L Representative immunofluorescent images of eEF2 ( magenta ) and PLA signals ( white ) in lentivirus ( LV ) - scramble - EGFP or LV - Eef2 shRNA - EGFP infected primary cortical neurons ( green , DIV 14 ) . Scale bar , 10 μm . M Quantification of mean fluorescent intensity of eEF2 in EGFP + cells in scramble and Eef2 shRNA groups . 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . N Quantification of mean fluorescent intensity of PLA signals in EGFP + cells in the two groups . The PLA signals represent newly synthesized GluA2 . 64 cells for scramble group , 51 cells for Eef2 shRNA group from 3 independent experiments . Mann - Whitney test . * * * * p < 0 . 0001 . Data information : All data are shown as means ± s . e . m . All the tests are two - tailed .

O O B-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L Representative immunofluorescent images of eEF2 (magenta) and PLA signals (white) in lentivirus (LV)-scramble-EGFP or LV-Eef2 shRNA-EGFP infected primary cortical neurons (green, DIV 14). Scale bar, 10 μm. M Quantification of mean fluorescent intensity of eEF2 in EGFP+ cells in scramble and Eef2 shRNA groups. 64 cells for scramble group and 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. N Quantification of mean fluorescent intensity of PLA signals in EGFP+ cells in the two groups. The PLA signals represent newly synthesized GluA2. 64 cells for scramble group, 51 cells for Eef2 shRNA group from 3 independent experiments. Mann-Whitney test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.


A PF - 4778574 ( PF ) directly potentiates EPSC amplitude , dashed line represents baseline value of normalized EPSC amplitude . Bottom : representative EPSC traces from baseline ( 1 ) , maximal potentiation ( 2 ) , and recovery ( 3 ) . B , C Quantifications of EPSC peak amplitude ( B ) and decay time ( C ) after PF ( 6 cells from 3 HET mice ) . ( B ) Wilcoxon signed - rank test , * p = 0 . 0313 ( PF v . s . baseline ) and p = 0 . 1600 ( Wash out v . s . baseline ) ; ( C ) Paired t test , * * * * p < 0 . 0001 ( PF v . s . baseline ) and p = 0 . 4423 ( Wash out v . s . baseline ) . Data information : Data are shown as means ± s . e . m . ,

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PF-4778574 (PF) directly potentiates EPSC amplitude, dashed line represents baseline value of normalized EPSC amplitude. Bottom: representative EPSC traces from baseline (1), maximal potentiation (2), and recovery (3). B, C Quantifications of EPSC peak amplitude (B) and decay time (C) after PF (6 cells from 3 HET mice). (B) Wilcoxon signed-rank test, *p=0.0313 (PF v.s. baseline) and p=0.1600 (Wash out v.s. baseline); (C) Paired t test, ****p<0.0001 (PF v.s. baseline) and p=0.4423 (Wash out v.s. baseline). Data information: Data are shown as means ± s.e.m., data


D PF rescued AMPAR - EPSC in L5 neurons from HET mice . 25 - 30 % increase at strongest stimulation ( 7 cells from 3 HET mice ) , paired t test , * * p = 0 . 0014 . Bottom : representative traces of the effects of PF on AMPAR - EPSC evoked by different stimulus intensities . Data information : Data are shown as means ± s . e . m .

O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

D PF rescued AMPAR-EPSC in L5 neurons from HET mice. 25-30% increase at strongest stimulation (7 cells from 3 HET mice), paired t test, **p=0.0014. Bottom: representative traces of the effects of PF on AMPAR-EPSC evoked by different stimulus intensities. Data information: Data are shown as means ± s.e.m.


E Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) in the Three - chamber test performed by Eef2 HET mice intraperitoneal administered with PF - 4778574 or DMSO ( vehicle control ) . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . F Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 7335 . WT , n = 9 mice ; HET , n = 8 mice . G Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0492 . WT , n = 9 mice ; HET , n = 8 mice . Data information : Data F , G , are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O 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-ORGANISM O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O 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-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative heatmaps of the two social tasks (left, social approach; right, social novelty) in the Three-chamber test performed by Eef2 HET mice intraperitoneal administered with PF-4778574 or DMSO (vehicle control). E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. F Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.7335. WT, n=9 mice; HET, n=8 mice. G Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0492. WT, n=9 mice; HET, n=8 mice. Data information: Data F, G, are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.


