction of cleaved Caspase-3 compared to TC treated cells. Fl, full length. DMSO, Dimethyl sulfoxide. MtOH, Methanol. Gapdh, Glyceraldehyde 3-phosphate dehydrogenase. The displayed blot was cropped for better visualization. The boxes exemplify different regions of the same gel. The uncropped blots can be found as Supplementary Fig. .

Article Snippet: The following primary antibodies were used in this study: cleaved Caspase-3 (Asp175) Antibody #9661 (Cell Signaling Technology), 1:1.000; GAPDH (D16H11) XP® Rabbit mAb#5174 (Cell Signaling Technology), 1:1.000.

Techniques: Viability Assay, Activity Assay, Western Blot

Journal: Cancers

Article Title: Evaluation of Electrochemotherapy with Bleomycin in the Treatment of Colorectal Hepatic Metastases in a Rat Model

doi: 10.3390/cancers15051598

Figure Lengend Snippet: ( A – D ) Immunohistochemical analysis of cleaved caspase-3 in animals treated with ECT ( A ), rEP ( B ), BLM ( C ), or Sham ( D ). Apoptotic cells (arrows) are stained red. Scale bars: 50 µm. ( E ) The diagram displays the mean number of positive cells in the tumor tissue per HPF. Data are given as mean ± SEM; § p < 0.05 vs. Sham.

Article Snippet: For the immunohistochemical detection of apoptotic cells, sections were stained with a rabbit polyclonal anti-cleaved caspase-3 antibody (1:100, Cell Signaling Technology, Frankfurt, Germany).

Techniques: Immunohistochemical staining, Staining

Journal: Renal Failure

Article Title: Forecast and verification of the active compounds and latent targets of Guyuan decoction in treating frequently relapsing nephrotic syndrome based on network pharmacology

doi: 10.1080/0886022X.2023.2184654

Figure Lengend Snippet: Compound-major target molecular docking. (A–C) The interaction mode of AKT1 with luteolin, wogonin, and kaempferol, respectively. (D–F) represented the interaction mode of CASP3 with luteolin, wogonin, and kaempferol, respectively.

Article Snippet: The membranes were sequentially soaked in blocking solution to reduce nonspecific binding and incubated with primary antibodies against Cleaved (Clea)-caspase-3 (#9661, 1:1000, 17, 19 kDa, Cell Signaling Technology, USA), AKT1 (ab179463, 1:10,000, 56 kDa, abcam, UK), phosphorylated (p)-AKT1 (ab81283, 1:10,000, 56 kDa, abcam, UK), and GAPDH (ab9485, 1:2500, 37 kDa, abcam, UK) at 4 °C overnight.

Techniques:

Journal: Renal Failure

Article Title: Forecast and verification of the active compounds and latent targets of Guyuan decoction in treating frequently relapsing nephrotic syndrome based on network pharmacology

doi: 10.1080/0886022X.2023.2184654

Figure Lengend Snippet: Effects of luteolin (LUT) treatment on the viability and apoptosis of ADR-treated MPC-5 cells as well as the protein expressions of AKT1 and CASP3. (A) The viability of MPC-5 cells in the LUT (0, 100, 150, 200, 250 μM) groups was determined by cell counting kit-8 (CCK-8) assay. (B) CCK-8 assay was also performed again to detect the cell viability in the control, adriamycin (ADR), ADR + LUT-100, ADR + LUT-150, and ADR + LUT-200 groups. (C–D) The apoptosis rate of MPC-5 cells in the control, ADR, ADR + LUT-100, ADR + LUT-150, and ADR + LUT-200 groups was measured by flow cytometry. (E–F) The protein expressions of cleaved (Clea)-caspase-3, AKT1, and phosphorylated (p)-AKT1 were determined by Western blot, with GAPDH serving as the loading control.

