Journal: Bioactive materials

Article Title: An Injectable silk-based hydrogel as a novel biomineralization seedbed for critical-sized bone defect regeneration.

doi: 10.1016/j.bioactmat.2024.01.024

Figure Lengend Snippet: Fig. 6. The potential mechanism involved in the osteogenesis induced by the seedbed hydrogel. A) Venn diagram and volcano plot of the upregulated genes influenced by the seedbed hydrogel. B) Relative mRNA expression of SLC13A3 and MKX in BMSCs (n = 3). C) Expression of proteins SLC13A3 and MKX in BMSCs (normalization using GAPDH). D) Schematic diagram of the function of SLC13A3 and its inhibitor. E) Succinate uptake of BMSCs (Scale bar = 100 μm). F) ALP staining of BMSCs with or without inhibition of SLC13A3 (Scale bar = 5 mm). Error bars, mean ± standard deviation, **p < 0.01, ***p < 0.001.

Article Snippet: The PVDF membrane was incubated with primary antibodies against COL1A1 (1:1000 dilution, E8I9Z, Cell Signaling Technology, USA), OPN (1:1000 dilution, AKm2A1, Santa Cruz, USA), SLC13A3 (1:500 dilution, 26184- 1-AP, Proteintech, China), MKX (1:500 dilution, sc-515878, Santa Cruz, USA), Akt and phospho-Akt (1:1000 dilution, 9272S, 9271T, Cell Signaling Technology, USA) and GAPDH (1:1000 dilution, D16H11, Cell Signaling Technology, USA), respectively, and further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Staining, Inhibition, Standard Deviation

Journal: Bioactive materials

Article Title: An Injectable silk-based hydrogel as a novel biomineralization seedbed for critical-sized bone defect regeneration.

doi: 10.1016/j.bioactmat.2024.01.024

Figure Lengend Snippet: Fig. 7. The intracellular signaling pathway involved in the osteogenesis induced by the seedbed hydrogel. A) KEGG analysis of upregulated genes induced by the biomineralization seedbed. B) GSEA analysis of PI3K-Akt signaling pathway related features in the biomineralization seedbed. C) Western blotting of the activation of Akt and p-Akt in BMSCs (normalization using GAPDH). D) ALP staining of BMSCs with or without inhibition of PI3K-Akt signaling pathway (Scale bar = 5 mm). E) Western blotting of the activation of Akt and p-Akt in BMSCs with a gradient concentration of exogenous succinate or with the biomineralization seedbed. F) Western blotting of the activation of Akt and p-Akt with or without the inhibition of SLC13A3. G) The calcium distribution in mitochondria and cytoplasm in BMSCs (Scale bar = 100 μm). H) Schematic diagram of the regulation of the biomineralization hydrogel seedbed. Error bars, mean ± standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The PVDF membrane was incubated with primary antibodies against COL1A1 (1:1000 dilution, E8I9Z, Cell Signaling Technology, USA), OPN (1:1000 dilution, AKm2A1, Santa Cruz, USA), SLC13A3 (1:500 dilution, 26184- 1-AP, Proteintech, China), MKX (1:500 dilution, sc-515878, Santa Cruz, USA), Akt and phospho-Akt (1:1000 dilution, 9272S, 9271T, Cell Signaling Technology, USA) and GAPDH (1:1000 dilution, D16H11, Cell Signaling Technology, USA), respectively, and further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Western Blot, Activation Assay, Staining, Inhibition, Concentration Assay, Standard Deviation

Journal:

Article Title: Poliovirus Induces Bax-Dependent Cell Death Mediated by c-Jun NH 2 -Terminal Kinase ▿

doi: 10.1128/JVI.02690-06

Figure Lengend Snippet: PV-mediated MOMP in neuronal cells is Bid and Bim independent. (A) Time course of caspase-8 and Bid processing in PV-infected IMR5 cells. (Top) At the indicated times p.i., whole-cell extracts were subjected to immunoblot analysis with anti-caspase-8 (Pro-C8) and anti-Bid antibodies. Actin was used as a control for protein loading. (Bottom) Caspase-8 activation in PV-infected IMR5 cells. Caspase-8 activation was determined in mock- and PV-infected IMR5 cells (14 h p.i.) by flow cytometry using a fluorescein-labeled inhibitor (FAM-LETD-fmk) that binds specifically to active caspase-8, as described in Materials and Methods. Histograms representative of two independent experiments are shown. The percentages of cells positive for activated caspase-8 are indicated. (B) The broad-spectrum caspase inhibitor z-VAD-fmk does not inhibit cytochrome c release during PV infection. Mock- and PV-infected IMR5 cells were left untreated or treated with 100 μM z-VAD-fmk for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Cytochrome c (Cyt c) was assayed in cytosolic extracts by Western blot analysis (14 h p.i.). Actin was used as a control for protein loading. (C) No inhibition of cytochrome c release after knockdown of Bim expression in PV-infected cells. (Top) IMR5 cells were transfected with Bim siRNA or nontargeted control siRNA. Bim protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected and Bim siRNA-transfected cells. Actin was used as a protein loading control. (Bottom) Cells were uninfected or were infected with PV 72 h after transfection, and cytochrome c (Cyt c) release was analyzed in cytosolic fractions by Western blotting 8 h p.i. Actin was used as a protein loading control. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin.

Article Snippet: Small interfering RNAs (siRNAs) that target human Bim (6461) and nontargeted siRNA (6201) were purchased from Cell Signaling. (ii) Cell lines, virus stock, and viral infection.

Techniques: Infection, Western Blot, Control, Activation Assay, Flow Cytometry, Labeling, Concentration Assay, Adsorption, Inhibition, Knockdown, Expressing, Transfection

Journal: Journal of Ovarian Research

Article Title: Lipoic acid decreases Mcl-1, Bcl-x L and up regulates Bim on ovarian carcinoma cells leading to cell death

doi: 10.1186/s13048-015-0165-z

Figure Lengend Snippet: siBim attenuates lipoic acid induced-apoptosis 72 h after transfection. a : The cells were treated following protocol of exposure regarding the treatment by lipoic acid (1 mM) administered 24 h after transfection with either 20nM nonspecific siRNA control (siCRTL) or siBim, as described in materials and methods section. b : Bim protein expression level was assessed in control or treated-cells at 72 h post-transfection of IGROV1 (left panel) and IGROV1-R10 (right panel) by western blot. Actin protein is used as a loading control. Actin is a same actin that in Fig. (4e). This blot was performed in the same experiment as that of blot in Fig. 4e. Western blots shown come from one experiment representative of at least three independent experiments and cell lysates. c - d : Morphological features of the cells observed by photon microscopy (left column of each panel) and nuclear features of the cells after DAPI staining (middle column of each panel) were then studied, Bars: 20 μm. DNA content histograms obtained by flow cytometry (right column of each panel) after a 48 h of LA treatment in IGROV1 [c] and IGROV1-R10 ( d ) cell lines were studied. For each condition, the percentage of sub-G1 and G0-G1 phases is indicated. e : Bcl-x L , Mcl-1, caspases-3 (pro and cleaved forms) protein expression levels were assessed in control or treated-cells at 72 h post-transfection of IGROV1 (left panel) and IGROV1-R10 (right panel) by western blot. Actin protein is used as a loading control. Actin is a same actin that in Fig. (4b). This blot was performed in the same experiment as that of blot in Fig. 4b. Western blots shown are from one experiment representative of at least three independent experiments and cell lysates

Article Snippet: Primer and probe sequences for real-time detection of Bim mRNA (assay ID#Hs00708019_s1), Mcl-1 mRNA (assay ID#HS 001 720 36_m1) and endogenous control gene GAPDH mRNA [ ] were purchased form Applied Biosystems.

Techniques: Transfection, Control, Expressing, Western Blot, Microscopy, Staining, Flow Cytometry