I Representative heatmaps of the two social tasks ( left , social approach ; right , social novelty ) performed by Eef2 HET mice with mPFC infusion of PF - 4778574 or DMSO ( vehicle control ) in the Three - chamber test . E : empty cup ; S1 : stranger # 1 contained cup ; S2 : stranger # 2 contained cup . J Discrimination score of social approach performed by two groups of mice , calculated by the difference of time spent in sniffing S1 and E . Unpaired t test . p = 0 . 3984 . n = 11 mice for each group . K Discrimination score of social novelty performed by two groups of mice , calculated by the difference of time spent in sniffing S2 and S1 . Unpaired t test . * p = 0 . 0489 . n = 11 mice for each group . Data information : Data of J and K are shown as interquartile range , with line across the box indicating median , whiskers show the highest and lowest values . n . s . , not significant . All the t tests are two - tailed .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-ORGANISM O B-TISSUE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O 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-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Representative heatmaps of the two social tasks (left, social approach; right, social novelty) performed by Eef2 HET mice with mPFC infusion of PF-4778574 or DMSO (vehicle control) in the Three-chamber test. E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. J Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.3984. n=11 mice for each group. K Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0489. n=11 mice for each group. Data information: Data of J and K are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.


Three independent samples ( Exp 1 to 3 ) of EV secreted by labeled 3T3 - F442A cells were analyzed by mass spectrometry ( in duplicate injections , Inj1 / 2 ) . The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated .

O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.


Two human ( SKMEL28 and 1205Lu ) and a murine ( B16BL6 ) melanoma cell lines were exposed , or not , to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) , then FAO was measured ( n = 5 for B16BL6 , SKMEL28 HFD EV and 1205Lu ND EV and n = 6 for other conditions ) .

O B-ORGANISM O B-CELL_LINE O B-CELL_LINE O O O B-ORGANISM O B-CELL_LINE O B-DISEASE O O O O O O O O O O O B-SUBCELLULAR O O B-ORGANISM B-CELL_TYPE O O O B-ORGANISM O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O B-CELL_LINE O B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O O

Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).


Volcano plot of mass spectrometry - based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice ( HFD ) , as compared to those from lean mice ( ND ) . The dashed lines indicate cut - off values and points colored in grey indicate proteins that display non - significant fold - change by Welch t - test between both conditions ( n = 3 for ND , 4 for HFD ) . Proteins involved in FAO are indicated by yellow dots .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE B-SUBCELLULAR O B-DISEASE B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O

Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots.


Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice ( top panel ) and from human individuals with varying BMI ( normal weight , NW ; overweight , OW and obese , OB ) ( bottom panel ) . For each blot , extracts from 3 independent batches of murine samples or 3 independent individuals for human samples ( 1 - 3 ) are shown . Flotillin 1 ( FLOT1 ) is used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O B-DISEASE O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O B-DISEASE O B-DISEASE O B-DISEASE O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O

Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control.


Western blot analysis of the indicated FAO enzymes in melanoma cells treated , or not , with EV from lean ( ND ) and obese ( HFD ) mice . Tubulin ( TUB ) is used as a loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-DISEASE O O O O O O O B-SUBCELLULAR O O O O O O B-DISEASE O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O

Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.


RT - qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice for 48 h . Results are expressed relative to the corresponding value for control cells ( n = 3 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O B-DISEASE O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3).


Analysis of FAO levels in 1205Lu cells exposed to 3T3 - F442A EV and treated , or not , with cycloheximide ( CHX ) ( n = 5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).


Lipids were extracted from EV secreted by 3T3 - F442A pre - adipocytes and differentiated 3T3 - F442A adipocytes ( respectively preAd - 3T3 - EV and Ad - 3T3 - EV ) , and FA content was measured ( n = 6 ) .

B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O B-CELL_LINE O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_LINE O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O

Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).


Indicated melanoma cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 ( 3T3 - FL C16 - EV ) and , 24h later , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy .

O B-DISEASE O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O

Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.


Left panel , indicated cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated , or not , with Etomoxir for 24h . Then , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy . Right panel , quantification of BODIPY FL C16 staining area per cell ( n = 3 ) .

O O O O O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O

Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).


