A Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 7 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm B Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 7 . ( n = 30 fields of view from n = 3 animals ) , two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at postnatal day 21 . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm D Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at postnatal day 21 . ( n = 27 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD ) . E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 3 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm F Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 3 months of age . ( n = 52 fields of view from n = 5 animals ) , two tailed t - test , error bars = + / - SD . G Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and Laminin ( LAMA1 ) antibodies at 10 months of age . Arrowheads indicate PAX7 + satellite cells . Scale bar = 200μm H Quantification of the number of PAX7 + cells per unit area ( mm2 ) in TA muscle cross - sections from Myf6 - KO and WT counterparts at 10 months of age . ( n = 60 fields of view from n = 3 animals ) , two tailed t - test , error bars = + / - SD .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 7. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm B Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 7. (n=30 fields of view from n=3 animals), two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at postnatal day 21. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm D Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at postnatal day 21. (n=27 fields of view from n=3 animals) , two tailed t-test, error bars = +/- SD). E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 3 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm F Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 3 months of age. (n=52 fields of view from n=5 animals), two tailed t-test, error bars = +/- SD. G Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and Laminin (LAMA1) antibodies at 10 months of age. Arrowheads indicate PAX7+ satellite cells. Scale bar=200μm H Quantification of the number of PAX7+ cells per unit area (mm2) in TA muscle cross-sections from Myf6-KO and WT counterparts at 10 months of age. (n=60 fields of view from n=3 animals), two tailed t-test, error bars = +/- SD.


Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 3 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm J Quantification of the number of MuSCs per fiber between 3 month - old Myf6 - KO ( n = 41 fibers from n = 3 mice ) and WT ( n = 31 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / - SD K Immunofluorescent analysis of myofiber - associated MuSCs isolated from the EDL muscles of 10 month - old Myf6 - KO and WT counterparts . Fibers were stained with PAX7 antibody and counterstained with DAPI at time T = 0 hours post fiber isolation . Scale bar = 25μm L Quantification of the number of MuSCs per fiber between 10 month - old Myf6 - KO ( n = 38 fibers from n = 2 mice ) and WT ( n = 67 fibers from n = 3 mice ) counterparts . Two - tailed t - test , error bars = + / -

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm J Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO (n=41 fibers from n=3 mice) and WT (n=31 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD K Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm L Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO (n=38 fibers from n=2 mice) and WT (n=67 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD


Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7 . Scale bar = 100μm , arrowheads highlight select MuSC B Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at postnatal day 7 ( n = 15 fields of view from n = 3 animals ) Two - tailed t - test , error bars = + / - SD . C Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21 . Scale bar = 100μm , arrowheads highlight select MuSC D Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO ( n = 28 fields of view from n = 3 animals ) and WT mice ( n = 31 fields of view from n = 3 animals ) at postnatal day 21 . Two - tailed t - test , error bars = + / - SD E Immunofluorescent analysis of TA muscle cross - sections from Myf6 - KO and WT mice with PAX7 and KI67 antibodies at 3 months of age . Scale bar = 100μm , arrowheads highlight select MuSC F Quantification of the percentage of KI67 + over PAX7 + MuSCs between Myf6 - KO and WT mice at 3 months of age ( n = 30 fields of view from n = 5 mice ) . Two - tailed t - test , error bars = + / -

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 7. Scale bar=100μm, arrowheads highlight select MuSC B Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at postnatal day 7 (n=15 fields of view from n=3 animals) Two-tailed t-test, error bars = +/- SD. C Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at postnatal day 21. Scale bar=100μm, arrowheads highlight select MuSC D Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO (n=28 fields of view from n=3 animals) and WT mice (n=31 fields of view from n=3 animals) at postnatal day 21. Two-tailed t-test, error bars = +/- SD E Immunofluorescent analysis of TA muscle cross-sections from Myf6-KO and WT mice with PAX7 and KI67 antibodies at 3 months of age. Scale bar=100μm, arrowheads highlight select MuSC F Quantification of the percentage of KI67+ over PAX7+ MuSCs between Myf6-KO and WT mice at 3 months of age (n=30 fields of view from n=5 mice). Two-tailed t-test, error bars = +/- SD


H ( Left ) Heat Map of up and down - regulated genes in the p38 MAPK pathway ( WP400 ) in Myf6 - KO and WT satellite cells . ( Right ) Gene set enrichment analysis of p38 MAPK pathway genes . In each plot , the blue curve represents the gene set enrichment score profile ( Subramanian et al . , 2005 ) across p38 MAPK pathway genes that are ranked by their differential expression . The grey curve represents the null expectation . P - values are based on permutation test ( 10 , 000 permutations ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H (Left) Heat Map of up and down-regulated genes in the p38 MAPK pathway (WP400) in Myf6-KO and WT satellite cells. (Right) Gene set enrichment analysis of p38 MAPK pathway genes. In each plot, the blue curve represents the gene set enrichment score profile (Subramanian et al., 2005) across p38 MAPK pathway genes that are ranked by their differential expression. The grey curve represents the null expectation. P-values are based on permutation test (10,000 permutations).


