Journal: bioRxiv
Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration
doi: 10.1101/2025.03.06.641871
Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.
Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).
Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test