Lipids were extracted from EV secreted by adipocytes from lean ( ND ) and obese ( HFD ) mice ( n = 5 ) ( left panel ) or from human adipose tissue samples from patients with varying BMI ( right panel ) and fatty acid content was measured ( n = 15 ) .

B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O B-DISEASE O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).


Indicated cells were exposed , or not , to adipocyte EV from primary adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) for 24h . Then , cells were fixed , stained with BODIPY and counterstained with DAPI . Left panel , confocal microscopy observation . Right panel , quantification of BODIPY staining area per cell ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

O O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O

Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).


Transmission electron micrographs of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) . Scale bar represents 1µm . A zoomed crop of the area with autophagic structures containing lipids ( indicated by an arrow ) is shown ( scale bar : 0 . 5µm ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).


1205Lu cells were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 in the presence , or not , of Lalistat 2 . Then , live cells were stained with the LysoTracker probe and observed by confocal microscopy . Arrows indicate colocalization .

B-CELL_LINE O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O

1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.


1205Lu were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 and treated , or not , with Lalistat 2 . Then , cells were fixed and counterstained with DAPI before observation by confocal microscopy . Quantification of BOBIPY FL C16 staining per cell is shown beside ( n = 4 ) .

B-CELL_LINE O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O

1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Lalistat 2 ( Lal ) . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 5 ) or cell motility was tracked by video microscopy ( right panel ; n = 40 cells per group ) .

B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).


1205Lu cells were exposed , or not , to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) with , or without , Lalistat 2 ( Lal ) . Cells were then fixed , stained with BODIPY and counterstain with DAPI before observation by confocal microscopy . Quantification of BODIPY staining per cell is shown on the right ( n = 5 ) .

B-CELL_LINE O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O B-ORGANISM O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O

1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).


1205Lu cells were exposed , or not , to adipocyte EV from ND or HFD mice with , or without , Lalistat 2 ( Lal ) . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

B-CELL_LINE O O O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O

1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).


1205Lu cells exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 , were stained with a MitoTracker probe , fixed and observed by confocal microscopy . Arrows indicate the presence of mitochondria in membrane protrusions . On the images to the right , the outline of cells is shown in dotted lines . Scale bars : 20µm . Right panel , quantification of the distance between mitochondria and nuclei ( n = 15 ) .

B-CELL_LINE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O

1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).


Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated , or not , with EV from 3T3 - F442A adipocytes ( 3T3 - EV ) . Tubulin ( TUB ) is used as a loading control .

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Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 6 ) . Right panel , 3T3 - F442A EV were added to 1205Lu cells , which were immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) ( right panel ) .

B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).


1205Lu cells were transfected , or not , with 2 different siRNA targeted against DRP1 ( siDRP1 # 1 or 2 ) or an untargeted siRNA ( siUT ) . Top , 48h after transfection , protein extracts were prepared and DRP1 expression was evaluated by western blot . Bottom , 36h post transfection , cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and cell motility was tracked by video microscopy ( n = 40 cells per group ) .

B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O 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-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O

1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).


1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) and immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

B-CELL_LINE O O O O B-CELL_TYPE B-SUBCELLULAR O O B-ORGANISM O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O

1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).


Left panel , indicated melanoma cells exposed to 3T3 - F442A EV were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 6 ) .

O O O O B-DISEASE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).


Left panel , melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

O O O B-DISEASE O O O B-SUBCELLULAR O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O

Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).


1205Lu cells exposed to 3T3 - F442A EV were stained with a MitoTracker probe . Then , cells were fixed and stained with BODIPY before observation by confocal microscopy . A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown ( scale bar : 5µm ) .

B-CELL_LINE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O

1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).


Transmission electron microscope observations of 1205Lu cells exposed , or not , to 3T3 - F442A EV . Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O

Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right.


Number of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) ( n = 28 for control and n = 27 for 3T3 - EV ) . Area of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( n = 19 for control and n = 25 for 3T3 - EV ) .

O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O B-CELL_LINE O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_LINE O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O B-SUBCELLULAR O O

Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV).


Left panel , 1205Lu melanoma cells were exposed , or not , to 3T3 - F442A EV . Then , live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy . Right panel , quantification of the percentage of cells presenting lysosome within membrane protrusions ( n = 3 ) .

O O O B-CELL_LINE B-DISEASE O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).


TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 449 , 450 and 459 for HADHA , HADHB and ECHS1 respectively ) .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-ORGANISM O B-DISEASE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).