Immunofluorescent analysis of WT and Myf6 - KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI . Scale bar = 200μm J Quantification of the fusion index between Myf6 - KO ( n = 54 fields of view from n = 3 mice ) and WT ( n = 81 fields of view from n = 4 mice ) 7DM myotubes . Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view . Two - tailed t - test , error bars = + / -

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Immunofluorescent analysis of WT and Myf6-KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI. Scale bar=200μm J Quantification of the fusion index between Myf6-KO (n=54 fields of view from n=3 mice) and WT (n=81 fields of view from n=4 mice) 7DM myotubes. Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view. Two-tailed t-test, error bars = +/- SD


EDL myofibers isolated from WT and Myf6 - KO mice fixed at time T = 0 and stained with PAX7 , MYOD and DAPI . Scale bar = 30μm L Quantification of the percentage of MYOD + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 88 myofibers ) vs WT ( n = 86 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD M Quantification of the average percentage of MYOD + / PAX7 + satellite cells per mouse in Myf6 - KO and WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

EDL myofibers isolated from WT and Myf6-KO mice fixed at time T=0 and stained with PAX7, MYOD and DAPI. Scale bar=30μm L Quantification of the percentage of MYOD+/PAX7+ satellite cells per fiber in Myf6-KO (n= 88 myofibers) vs WT (n= 86 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD M Quantification of the average percentage of MYOD+/PAX7+ satellite cells per mouse in Myf6-KO and WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Staining for phospho - p38 MAPK on EDL myofibers cultured for 48 hours from Myf6 - KO and WT mice . Scale bar = 30μm O Quantification of the percentage of phospho - p38 + / PAX7 + satellite cells per fiber in Myf6 - KO ( n = 56 myofibers ) vs WT ( n = 67 myofibers ) myofibers . Two - tailed t - test , error bars = + / - SD P Quantification of the average percentage of phospho - p38 + / PAX7 + satellite cells per mouse in Myf6 - KO vs WT myofibers ( n = 3 mice ) . Two - tailed t - test , error bars = + / -

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Staining for phospho-p38 MAPK on EDL myofibers cultured for 48 hours from Myf6-KO and WT mice. Scale bar=30μm O Quantification of the percentage of phospho-p38+/PAX7+ satellite cells per fiber in Myf6-KO (n=56 myofibers) vs WT (n=67 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD P Quantification of the average percentage of phospho-p38+/PAX7+ satellite cells per mouse in Myf6-KO vs WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD


Western blot analysis of C2C12 myoblasts treated with EGF , LIF , or EGF + LIF while in differentiation media and stained with phosphor - p38 , total p38 , myogenin , MF20 and GAPDH . Cells were treated with EGF ( 50ng / ml ) , LIF ( 1000U / ml ) or EGF + LIF after 48 hours in differentiation media , and every 6 hours following initial treatment , followed by cell lysis at 72 hours . R Phospho - p38 western blot signal intensity normalized to total p38 signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / - SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control , EGF - treated , LIF - treated and EGF + LIF - treated C2C12s . Signal intensity was measured by placing ROIs over western blot bands using LI - COR Image Studio . ( n = 3 technical replicates ) Two - tailed t - test , error bars = + / -

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of C2C12 myoblasts treated with EGF, LIF, or EGF+LIF while in differentiation media and stained with phosphor-p38, total p38, myogenin, MF20 and GAPDH. Cells were treated with EGF (50ng/ml), LIF (1000U/ml) or EGF+LIF after 48 hours in differentiation media, and every 6 hours following initial treatment, followed by cell lysis at 72 hours. R Phospho-p38 western blot signal intensity normalized to total p38 signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD S Myogenin western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD T MF20 western blot signal intensity normalized to GAPDH signal intensity in control, EGF-treated, LIF-treated and EGF+LIF-treated C2C12s. Signal intensity was measured by placing ROIs over western blot bands using LI-COR Image Studio. (n=3 technical replicates) Two-tailed t-test, error bars = +/- SD


EDL myofibers treated repeatedly with recombinant EGF protein ( 100ng / ml ) at 0h , 24h , 32h , 40h , and 48h in culture , and fixed at 48 hours , compared to untreated controls . V Quantification of the number of PAX7 + cells per fiber in EGF - treated ( n = 19 fibers from n = 1 mouse ) and untreated control EDL myofibers ( n = 16 fibers from n = 1 mouse ) after 48 hours in culture . Two - tailed t - test , error bars = + / -

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EDL myofibers treated repeatedly with recombinant EGF protein (100ng/ml) at 0h, 24h, 32h, 40h, and 48h in culture, and fixed at 48 hours, compared to untreated controls. V Quantification of the number of PAX7+ cells per fiber in EGF-treated (n=19 fibers from n=1 mouse) and untreated control EDL myofibers (n=16 fibers from n=1 mouse) after 48 hours in culture. Two-tailed t-test, error bars = +/- SD


B Representative images of bioluminescent signal from hindlimbs of Myf6 - KO and WT counterparts at day 1 , 7 , 14 , and 21 post transplantation . Scale bar =

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

B Representative images of bioluminescent signal from hindlimbs of Myf6-KO and WT counterparts at day 1, 7, 14, and 21 post transplantation. Scale bar=1cm


C Time course of average bioluminescent signal ( total flux in radiance ) in Myf6 - KO and WT mice at 1 , 7 , 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs ( mean across 4 - 5 animals per group ± SEM at each time point , two - tailed t - test ) .

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C Time course of average bioluminescent signal (total flux in radiance) in Myf6-KO and WT mice at 1, 7, 14 and 21 days post MuSC transplantation showing engraftment dynamics of donor MuSCs (mean across 4-5 animals per group ± SEM at each time point, two-tailed t-test).


D Bioluminescent signal ( radiance ) in Myf6 - KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation . ( n = 4 - 5 animals per group ± SD ) two - tailed t - test , bars represent mean + / - SD .

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D Bioluminescent signal (radiance) in Myf6-KO and WT mice at each time point normalized to the bioluminescent signal at day 1 post MuSC transplantation. (n=4-5 animals per group ± SD) two-tailed t-test, bars represent mean +/- SD.


