Journal: Frontiers in Pharmacology

Article Title: Baicalein Attenuates Pyroptosis and Endoplasmic Reticulum Stress Following Spinal Cord Ischemia-Reperfusion Injury via Autophagy Enhancement

doi: 10.3389/fphar.2020.01076

Figure Lengend Snippet: Baicalein enhances autophagy after spinal cord ischemia-reperfusion injury (SCIR). (A) Immunofluorescence staining for LC3II and NeuN colocalization at the spinal cord lesion after SCIR (Scan bar = 25μm). (B) Immunofluorescence staining for p62 and NeuN colocalization at the spinal cord lesion after SCIR (Scan bar = 25μm). (C) The quantitative percentage of the LC3II positive neurons at the spinal cord lesion in each group. (D) The quantitative percentage of the p62 positive neurons in each group. (E) Western blotting for Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II, and p62 expression levels in the Sham, SCIR+Vehicle, and SCIR+Baicalein groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II, and p62 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. * p < 0.05 and ** p < 0.01, vs. Sham group. ## p < 0.01, vs. SCIR+Vehicle group.

Article Snippet: The rabbit monoclonal anti-phosphoinositide-3-kinase (VPS34) and anti-cathepsin D (CTSD) antibodies were acquired from the Proteintech Group (12452-1 and 21327-1; Chicago, IL, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Baicalein Attenuates Pyroptosis and Endoplasmic Reticulum Stress Following Spinal Cord Ischemia-Reperfusion Injury via Autophagy Enhancement

doi: 10.3389/fphar.2020.01076

Figure Lengend Snippet: Inhibition of autophagy reverses the effects of baicalein on endoplasmic reticulum (ER) stress, pyroptosis, and functional recovery after SCIR. (A) Western blotting for the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 expression levels in the SCIR+Baicalein, and SCIR+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (B) The optical density values of the Beclin1, ATG5, VPS34, CTSD, ATP6V1B2, LC3II and p62 expression levels were quantified and analyzed in each group. (C, D) Western blotting for the NLRP3, GSDMD, C-CASP1, GRP78, PDI, ATF4, CHOP, and CASP12 expression levels in the SCIR+Baicalein and SCIR+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (E, F) The optical density values of GRP78, PDI, ATF4, CHOP, CASP12, NLRP3, GSDM, and C-CASP1expression levels were quantified and analyzed in the both groups. (G, H) Enzyme-linked immunosorbent assay (ELISA) results for IL1βand IL18 expression levels in the SCIR+Baicalein and SCIR+3MA groups. (I, J) The BMS scores of the mice in each group at 24 h and 7 days after SCIR. The values are expressed as the means ± SEM, n=5 per group. * p < 0.05 and ** p < 0.01, vs. SCIR+Baicalein group.

Article Snippet: The rabbit monoclonal anti-phosphoinositide-3-kinase (VPS34) and anti-cathepsin D (CTSD) antibodies were acquired from the Proteintech Group (12452-1 and 21327-1; Chicago, IL, USA).

Techniques: Inhibition, Functional Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Alternative polyadenylation upon CPSF6 knock-out enhances HIV-1 infection in primary T cells

doi: 10.1371/journal.ppat.1013745

Figure Lengend Snippet: 3’ UTRs are designated shortened (blue) for Compositional Fold Change (cFC) < -0.25 and -log 10 (adjusted p-value) < 0.05 and lengthened (red) for cFC > 0.25 and -log 10 (adjusted p-value) < 0.05. Dots represent individual altered 3’ UTRs. b, Tracks show read coverage of TGFBR1 in RNA-Seq data from CPSF6 knock-out and NT control cells from one representative biological replicate (donor E), visualized using Integrative Genomics Visualizer. 3’ UTRs show evidence of shortening in response to CPSF6 knock-out. c, Charts show functional enrichment (Metascape) analysis of top pathways enriched in genes with shortened 3’ UTRs (left, blue) and lengthened 3’ UTRs (right, red) in response to CSPF6 knock-out in primary CD4+ T cells as assessed by REPAC analysis in 3 biological replicates (donors E-G). d, Violin plot shows comparison of cFC values from REPAC analysis in all tested genes as compared to genes in the indicated pathways (GO:0042110:T Cell activation, R-HSA-1280215: Cytokine Signaling in Immune system, R-HSA-913531: Interferon Signaling) in 3 biological replicates (donors E-G). e, Scatterplot shows cFC of all tested APA sites in REPAC analysis of CPSF6 knock-out primary CD4+ T cells in as compared to NT controls plotted against differential expression data as shown in in data aggregated from 3 biological replicates (donors E-G). 3’ UTRs are designated shortened (blue) for cFC < -0.25 and -log 10 (adjusted p-value) < 0.05 and lengthened (red) for cFC > 0.25 and -log 10 (adjusted p-value) < 0.05. Dots represent individual altered 3’ UTRs. f, Immunoblot shows knock-out of CPSF5 in protein lysates harvested at day 4 post-editing in 3 biological replicates (donor Z-AB). g, HIV-1 infectivity (% GFP positive cells normalized to NT control per donor) at day 5 post-challenge with HIV-1 NL4-3 nef:IRES:GFP in indicated knock-out primary CD4+ T cells from 3 biological replicates (donors Z-AB) as assessed by flow cytometry. Each bar represents the average of technical triplicates + /- SD with individual data points shown. Statistics were calculated relative to the NT control per condition by one-way ANOVA with Dunnet’s test for multiple comparisons; * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Primary antibodies used in this study are as follows: CPSF6 (Rabbit, 1:3000 in 5% BSA, Novus, #NBP1–85676), CPSF5 (Rabbit, 1:5000 in 4% milk, Proteintech, #10322–1-AP), CYPA (Rabbit, 1:12000 in 4% milk, Cell Signaling, #2175), IFNAR1 (Rabbit, 1:1000 in 4% milk, abcam, #ab124764 [clone EPR6244]), MX1 (Rabbit, 1:1000 in 5% BSA, Cell Signaling, #37849S [clone D3W7I]), IFIT1 (Rabbit, 1:1000 in 4 milk, Cell Signalling #14769 [clone D2X9Z], TRIM5α (1:1000 in 4% milk, Cell Signaling, #14326 [clone D6Z8L]), and β-actin (Mouse, 1:10000 in 4% milk, Cell Signaling, #3700S [clone 8H10D10]).

Techniques: RNA Sequencing, Knock-Out, Control, Functional Assay, Comparison, Activation Assay, Quantitative Proteomics, Western Blot, Infection, Flow Cytometry