Article Snippet: The membranes were sequentially soaked in blocking solution to reduce nonspecific binding and incubated with primary antibodies against Cleaved (Clea)-caspase-3 (#9661, 1:1000, 17, 19 kDa, Cell Signaling Technology, USA), AKT1 (ab179463, 1:10,000, 56 kDa, abcam, UK), phosphorylated (p)-AKT1 (ab81283, 1:10,000, 56 kDa, abcam, UK), and GAPDH (ab9485, 1:2500, 37 kDa, abcam, UK) at 4 °C overnight.

Techniques: Cell Counting, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: The American Journal of Pathology

Article Title: Lung remodeling regions in long-term Covid-19 feature basal epithelial cell reprogramming

doi: 10.1016/j.ajpath.2023.02.005

Figure Lengend Snippet: Cell proliferation and apoptosis in lung remodeling regions in Covid-19. A, Immunostaining for Ki-67 plus CD68 with DAPI counterstaining in lung sections from Covid-19 patients and non-disease controls (ND). Scale bar=100 μm; Original magnification, x4 (inset). B , Quantitation of staining for conditions in (A). C , Immunostaining for active caspase-3 with DAPI counterstaining in lung sections for conditions in (A). Scale bar=100 μm. D , Quantitation of staining for conditions in (C). E , Immunostaining for active-caspase-3 plus HT2-280 or CD68 with DAPI counterstaining for conditions in (A). Scale bar=50 μm; Original magnification, x3 (inset). F , Quantitation of staining for conditions in (E). G , Immunostaining for KRT5 plus CXCL17 with DAPI counterstaining for conditions in (A). h, Scale bar=50 μm; Original magnification, x3 (inset). H, Quantitation of staining for conditions in (G). Data are representative of 5 patients and 5 control subjects per staining condition. Values represent mean and s.e.m; * P <0.05 (n=5 patients or subjects per group).

Article Snippet: Immunostaining was performed using the following primary antibodies: rabbit anti-ACE-2 polyclonal Ab (pAb) (ab65863, Abcam), mouse anti-ACE-2 mAb (clone 171606, R&D Systems), rabbit anti-KRT5 pAb (ab53121, Abcam) and mAb (clone EP1601Y, ab52635, Abcam), rabbit anti-AQP3 pAb (ab125219, Abcam), mouse anti-CD68 mAb (clone Kp-1, Sigma-Aldrich), rabbit anti-CD163 mAb (clone D6U1J, Cell Signaling), rabbit anti-CD31 mAb (clone EPR17259, Abcam), rabbit anti-CollagenIV (CollIV) mAb (clone EPR20966, Abcam), mouse anti-SCGB1A1 mAb (clone E-11, Santa Cruz), mouse anti-acetylated Tubulin (clone 6-11B-1, Sigma-Aldrich), mouse anti-MUC5AC mAb (clone 45M1, ThermoFisher Scientific and Santa Cruz Biotechnology), mouse anti-HT2-280 pAb (TB-27AHT2-280, Terrace Biotech), rabbit anti-surfactant protein C (SFTPC) pAb (ab90716, Abcam), rabbit anti-podoplanin (PDPN) mAb (clone EPR7072, Abcam), rabbit anti-MUC5B pAb (ab87276, Abcam), rabbit anti-Ki-67 mAb (clone D2H10, Cell Signaling), rabbit active (cleaved) caspase-3 mAb (clone 5A1E, Cell Signaling), and mouse anti-CXCL17 mAb (clone 422204, R&D Systems).

Techniques: Immunostaining, Quantitation Assay, Staining

Journal: Communications Biology

Article Title: ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation

doi: 10.1038/s42003-023-04611-w

Figure Lengend Snippet: a MCF10A-5E cells that transiently expressed ANKLE1 form larger spheroids. B MCF10A-5E cells that transiently expressed ANKLE1 form less circular spheroids. Note that the symbols to the left are examples of circularity at the indicated y-value. c Representative confocal microscopy images (scale bar = 10 μm) of spheroids stained for DNA, cleaved Caspase 3, and f-Actin reveal the effect of transient ANKLE1 expression. d , e Spheroids derived from MCF10A-5E cells transiently transfected with ANKLE1 contain more intact nuclei ( d ) and exhibit less cleaved Caspase 3 staining ( e ) inside the spheroid. f Western blot shows a lower level of cleaved PARP in protein extracts from spheroids derived from MCF10A-5E cells transiently expressing ANKLE1. We used densitometry to quantify the percent of cleaved PARP relative to the control.