E - F Immunostaining of TA cross - sections of injured muscle from 3 month - old WT ( E ) and Myf6 - KO ( F ) mice stained with laminin ( LAMA1 ) and PAX7 antibodies . Acute muscle injury was performed by injection of Cardiotoxin ( CTX ) into the TA muscle of Myf6 - KO and WT mice ( n = 5 animals per group ) . Arrowheads indicate PAX7 + MuSCs . Scale bar = 100μm G Quantification of the number of PAX7 + cells per unit area of TA muscle cross sections between Myf6 - KO and WT mice ( n = 5 animals per group ) two - tailed t - test , bars represent mean + / -

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E-F Immunostaining of TA cross-sections of injured muscle from 3 month-old WT (E) and Myf6-KO (F) mice stained with laminin (LAMA1) and PAX7 antibodies. Acute muscle injury was performed by injection of Cardiotoxin (CTX) into the TA muscle of Myf6-KO and WT mice (n=5 animals per group). Arrowheads indicate PAX7+ MuSCs. Scale bar=100μm G Quantification of the number of PAX7+ cells per unit area of TA muscle cross sections between Myf6-KO and WT mice (n= 5 animals per group) two-tailed t-test, bars represent mean +/- SD


H Immunofluorescent analysis of Myf6 - KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies . Scale bar = 20μm IQuantification of the number of PAX7 + MuSCs per fiber in Myf6 - KO ( n = 27 fibers from n = 3 mice ) and WT ( n = 29 fibers from n = 3 mice ) mice after 48 hours in culture . Two - tailed t - test , error bars = + / - SD .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Immunofluorescent analysis of Myf6-KO and WT EDL myofibers cultured in growth media for 48 hours and stained with PAX7 and MYOD antibodies. Scale bar=20μm IQuantification of the number of PAX7+ MuSCs per fiber in Myf6-KO (n=27 fibers from n=3 mice) and WT (n=29 fibers from n=3 mice) mice after 48 hours in culture. Two-tailed t-test, error bars = +/- SD.


J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF ( 1000U / ml ) or EGF ( 80ng / ml ) every 8 hours . Fibers were stained with antibodies against PAX7 and MyoD . Scale bar = 30μm K Quantification of the number of PAX7 + and MYOD + cells on the EDL myofiber treated with recombinant LIF ( n = 80 fibers ) , EGF ( n = 50 fibers ) or vehicle control ( n = 61 fibers ) . Two - tailed t - test , bars represent mean + / -

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J Immunofluorescence staining of WT EDL myofibers cultured in growth media for 48 hours and treated with LIF (1000U/ml) or EGF (80ng/ml) every 8 hours. Fibers were stained with antibodies against PAX7 and MyoD. Scale bar=30μm K Quantification of the number of PAX7+ and MYOD+ cells on the EDL myofiber treated with recombinant LIF (n=80 fibers), EGF (n=50 fibers) or vehicle control (n=61 fibers). Two-tailed t-test, bars represent mean +/- SD


EdU staining of cultured primary myoblasts isolated by FACS ( see materials and methods ) from Myf6 - KO and WT mice . Staining was analyzed after 6 , 12 and 24 hours of EdU incorporation . Scale bar = 100μm M Quantification of the percentage of EdU + myoblasts after 6 hours ( WT n = 75 fields of view from n = 3 mice , Myf6 - KO n = 72 fields of view from n = 3 mice ) , 12 hours ( WT n = 82 fields of view from n = 3 mice , Myf6 - KOn = 82 fields of view from n = 3 mice ) , and 24 hours ( WT n = 70 fields of view from n = 3 mice , Myf6 - KO n = 65 fields of view from n = 3 mice ) of EdU incorporation . Scale bar =

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EdU staining of cultured primary myoblasts isolated by FACS (see materials and methods) from Myf6-KO and WT mice. Staining was analyzed after 6, 12 and 24 hours of EdU incorporation. Scale bar=100μm M Quantification of the percentage of EdU+ myoblasts after 6 hours (WT n=75 fields of view from n=3 mice, Myf6-KO n=72 fields of view from n=3 mice), 12 hours (WT n=82 fields of view from n=3 mice, Myf6-KOn=82 fields of view from n=3 mice), and 24 hours (WT n=70 fields of view from n=3 mice, Myf6-KO n=65 fields of view from n=3 mice) of EdU incorporation. Scale bar=100μm


Representative images of a co - culture assay between WT myotubes and either WT or Myf6 - KO myoblasts . Briefly , WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6 - KO myoblasts for an additional 2 days , followed by immunostaining for MF20 and DAPI . Scale bar = 400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes , for 2 additional days ( n = 3 biological replicates ) . Two - tailed t - test , bars represent mean + / - SD .

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Representative images of a co-culture assay between WT myotubes and either WT or Myf6-KO myoblasts. Briefly, WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6-KO myoblasts for an additional 2 days, followed by immunostaining for MF20 and DAPI. Scale bar=400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes, for 2 additional days (n=3 biological replicates). Two-tailed t-test, bars represent mean +/- SD.


B OsPRR73 confers salinity stress tolerance in rice . The seedlings of Dongjin ( DJ , the wild type control ) , osprr73 , and the complementation line of osprr73 ( abbreviated as Com - L1 ) grown under 12 h light / 12 h dark conditions for 28 days ( left panel ) , transferred to 180 mM NaCl for 21 days ( middle panel ) and recovered for 12 and 24 days ( the two right panels ) respectively . Scale bar , 5 cm . C Statistical analysis of the survival rate of DJ , osprr73 and Com - L1 plants in ( B ) after recovery 12 days . Data are presented as mean ± SD . ( n from 4 biological replicates , and 24 plants were tested in each of biological replicates ) . ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test .