BODIPY staining of neutral lipid stores in the indicated melanoma cell lines , which present increasing degrees of aggressiveness ( from left to right ) . DAPI was used to counterstain nuclei . Scale bar : 20µm .

B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O

BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.


FAO in the indicated melanoma cell lines was measured . Bars and error bars represent means ± SEM ( n = 5 ) ; statistically significant by one - way ANOVA with post hoc Tukey ' s test , * * * P < 0 . 001 . If not indicated , non - significant .

B-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.


TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 459 ) .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-ORGANISM O B-DISEASE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).


( A ) RT - PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages . NC indicates the no template control .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O

(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control.


( B ) Time - course expression of sinhcaf revealed by whole - mount in situ hybridization . Scale bars , 100

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ


( C ) RT - PCR analysis for spatial expression of sinhcaf mRNA in adult tissues .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O

(C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues.


( E ) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf - / - mutant embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( F ) Fluorescence microscopy of 10hpf Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Scale bar , 400 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O

(F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm.


( G ) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg ( CMV : EGFP ; CMV : sinhcaf ) embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by Holm - Sidak post - hoc test .

O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.


( H ) Percentage of male zebrafish in WT ( n = 274 ) , MZsinhcaf - / - ( n = 166 ) , Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 158 ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 135 ) adult zebrafish . Data shown are mean ± SEM ( n = the number of fish analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

O O O O O O B-ORGANISM 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-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( A and B ) Whole - mount in situ hybridization was performed in WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos with PGCs markers nanos3 ( A ) and vasa ( B ) . Scale bars , 100 μm . ( C ) Quantitative analysis of PGCs in bud stage WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Data shown are mean ± SEM ( n = 10 , the number of embryos analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM B-TISSUE O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 and B) Whole-mount in situ hybridization was performed in WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos with PGCs markers nanos3 (A) and vasa (B). Scale bars, 100 μm. (C) Quantitative analysis of PGCs in bud stage WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Data shown are mean ± SEM (n = 10, the number of embryos analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


( D ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos , dorsal views . Arrowheads point to PGCs . Scale bars , 100 μm . ( E ) Quantitative analysis of PGCs in 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos . Data shown are mean ± SEM ( n = 13 , the number of embryos analyzed ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O

(D) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP) zebrafish embryos, dorsal views. Arrowheads point to PGCs. Scale bars, 100 μm. (E) Quantitative analysis of PGCs in 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP)zebrafish embryos. Data shown are mean ± SEM (n = 13, the number of embryos analyzed). Statistical analysis was performed using unpaired two-tailed Student's t-test.


( A ) Whole - mount in situ hybridization was performed in 4 - cell stage WT , MZsinhcaf - / - zebrafish embryos with germ plasm marker vasa . Red arrowheads point to cleavage furrows in the 4 - cell embryos . Scale bars , 100

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O

(A) Whole-mount in situ hybridization was performed in 4-cell stage WT, MZsinhcaf -/- zebrafish embryos with germ plasm marker vasa. Red arrowheads point to cleavage furrows in the 4-cell embryos. Scale bars, 100 μm


( B ) Relative mRNA levels of germ plasm genes vasa , dnd , nanos3 , dazl and buc in WT and MZsinhcaf - / - mutant eggs , respectively , as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by the Holm - Sidak post - hoc test ; NS , no significant difference .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA levels of germ plasm genes vasa, dnd, nanos3, dazl and buc in WT and MZsinhcaf -/- mutant eggs, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by the Holm-Sidak post-hoc test; NS, no significant difference.


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos . Scale bars , 100 μm .

O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O

(C) Maximal intensity projection of a confocal z-stack of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. White arrowheads point to cleavage furrows of 4-cell embryos. Scale bars, 100 μm.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data shown are mean ± SEM ( n = 12 , the number of embryos analyzed ) .

O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O 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-TISSUE O O O

(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. Statistical analysis was performed using unpaired two-tailed Student's t-test. Data shown are mean ± SEM (n = 12, the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test.


( F ) Western blots for GFP - Buc expression in 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD

(F) Western blots for GFP-Buc expression in 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA


( A , B and C ) Co - immunoprecipitation of zebrafish Sinhcaf - GFP with HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) in HEK293Tcells transfected with both Sinhcaf - GFP and HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) plasmids .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O

(A, B and C) Co-immunoprecipitation of zebrafish Sinhcaf-GFP with HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) in HEK293Tcells transfected with both Sinhcaf-GFP and HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) plasmids.