Article Snippet: Cells or spheroids were fixed with 4% PFA (20% Paraformaldehyde Solution (Electron Microscopy Science)) for 1 h. Spheroids were blocked and permabilized with 0.5% Triton, 100 mM Glycine, 10% FBS in PBS for 30 min, then stained overnight with Laminin-5 Alexa Fluor 488 Conjugated antibodies (Milipore), Cleaved Caspase-3 (Asp175) (D3E9) Alexa Fluor 467 antibodies (Cell Signaling) and ActinRed 555 (ReadyProbes, Invitrogen) overnight in 0.2% Triton, 10% FPS in PBS.

Techniques: Confocal Microscopy, Staining, Expressing, Derivative Assay, Transfection, Western Blot

Journal: eLife

Article Title: Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

doi: 10.7554/eLife.80179

Figure Lengend Snippet: ( A ) Posterior midgut views of wild type flies fed with water (Ctrl), Btk ∆Cry or Btk SA11 spores or crystals. The R4 region we analyzed in all this study is detoured in orange. ×5 magnification. Scale bar = 200 µm. ( B ) Upper panels: myo1A>GFP midguts labeled with the anti-cleaved Caspase 3 antibody (Casp3, red). ECs are labelled by the GFP (green). Lower panels: single anti-cleaved Caspase 3 channel. Red arrows point cleaved Caspase-3-positive cells. Scale bare = 20 µm. ( C ) Monitoring of the ISC daughter cell commitment in posterior midguts of Dl-ReDDM flies. Drosophila were fed with water, Btk ∆Cry spores or Btk SA11 spores. The experimental design is shown below the panels. Anti-Pros (blue) marks the EEPs and EEs. Red arrows point to EBs or ECs (expressing only the RFP). Pink arrows point the EEPs (expressing the GFP, the RFP and Pros) ×40 magnification. Scale bar = 20 µm ( D ) myo1A>GFP posterior midguts of Drosophila fed with water (control), Btk ∆Cry spores, Btk SA11 spores or crystals at 1, 3, and 5 days PI. GFP marks the ECs. ×40 magnification. Scale bar = 20 µm.

Article Snippet: antibody , Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) antibody , Cell Signalling , Cat# 9661 RRID: AB_2341188 , 1/600.

Techniques: Labeling, Expressing

Journal: eLife

Article Title: Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

doi: 10.7554/eLife.80179

Figure Lengend Snippet: ( A ) EC apoptosis was monitored by expressing the Caspase 3 sensor (Casp:: GFP) using the myo1A-GAL4 EC driver ( myo1A>Casp::GFP ). With this transgenic combination, the GFP is detectable only when the Caspase 3 is activated in ECs. Left panel: ×40 magnification of a R4 subregion. Green stars mark GFP-positive dying ECs. Scale bar = 20 µm. Right panel: quantification of dead ECs 24 hr post ingestion (PI) in the posterior midgut (R4 region). ( B ) Quantification of mitoses using the anti-PH3 antibody in the whole midgut 24 hr PI. ( C ) ISC density in the R4 region of esg >GFP flies 24, 72, and 120 hr PI. ( D ) EC density in the R4 region of myo1A>GFP flies 24, 72, and 120 hr PI. Data is reported as mean ± SEM. ns = not significant; * (p≤0.05); ** (p≤0.01), *** (p≤0.001). Figure 1—source data 1. Cell type counting.

Article Snippet: antibody , Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) antibody , Cell Signalling , Cat# 9661 RRID: AB_2341188 , 1/600.