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B OsPRR73 confers salinity stress tolerance in rice. The seedlings of Dongjin (DJ, the wild type control), osprr73, and the complementation line of osprr73 (abbreviated as Com-L1) grown under 12 h light/12 h dark conditions for 28 days (left panel), transferred to 180 mM NaCl for 21 days (middle panel) and recovered for 12 and 24 days (the two right panels) respectively. Scale bar, 5 cm. C Statistical analysis of the survival rate of DJ, osprr73 and Com-L1 plants in (B) after recovery 12 days. Data are presented as mean ± SD. (n from 4 biological replicates, and 24 plants were tested in each of biological replicates). (***) P ≤ 0.001 were generated by student's t-test.


E Null mutant of osprr73 in Nipponbare ( NIP ) background by genome editing displayed hypersensitivity to NaCl treatment . Scale bar , 5 cm . F Survival rate of NIP ( wild type of CRISPR lines ) and osprr73 - C ( C stands for CRISPR ) plants in ( E ) after recovery 9 days . Data are presented as mean ± SD . n = 4 biological replicates , 24 plants for each biological replicate . ( * ) P ≤ 0 . 05 and ( * * * ) P ≤ 0 . 001 were generated by student ' s t - test .

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E Null mutant of osprr73 in Nipponbare (NIP) background by genome editing displayed hypersensitivity to NaCl treatment. Scale bar, 5 cm. F Survival rate of NIP (wild type of CRISPR lines) and osprr73-C (C stands for CRISPR) plants in (E) after recovery 9 days. Data are presented as mean ± SD. n = 4 biological replicates, 24 plants for each biological replicate. (*) P ≤0.05 and (***) P ≤ 0.001 were generated by student's t-test.


Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 .

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Quantiﬁcation of chlorophyll content of WT and osprr73 mutants Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.


Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days . Data are presented as mean ± SD . n = 3 , biological replicates , and asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 , ( * * * ) P ≤ 0 . 001 .

O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantiﬁcation of electrolyte leakage of WT and osprr73 mutants with or without 180 mM NaCl treatment for 7 days. Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.


A , B The Na + and K + contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days . Na + ( A ) and K + ( B ) contents were measured with ICP method . Data represent means ± SD , ( n = 3 , biological replicates ) . ( * * ) P ≤ 0 . 001 and ( * ) P ≤ 0 . 001 indicate significant difference by student t - test . The abbreviation of n . s . stands for no significant . C Ratio of Na + to K + was calculated with their respective content in ( A ) and ( B ) . Data represent mean ± SD ( n = 3 , biological replicates ) . For each of biological replicates , 6 individual plants were measured . The asterisk represents significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , and n . s . indicates no significant .

O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B The Na+ and K+ contents in the shoots and roots of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days. Na+ (A) and K+ (B) contents were measured with ICP method. Data represent means ± SD, (n = 3, biological replicates). (**) P ≤ 0.001 and (*) P ≤ 0.001 indicate significant difference by student t-test. The abbreviation of n.s. stands for no significant. C Ratio of Na+ to K+ was calculated with their respective content in (A) and (B). Data represent mean ± SD (n = 3, biological replicates). For each of biological replicates, 6 individual plants were measured. The asterisk represents significant difference among means by student's t-test with (*) P ≤0.05, and n.s. indicates no significant.


D Four - weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d ( middle panel ) and recovered for additional 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rates with Na2SO4 treatment in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants for each biological replicate . ( * * * ) P ≤ 0 . 001 indicates significant difference by student t - test . F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . G Statistical analysis of survival rates with MgCl2 treatment in ( F ) . The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days . Data represent means ± SD . n = 2 biological replicates , 24 plants of each replicate . H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days ( middle panel ) and recovered for 7 days ( right panel ) . Scale bar , 5 cm . I The survival rate of DJ and osprr73 plants in mannitol stress . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . The abbreviation of n . s . stands for no significant generated by student t - test .

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D Four-weeks old seedlings of DJ and osprr73 were treated with 90 mM Na2SO4 for 14 d (middle panel) and recovered for additional 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rates with Na2SO4 treatment in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants for each biological replicate. (***) P ≤ 0.001 indicates significant difference by student t-test. F The seedlings of DJ and osprr73 mutant were treated with 90 mM MgCl2 for 14 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. G Statistical analysis of survival rates with MgCl2 treatment in (F). The survival rate of DJ and osprr73 plants in MgCl2 stress were calculated after recovery 7 days. Data represent means ± SD. n = 2 biological replicates, 24 plants of each replicate. H The seedlings of DJ and osprr73 mutant were treated with 180 mM mannitol for 36 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. I The survival rate of DJ and osprr73 plants in mannitol stress. Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. The abbreviation of n.s. stands for no significant generated by student t-test.


A , B The expression level of OsHKT2 ; 1 and OsHKT1 ; 5 in osprr73 mutant and the wild control Dongjin ( DJ ) plants with or without salinity stress in multiple time points . DJ Data represent means ± SD . n = 3 , technical replicates . The representative data is from at least two individual biological replicates . The asterisks represent significant difference among means by student ' s t - test with ( * ) P ≤ 0 . 05 , ( * * ) P ≤ 0 . 01 and ( * * * ) P ≤ 0 . 001 .

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A, B The expression level of OsHKT2;1 and OsHKT1;5 in osprr73 mutant and the wild control Dongjin (DJ) plants with or without salinity stress in multiple time points. DJ Data represent means ± SD. n = 3, technical replicates. The representative data is from at least two individual biological replicates. The asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01 and (***) P ≤ 0.001.


