Fig. 7: Viruses regulate migrasome formation via AMFR-TSPAN4 axis.
From: O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release
a Amfr WT and KO HeLa cells stably expressing TSPAN4-Flag were infected with or without VSV-GFP at an MOI of 1 for 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. b, c Amfr KO NRK cells stably expressing TSPAN4-mCherry were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, TSPAN4-mCherry; yellow, Merge. Number of migrasomes from the cells in (b); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. d Amfr KO HeLa cells stably expressing TSPAN4-Flag were transfected with or without AMFR-Myc or AMFR3CS-Myc for 24 h and then infected with VSV-GFP at an MOI of 1 for another 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e, f Amfr KO NRK cells stably expressing ITGA5-mCherry with or without AMFR-Halo or AMFR3CS-Halo were infected with VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, ITGA5-mCherry; white, Halo, AMFR-Halo, and AMFR3CS-Halo. Number of migrasomes from the cells in (e); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. g HeLa cells stably expressing TSPAN4-Flag or TSPAN46KR-Flag were infected with or without VSV-GFP at an MOI of 1 for 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. h, i NRK cells stably expressing TSPAN46KR- mCherry were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Green, VSV-GFP; red, TSPAN46KR-mCherry; yellow, Merge. Cells were imaged by confocal microscopy. Number of migrasomes from (h); n = 149 no infection, n = 149 VSV-GFP from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. j HeLa cells stably expressing TSPAN4-Flag were transfected with or without OGT-Myc or OGTK852M-Myc for 24 h and then infected with or without VSV-GFP at an MOI of 1 for another 12 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 of three independent experiments normalized to Tubulin, was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. k, l NRK cells stably expressing ITGA5-mCherry transfected with or without OGT-Halo or OGTK852M-Halo were infected with or without VSV-GFP at an MOI of 0.2 for 12 h. Cells were imaged by confocal microscopy. Green, VSV-GFP; red, ITGA5-mCherry; blue, OGT-Halo and OGTK852M-Halo. Number of migrasomes from the cells in (k); n = 150 cells per group from three independent experiments. Scar bar represents 10 μm. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. m Proposed model for virus infection dynamically regulates migrasome formation via AMFR O-GlcNAcylation and TSPAN4 ubiquitination.