Techniques: Expressing, Transgenic Assay

Journal: eLife

Article Title: Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

doi: 10.7554/eLife.80179

Figure Lengend Snippet:

Article Snippet: antibody , Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) antibody , Cell Signalling , Cat# 9661 RRID: AB_2341188 , 1/600.

Techniques: Isolation, Recombinant, Software, Microscopy

Journal: Cell Death & Disease

Article Title: LncGMDS-AS1 promotes the tumorigenesis of colorectal cancer through HuR-STAT3/Wnt axis

doi: 10.1038/s41419-023-05700-8

Figure Lengend Snippet: A Cell viability was determined by CCK-8 assay with HCT116 and SW620 cells stably transduced with control shRNA (shCtrl) or GMDS-AS1 shRNAs (shRNA1 or shRNA2). B Colony formation assays with HCT116 and SW620 cells stably transduced with control shRNA or GMDS-AS1 shRNAs. Above, representative images; below, quantification. C CCK-8 assays of control and stable GMDS-AS1-expressing RKO cells. D Colony formation assays with control and stable GMDS-AS1-expressing RKO cells. Above, representative images; below, quantification. E The percentage of apoptotic HCT116 and SW620 cells was estimated by flow cytometry using APC-labeled Annexin V and 7-AAD staining. Representative result based on three independent experiments. F Immunoblot analysis of the apoptosis hallmarks cleaved PARP and Caspase-3 in HCT116 and SW620 cells stably transduced with a control shRNA or GMDS-AS1 shRNA. G Cell cycle evaluation of HCT116 and SW620 cells stably transduced with control shRNA or GMDS-AS1 shRNA by EdU/7-AAD double-staining with flow cytometry. Left: flow plots. Right: quantification. H Representative image of xenograft tumors excised from nude mice subcutaneously injected with control or GMDS-AS1 KD HCT116 cells ( n = 8). The small pieces of mouse tails represent the mice that failed to form tumors. I , J Tumor volume and tumor weight of the mice shown in H were measured. K Representative image of xenograft tumors excised from nude mice subcutaneously injected with control or GMDS-AS1 OE RKO cells ( n = 6). L , M Tumor volume and tumor weight of the mice shown in K were measured. A – D Values are expressed as the means ± SEM, n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05 by two-tailed Student’s t test.

Article Snippet: The following antibodies were used: an anti-p-STAT3 antibody (Y705) (Cy6566, Abways), an anti-STAT3 antibody (sc-482, Santa Cruz), an anti-HuR rabbit polyclonal antibody (11910-1-AP, Proteintech), an anti-HuR mouse monoclonal antibody (mAb; 66549-1-Ig, Proteintech), an anti-β-actin antibody (30101ES60, Yeasen Biotech Co.), an anti-BCL2 antibody (12789-1-AP, Proteintech), an anti-C-MYC antibody (10828-1-AP, Proteintech), an anti-cyclin D1 antibody (sc-753, Santa Cruz), an anti-PARP antibody (#9542, CST), an anti-cleaved caspase-3 antibody (#9661, CST), an anti-caspase-3 antibody (#9662, CST), an anti-Flag antibody (F1804, Sigma), an anti-Ub (P4D1) mouse mAb (#3936, CST), an anti-K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (#8081, CST), an anti-β-TrCP antibody (sc-390629, Santa Cruz), anti-rabbit IgG (sc-2027, Santa Cruz), anti-mouse IgG (sc-2025, Santa Cruz), peroxidase-conjugated goat anti-rabbit IgG (33101ES60, Yeasen Biotech Co.), and peroxidase-conjugated goat anti-mouse IgG (33201ES60, Yeasen Biotech Co.).

Techniques: CCK-8 Assay, Stable Transfection, Transduction, shRNA, Expressing, Flow Cytometry, Labeling, Staining, Western Blot, Double Staining, Injection, Two Tailed Test