C Transient expression assay in the infiltrated leaves of N . benthamiana showing OsPRR73 can repress OsHKT2 ; 1 transcription . Left panel showing the bioluminescence signal of OsHKT2 ; 1 and OsPRR73 . Bioluminescence of co - transformation of either GFP or 35S : GFP - OsPRR73 ( GFP - OsPRR73 ) with OsHKT2 ; 1pro : LUC in N . benthamiana . D Quantiﬁcation of bioluminescence intensity of OsHKT2 ; 1pro : LUC shown in ( C ) . Data represent means ± SD . n = 10 . The asterisks indicate the significant difference by student ' s t - test with ( * * * ) P ≤ 0 . 001 . At least two biological replicates were conducted with similar results .

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C Transient expression assay in the infiltrated leaves of N. benthamiana showing OsPRR73 can repress OsHKT2;1 transcription. Left panel showing the bioluminescence signal of OsHKT2;1 and OsPRR73. Bioluminescence of co-transformation of either GFP or 35S: GFP-OsPRR73 (GFP-OsPRR73) with OsHKT2;1pro: LUC in N. benthamiana. D Quantiﬁcation of bioluminescence intensity of OsHKT2;1pro: LUC shown in (C). Data represent means ± SD. n = 10. The asterisks indicate the significant difference by student's t-test with (***) P ≤ 0.001. At least two biological replicates were conducted with similar results.


E ChIP - qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2 ; 1 promoter by OsPRR73 , compared to wild type DJ controls . The amplicon of Ubiquitin was taken as a negative control . Two - week - old seedlings were harvested at ZT12 . Data represent means ± SD ( n = 3 , technical repeats ) . The experiments were performed at least two biological replicates with similar result . Top scheme indicates the locations of amplicons for ChIP analysis . The asterisks represent significant difference among means by student ' s t - test with ( * * ) P ≤ 0 . 01 .

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E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01.


F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2 ; 1 promoter . Unlabelled probes were used as competitor with indicated folds . MBP alone was used as negative control . Arrowhead marks the shifted positive bands .

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F EMSA assay with the OsPRR73 incubated with the probes designed for the S4 region of OsHKT2;1 promoter. Unlabelled probes were used as competitor with indicated folds. MBP alone was used as negative control. Arrowhead marks the shifted positive bands.


A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo . CoIP was conducted with GFP - Trap beads , and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated .

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A Coimmunoprecipitation assay showing the physical interaction between OsPRR73 and HDAC10 in vivo. CoIP was conducted with GFP-Trap beads, and GFP and FLAG antibodies were used to detect OsPRR73 and HDAC10 in immunoprecipitants as indicated.


B Split luciferase complementation ( SLC ) assay showing OsPRR73 interacts with HDAC10 in the co - infiltrated N . benthamiana leaves .

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B Split luciferase complementation (SLC) assay showing OsPRR73 interacts with HDAC10 in the co-infiltrated N.benthamiana leaves.


C Bimolecular - fluorescence complementation ( BiFC ) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus . BF represents bright field . Scale bar , 40 µm .

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C Bimolecular-fluorescence complementation (BiFC) assay shows the interaction between OsPRR73 and HDAC10 occurs in nucleus. BF represents bright field. Scale bar, 40 µm.


D N - terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10 . Upper panel shows the schematic illustration of full - length and the truncated OsPRR73 proteins . CoIP was performed with GFP - Trap beads , and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively .

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D N-terminus of OsPRR73 containing PR domain is required and sufficient for interacting with HDAC10. Upper panel shows the schematic illustration of full-length and the truncated OsPRR73 proteins. CoIP was performed with GFP-Trap beads, and immunoprecipitants were detected by FLAG for HADC10 and GFP for truncated proteins of OsPRR73 respectively.


E , F Chromatin Immunoprecipitation - qPCR ( ChIP - qPCR ) assay to detect the enriched promoter regions of OsHKT2 ; 1 by using the H3K9ac ( E ) and H3 ( F ) antibodies . Two - week - old seedlings were harvested at ZT12 . The locations of amplicons were described in Figure 4E . Data represent means ± SD . n = 3 technical replicates . The asterisk indicates the significant difference by student ' s t - test with ( * ) P ≤ 0 . 05 . The experiments were repeated at least twice with similar result .

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E, F Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay to detect the enriched promoter regions of OsHKT2;1 by using the H3K9ac (E) and H3 (F) antibodies. Two-week-old seedlings were harvested at ZT12. The locations of amplicons were described in Figure 4E. Data represent means ± SD. n = 3 technical replicates. The asterisk indicates the significant difference by student's t-test with (*) P ≤ 0.05. The experiments were repeated at least twice with similar result.


D The oshkt2 ; 1 seedling is more tolerant to the treatment of NaCl . The oshkt2 ; 1 and NIP , its wild type , four - week - old seedlings grown under NaCl conditions for 0 days ( left panel ) , then were transferred to 200 mM NaCl for 28 days ( middle panel ) , and recovered for 7 days ( right panel ) . Scale bar , 5 cm . E Statistical analysis of survival rate in ( D ) . Data represent means ± SD . n = 3 biological replicates , 24 plants of each replicate . ( * ) P ≤ 0 . 05 was generated by student ' s t - test .

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D The oshkt2;1 seedling is more tolerant to the treatment of NaCl. The oshkt2;1 and NIP, its wild type, four-week-old seedlings grown under NaCl conditions for 0 days (left panel), then were transferred to 200 mM NaCl for 28 days (middle panel), and recovered for 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rate in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. (*) P ≤ 0.05 was generated by student's t-test.


A Malondialdehyde ( MDA ) content in leaves of 14 - d - old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition . Data represent means ± SD . n = 6 , biological replicates . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test .

