Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Macrophage-intrinsic MDA5-IRF5 axis drives HIV-1 icRNA-induced inflammatory responses

doi: 10.1101/2024.09.06.611547

Figure Lengend Snippet: THP-1/PMA macrophages were infected with Lailienv GFP/G (MOI 2) in the presence or absence of efaverinz (EFV, 1 µM), raltegravir (RAL, 30 µM), spironolactone (Spiro, 100 nM), and KPT335 (KPT, 1 µM). Cells and culture supernatants harvested at 3 dpi for (A) flow cytometry analysis to measure infection levels (¾GFP+) and (B) ELISA to measure IP-10 secretion. Infection levels (C) and IP-10 secretion (D) in THP-1/PMA macrophages infected with either WT (LailienvGFP/G) or HIV-1/M10 were determined at 3 dpi by flow cytometry and ELISA, respectively. (E) MAVS expression in THP1 cells transduced with shCTRL or shMAVS lentivectors was determined by western blot analy­ sis. (F, G) LaiiienvGFP/G infected THP-1/PMA macrophages and cell supernatants were harvested 3 dpi for flow cytometry analysis (F) and ELISA (G) to measure infection establishment (¾GFP+) and IP-10 secretion. Data is displayed as the means ± SEM with each dot representing an independent experiment. Statistical significance assessed via 1-way ANOVA with Dunnet’s multiple comparisons test (A-B), unpaired t-test (C-O, F-G). *: p < 0.05; **: p <0.01, ***: p < 0.001, ****: p < 0.0001, ns = not significant.

Article Snippet: In some experiments, THP-1/PMA macrophages were pre-treated (for 20 minutes) with efavirenz (1 μM, NIH AIDS Reagent Program), raltegravir (30 μM, Selleck Chemical #50-615-1), spironolactone (100 nM, Selleck Chemical, # S4054), and KPT 330 (1 μM, Selleck Chemical # 50-136-5156).

Techniques: Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Transduction, Western Blot