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A Malondialdehyde (MDA) content in leaves of 14-d-old DJ and osprr73 plants treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.


B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days . Scale bar , 1 cm .

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B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM NaCl treatment for 3 days. Scale bar, 1 cm.


C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants . Scale bar , 5 mm . - NaCl and + NaCl represented the untreated or treated plants by NaCl respectively .

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C H2O2 concentration was detected by using H2DCFDA in the root tip of DJ and osprr73 mutant plants. Scale bar, 5 mm. -NaCl and +NaCl represented the untreated or treated plants by NaCl respectively.


D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant . Data represent means ± SD . n ≥ 6 . The asterisks indicate the significant difference ( * * * ) P ≤ 0 . 001 by student ' s t - test .

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D Quantitative analysis of H2O2 concentration in the root of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.


E , F Measurement of SOD ( Superoxide dismutase ) ( E ) and CAT ( Catalase ) ( F ) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress . Data represent means ± SD . n = 3 technical replicates . Data are representative from two independent biological replicates with similar result . The asterisks indicate the significant difference ( * ) P ≤ 0 . 05 by student ' s t - test .

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E, F Measurement of SOD (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in shoots of DJ and osprr73 seedlings with or without NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.


scRNA - Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only ( GPI ) in primary CD4 + T - cells infected in vitro ( D , E ) or CD4 + T - cells of PLWH ( F ) . In panels ( D , E ) , cells were infected with VSVG - HIV - 1 - GFP and sorted for viral expression as detailed in ( Golumbeanu et al , 2018 ) . Following latency establishment , cells were left untreated or HIV - 1 expression was reactivated through suberoyl anilide hydroxamic acid ( SAHA ) or α - CD3 - CD28 engagement . Clusters 1 and 2 were identified by principal component analysis as described in ( Golumbeanu et al , 2018 ) . In panel ( F ) , CD4 + T - cells were isolated from total blood of PLWH under ART as described in ( Cohn et al , 2018 ) . Viral expression was reactivated by treatment with phytohemagglutinin ( PHA ) and cells were sorted using antibodies against Env and Gag . Sorted cells were then subjected to scRNA - Seq analysis . The expression level of the HUMAN - GLYCOLYSIS pathway in ( D ) was calculated as the average expression of genes comprising the gene list ; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test . For panels ( E , F ) , significance of GPI differential expression level between clusters ( E ) or between control and Env + Gag + conditions ( F ) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function . * * p < 0 . 01 , * * * p < 0 . 001 ; * * * p < 0 . 0001 . For panels ( D , E ) n = 1 donor , 43 cells ( untreated ) , 90 cells ( SAHA ) , 91 cells ( TCR ) , for panel ( F ) n = 3 donors , 109 cells ( control ) and 85 cells ( Gag + Env + ) .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only (GPI) in primary CD4+ T-cells infected in vitro (D,E) or CD4+ T-cells of PLWH (F). In panels (D, E), cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in (Golumbeanu et al, 2018). In panel (F), CD4+ T-cells were isolated from total blood of PLWH under ART as described in (Cohn et al, 2018). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env+ Gag+ conditions (F) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function. ** p< 0.01, *** p< 0.001; *** p< 0.0001. For panels (D,E) n = 1 donor, 43 cells (untreated), 90 cells (SAHA ), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag+ Env+).


Relative ratios of NADH / NAD + ( D ) and ATP / ADP ( E ) in latently infected and reactivated cells . Data were normalized using the matching uninfected control .

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Relative ratios of NADH/NAD+ (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.


Relative expression of HIV - 1 gag in cells infected with Tat - deficient HIV - 1pNL4 - 3 as compared to cells infected with wild type HIV - 1 pNL4 - 3 .

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Relative expression of HIV-1 gag in cells infected with Tat-deficient HIV-1pNL4-3 as compared to cells infected with wild type HIV-1 pNL4-3.


relative expression of the limiting rate enzyme of the pentose phosphate pathway , i . e . G6PD ( B ) in HIV - 1 infected as compared to mock infected cells .

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relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B) in HIV-1 infected as compared to mock infected cells.


relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways , i . e . TrxR1 ( C ) and GCLC ( D ) , in HIV - 1 infected as compared to mock infected cells .

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relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.


Reactivation from HIV - 1 latency ( A ) and relative cell viability ( B ) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin ( AF ; 500 nM ) , the GSH inhibitor buthionine sulfoximine ( BSO ; 250 μM ) , or a combination of the two . GFP - HIV - 1 expression was determined by FACS . Data are expressed as mean ± SD of three replicates and were analyzed by One - Way ANOVA followed by Tukey ' s post - test ( A ) or Two - Way ANOVA followed by Sidak ´ s post - test ( B ) . Solid lines represent the means .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reactivation from HIV-1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP-HIV-1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by One-Way ANOVA followed by Tukey's post-test (A) or Two-Way ANOVA followed by Sidak´s post-test (B). Solid lines represent the means.


Levels of integrated HIV - 1 DNA following treatment for 48 h with AF and / or BSO in CD4 + T - cells derived from PLWH under suppressive antiretroviral therapy . Live cells were sorted after treatment , and integrated DNA was measured by Alu - PCR . The latency reactivating agent SAHA was used as a reference compound ( Archin et al , 2012 ) . Data were analyzed by non - parametric Friedman ´ s test followed by Dunn ' s post - test .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4+ T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al, 2012). Data were analyzed by non-parametric Friedman´s test followed by Dunn's post-test.


EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation ( A - C ) ( A , C ) The successive pellets ( 2K , 10K , 100K ) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side - by - side with the lysate of 0 . 4 x 106 producing cells : C . Images from two different representative experiments are shown . Amount of proteins recovered in 2K , 10K and 100K pellets analyzed in stain - free gel images was similar between pellets : AU ( protein amount in a given pellet / total protein amount in 2K + 10K + 100K ) for 2K , 10K and 100K pellets were 1 . 13 ± 0 . 28 , 0 , 96 ± 0 . 10 and 0 . 91 ± 0 . 33 , respectively in control cells ( A ) , and 1 . 22 ± 0 . 29 , 0 , 84 ± 0 . 35 and 0 . 94 ± 0 . 30 , respectively in infected cells ( C ) ( mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation (A-C) (A, C) The successive pellets (2K, 10K, 100K) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side-by-side with the lysate of 0.4 x 106 producing cells: C. Images from two different representative experiments are shown. Amount of proteins recovered in 2K, 10K and 100K pellets analyzed in stain-free gel images was similar between pellets: AU (protein amount in a given pellet/total protein amount in 2K+10K+100K) for 2K, 10K and 100K pellets were 1.13±0.28, 0,96±0.10 and 0.91±0.33, respectively in control cells (A), and 1.22±0.29, 0,84±0.35 and 0.94±0.30, respectively in infected cells (C) (mean±SD).


EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD ) . ( B ) Whole - mount EM analysis showing representative images and size distribution of particles in 2K , 10K and 100K pellets ( Scale bar , 200 nm ) . The diameter ( nm ) of particles was determined with ImageJ for five different experiments . 387 , 363 and 385 particles were counted for 2K , 10K and 100K pellets , respectivelly .

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EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD). (B) Whole-mount EM analysis showing representative images and size distribution of particles in 2K, 10K and 100K pellets (Scale bar, 200 nm). The diameter (nm) of particles was determined with ImageJ for five different experiments. 387, 363 and 385 particles were counted for 2K, 10K and 100K pellets, respectivelly.


( A - D ) EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient ( D ) . ( D ) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non - infected and infected Jurkat cells and analysed by Western blot for the indicated proteins .

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(A-D) EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient (D). (D) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non-infected and infected Jurkat cells and analysed by Western blot for the indicated proteins.


( A ) Proteomic profiles of different proteins , showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells . Proteins with very similar profiles ( represented by the same colours ) are likely part of the same EV subtypes . Each profile consists of three independent data triplets ( F1 - F2 - F3A , F1 - F2 - F3B and F1 - F2 - F3C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C).


( C ) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1 - F2 - F3 subfractions as compared to expression in the total cell proteome . Solid horizontal lines indicate medians and dashed line indicate quartiles ( n = 3 ) . Mitochondria and ER markers are de - enriched , and plasma membrane markers are enriched in EV fractions , with progressively pronounced effects from F1 to F3 .

(C) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians and dashed line indicate quartiles (n=3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3.


EVs were purified by SEC from supernatants of Jurkat cells . ( A ) EVs were subjected to immuno - isolation with beads coupled to antibodies against CD81 or CD63 . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for CD63 , Syntenin - 1 , CD81 , ADAM10 and CD3G . A representative blot ( top panel ) and quantification ( mean ± SD ) of the proportion of signal in FT as compared with total ( PD + FT ) from four independent experiments ( bottom panel ) are shown .

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EVs were purified by SEC from supernatants of Jurkat cells. (A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown.


( B ) Representative images of double - immunogold labelling of EVs purified by SEC from Jurkat cells . Left panel , CD81 / CD63 ; right panel , CD81 / CD3E . Arrows show CD63 staining in CD81 + CD63 + EVs .

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(B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs.


EVs were purified by SEC from supernatants of Jurkat cells . ( C , D ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with 2 x 109 particles . Captured EVs were detected with either APC - labeled anti - CD81 , anti - CD63 or anti - CD3E . ( C ) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads ( log10 scale ) . shown . ( D ) Heat - map representation of the median APC fluorescence values for the different bead populations detected with anti - CD63 or anti - CD3E antibodies relative to the values detected with anti - CD81 .

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EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.


( B ) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions . Each profile consists of two independent data triplets ( F1 - F2 - F3A and F1 - F2 - F3B ) . Abundance profiles of the same groups of proteins showed the same clustering behaviour , although in this second dataset , the proportion of proteins recovered in the F2 was more variable . These differences , however , did not affect the results of the NNP or movement analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions. Each profile consists of two independent data triplets (F1- F2- F3A and F1- F2- F3B). Abundance profiles of the same groups of proteins showed the same clustering behaviour, although in this second dataset, the proportion of proteins recovered in the F2 was more variable. These differences, however, did not affect the results of the NNP or movement analysis.


( C ) Unbiased identification of significant translocation events triggered by HIV - 1 infection . Each protein is scored for magnitude of translocation ( M score , x - axis ) and reproducibility of translocation direction ( R score , y - axis ) across the two replicates . MR plot analysis reveals significant translocations in the top right quadrant . Proteins with M > 1 and R > 0 . 9 are candidate hits for changing localization ( estimated FDR = 8 % ) .

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(C) Unbiased identification of significant translocation events triggered by HIV-1 infection. Each protein is scored for magnitude of translocation (M score, x-axis) and reproducibility of translocation direction (R score, y-axis) across the two replicates. MR plot analysis reveals significant translocations in the top right quadrant. Proteins with M>1 and R>0.9 are candidate hits for changing localization (estimated FDR = 8%).


( A , B ) ( A ) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected ( red boxes ) Jurkat cells . The successive pellets ( 2K , 10K , 100K ) were analyzed by Western blot , using antibodies against SPN , MOV10 , SERINC3 and p24 ( HIV - 1 protein ) . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( A ) and quantifications in three to five independent experiments ( B ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet . In cell lysates from the same experiments , AU ( given protein / total amount of protein ) for SPN and MOV10 were 1 . 29 ± 0 . 15 and 0 . 84 ± 0 . 33 times the levels in control cells , respectively ( Mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD).


( C ) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV - 1 infected ( red boxes ) cells . Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN . SERINC3 was recovered in the middle fractions . ( T = top ; B = bottom ) . ( D , E ) Quantification of MOV10 , SPN , SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV - 1 infected samples . AU = band intensity in a given pellet / sum ( band intensity in all six pellets ) . ( F ) Quantification of SERINC3 signals in the different fractions from control and infected samples . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Lines represent the medians of three independent experiments .

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(C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom). (D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets). (F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments.


( G , H ) EVs from supernatant of HIV - infected Jurkat cells were subjected to immuno - isolation with beads coupled to antibody against SPN . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for SPN , MOV10 , CD63 , SERINC3 and viral p24 . A representative image ( G ) and quantification ( H ) Mean ± SD of the proportion of signal in PD as compared with total ( PD + FT ) in three independent experiments are shown .

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(G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown.


( I ) EVs remaining in the FT , or EVs secreted by the same number of cells but not subjected to immuno - isolation ( input ) were used to infect a reporter cell line . Mean ± SD of six independent experiments is shown .

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(I) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 ( A ) GFP + Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells . Results are expressed as a ratio to the number of GFP + cells after treatment with supernatant from infected control cells . . Mean ± SD of three independent experiments is shown .

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Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 (A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown.


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( B ) Number of particles secreted were quantified by NTA . Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells .

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Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells.


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( C ) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation . Six fractions were recovered and analyzed by Western blot for the presence of SPN , SERINC3 and p24 . ( T = top ; B = bottom ) . ( D ) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations ( exp 1 : open symbols , exp 2 : closed symbols ) . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets).


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed by Western blot with antibodies against SPN , and p24 . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( E )

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Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E)


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed and quantification of SPN and p24 signals ( F ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K + 10K + 100K from control cells .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells.


Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( G ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with EV samples . Captured EVs were detected with a mixture of APC - labeled anti - CD9 , anti - CD63 , and anti - CD81 . Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction . Results were relativized to the values obtained with control sample for each experiment . Dotted line represents values obtained for EVs isolated from control cell CM .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.


Immunostaining of H3K27ac in mouse germinal vesicle ( GV ) oocytes ( n = 8 ) , metaphase II ( MII ) oocytes ( n = 7 ) , zygotes ( n = 7 ) , early 2 - cell ( 2C ) ( n = 7 ) and late 2C ( n = 9 ) embryos . Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages . Scale bar : 20 μm . Data information : For boxplots in A the central band represents the median . The lower and upper edges of the box represent the first and third quartiles , respectively . The whiskers of the boxplot extend to 1 . 5 times inter - quartile range ( IQR ) .

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Immunostaining of H3K27ac in mouse germinal vesicle (GV) oocytes (n = 8), metaphase II (MII) oocytes (n = 7), zygotes (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages. Scale bar: 20 μm. Data information: For boxplots in A the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).


Immunostaining of H3K27ac in A - 485 treated embryos versus control ( DMSO treated ) at different time points . Scale bar : 20 μm .

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Immunostaining of H3K27ac in A-485 treated embryos versus control (DMSO treated) at different time points. Scale bar: 20 μm.


Developmental rate of embryos treated with A - 485 versus control .

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Developmental rate of embryos treated with A-485 versus control.


Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control ( DMSO treated ) at 28 hpi . Scale bar : 20 μm .

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Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control (DMSO treated) at 28 hpi. Scale bar: 20 μm.


Developmental rate of embryos treated with TSA at different time points ( 0 - 20 hpi and 8 - 28 hpi ) versus control .

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Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.


( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assays . * P < 0 . 05 ( one - way ANOVA ) .

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(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).


( B ) HT29 cells were infected with Shigella WT or ∆ ospD3 and incubated for 8 h . Infected cells were fixed and subjected to TUNEL and PI staining . Percentages of positive cells ( TUNEL , green ; PI , red ) are shown in graph at right . The nuclei were stained with DAPI ( blue ) . Scale bar : 100 μm . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


( C ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospD3 and incubated for 8 h . Infected cells were subjected to Giemsa staining . Arrows indicate cells in which the cytoplasm disappeared . Scale bar : 20 μm .

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(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.


( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Cell lysates were subjected to immunoblotting .

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(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.


( B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

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(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


C ) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

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C) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay


( D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


E ) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h , and then cell lysates were subjected to immunoblotting .

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(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.


( B ) 293T cells were transfected with Myc - tagged ospD expression plasmids . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 .

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(B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.


D ) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 .

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D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.


( E HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

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(E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


F ) HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays


( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 8 activation . Caspase - 8 activity is reported as relative light units ( RLU ) of infected samples , minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).


( B ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . Arrows indicate cleaved forms of caspase - 8 , PARP , GSDMD , and IL - 18 , respectively .

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(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.


( C ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assay . * P < 0 . 05 ; n . s . , not significant ( one - way ANOVA ) .

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(C) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).


( D ) HT29 cells were infected with Shigella WT or ∆ ospC1 mutant and incubated for 8 h . Infected cells were then fixed and stained with cleaved caspase - 8 ( green ) , rhodamine - phalloidin ( red ) , and DAPI ( blue ) . Percentages of positive cells are shown in the graph at right ( * P < 0 . 05 ; unpaired two - tailed Student ' s t - test ) . Scale bar : 100 μm .

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(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.