( D ) Relative mRNA level of sinhcaf in zebrafish PG , PV , EV , MV and FG follicles was measured using RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

O O O O O O O B-GENEPROD O B-ORGANISM B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-TISSUE I-TISSUE O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O

(D) Relative mRNA level of sinhcaf in zebrafish PG, PV, EV, MV and FG follicles was measured using RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( E and F ) Western blots for acetyl - histone H3 ( K9 ) ( Ace - H3 - K9 ) , histone H3 in WT ( E ) or MZsinhcaf - / - ( F ) PG , PV , EV , MV and FG follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O

(E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( G and H ) Statistical analysis of the relative levels of Ace - H3 - K9 in WT ( G ) or MZsinhcaf - / - ( H ) PG , PV , EV , MV and FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) .

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O

(G and H) Statistical analysis of the relative levels of Ace-H3-K9 in WT (G) or MZsinhcaf -/- (H) PG, PV, EV, MV and FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).


( I , J and K ) Western blots for Ace - H3 - K9 and H3 in WT and MZsinhcaf - / - EV ( I ) , MV ( J ) and FG ( K ) follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O

(I, J and K) Western blots for Ace-H3-K9 and H3 in WT and MZsinhcaf -/- EV (I), MV (J) and FG (K) follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( L ) Western blots for Ace - H4 - K5 , Ace - H3 - K18 and H3 in WT and MZsinhcaf - / - FG follicles . α - tubulin was used as a loading control . Data information : FG , full - grown stage ( late stage III )

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O

(L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III)


( M , N and O ) Statistical analysis of the relative levels of Ace - H3 - K9I n WT and MZsinhcaf - / - EV ( M ) , MV ( N ) and FG ( O ) follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III )

O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O

(M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)


( P and Q ) Statistical analysis of the relative levels of Ace - H3 - K18 ( P ) and Ace - H4 - K5 ( Q ) in WT and MZsinhcaf - / - FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : ; FG , full - grown stage ( late stage III )

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O

(P and Q) Statistical analysis of the relative levels of Ace-H3-K18 (P) and Ace-H4-K5 (Q) in WT and MZsinhcaf -/- FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: ; FG, full-grown stage (late stage III)


( A ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil : egfp ) , Tg ( piwil : egfp ) injected with kif26ab MO and Tg ( piwil : egfp ) injected with control MO embryos . ( B ) Quantitative analysis of PGCs in 24hpf Tg ( piwil : egfp ) ( n = 11 ) , Tg ( piwil : egfp ) injected with kif26ab MO ( n = 12 ) and Tg ( piwil : egfp ) injected with control MO ( n = 13 ) embryos . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE B-TISSUE O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O

(A) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil:egfp), Tg(piwil:egfp) injected with kif26ab MO and Tg(piwil:egfp) injected with control MO embryos. (B) Quantitative analysis of PGCs in 24hpf Tg(piwil:egfp) (n = 11), Tg(piwil:egfp) injected with kif26ab MO (n = 12) and Tg(piwil:egfp) injected with control MO (n = 13) embryos. Data shown are mean ± SEM (n = the number of embryos analyzed).


( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . White arrowheads point to cleavage furrows of 4 - cell embryos .

O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD B-SMALL_MOLECULE O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O

(C) Maximal intensity projection of a confocal z-stack of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. White arrowheads point to cleavage furrows of 4-cell embryos.


( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA ( n = 11 ) , GFP - Buc mRNA + RFP mRNA + Control MO ( n = 11 ) and GFP - Buc mRNA + RFP mRNA + kif26ab MO ( n = 10 ) . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD B-SMALL_MOLECULE 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) Relative GFP-Buc density at the cleavage furrow of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA (n = 11), GFP-Buc mRNA + RFP mRNA + Control MO (n = 11) and GFP-Buc mRNA + RFP mRNA + kif26ab MO (n = 10). Data shown are mean ± SEM (n = the number of embryos analyzed).


( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( F ) Western blots for GFP - Buc expression in 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (F) Western blots for GFP-Buc expression in 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


( G ) Maximal intensity projection of a confocal z - stack of MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O

(G) Maximal intensity projection of a confocal z-stack of MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. White arrowheads point to cleavage furrows of 4-cell embryos.


( H ) Relative GFP - Buc density at the cleavage furrow of MZsinhcaf - / - ( n = 12 ) or WT ( n = 13 ) 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . Data shown are mean ± SEM ( n = the number of embryos analyzed ) .

O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O

(H) Relative GFP-Buc density at the cleavage furrow of MZsinhcaf -/- (n = 12) or WT (n = 13) 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. Data shown are mean ± SEM (n = the number of embryos analyzed).


( I ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( J ) Western blots for GFP - Buc expression in MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


A . Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease ( MNase ) for three types of DNA wrapped around the CENP - A nucleosome ( red ) and the H3 nucleosome ( green ) . For α - satellite DNA with initial size of 171 bp , size ranges ( 141 - 171 ) corresponding to DNA lengths above NCP ( nucleosome core particle ) is presented . Data are presented as mean ( SD ) for each time point based on three independent experiments .

O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.


B . Native PAGE gel stained with Commassie blue showing complexes formed between CENP - A or H3 nucleosome and CENP - CCR . Lane 1 : CENP - A nucleosome , Lanes 2 - 4 : Increasing amounts of CENP - CCR are added to CENP - A nucleosome . Generation of a sharp band with slower mobility indicates formation of a specific CENP - A / CENP - CCR complex . Lane 5 : H3 nucleosome . Lanes 6 - 8 : Increasing amounts of CENP - CCR are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - CCR complexes . C . Same experiment as in ( B ) using CENP - Cmotif . Lane 1 . CENP - A nucleosome . Lanes 2 - 4 . Increasing amounts of CENP - Cmotif are added to CENP - A nucleosome . Upon binding CENP - Cmotif , CENP - A nucleosome migrates slower through the gel . Note only modest change in mobility due to small size of CENP - Cmotif , comparing to CENP - CCR in ( B ) . Lane 5 . H3 nucleosome . Lanes 6 - 8 . Increasing amounts of CENP - Cmotif are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - Cmotif complexes .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O

B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes.


D . Native gel showing CENP - CCR competing out CENP - Cmotif bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome , Lane 2 : CENP - A / CENP - Cmotif complex . Lane 3 - 6 : Increasing amounts of CENP - CCR are added to the pre - formed CENP - A / CENP - Cmotif complex . Formation of slower migrating bands indicates that longer CENP - CCR is replacing shorter CENP - Cmotif bound to the CENP - A nucleosome .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.


E . Native gel showing the inability of CENP - Cmotif to compete out CENP - CCR bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome . Lane 2 and 6 : CENP - CCR / CENP - A nucleosome complex . Lanes 3 - 5 : Increasing amounts of CENP - Cmotif are added to the pre - formed CENP - A / CENP - CCR complex . Formation of smear at high amounts of CENP - Cmotif added , indicates that CENP - Cmotif , at high concentrations , non - specifically binds CENP - A / CENP - CCR complex rather than replacing bound CENP - CCR . Lane 7 - 9 : Increasing amounts of CENP - CCR - short are added to the pre - formed CENP - A / CENP - CCR complex . Formation of bands with higher mobility indicates that smaller CENP - CCR - short is effectively replacing bigger CENP - CCR bound to CENP - A nucleosome .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.


G . Native gels showing the impaired ability of mutated CENP - CCR to form complexes with the CENP - A nucleosome . The molar ratio of CENP - CCR / CENP - A nucleosomes is shown above each lane .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O

G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.


A . Graph showing the relative abundance of undigested DNA ( 145 bp ) as a function of time during digestion with micrococcal nuclease ( MN - ase ) for the CENP - A nucleosome ( red ) , the CENP - A / CENP - CCR complex ( blue ) and the CENP - A / CENP - Cmotif complex ( black ) .

O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).


D . Same type of data as in ( A ) for the CENP - A nucleosome assembled with tailless H2A ( H2A , 1 - 109 ) alone ( pink ) and in complex with CENP - CCR ( light blue ) .

O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).


( A ) Representative single immunofluorescent staining for dsDNA at 6 h , 24 h , and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain . Scale bars , 5 μm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE O B-DISEASE O O B-TISSUE O O O O O O B-TISSUE O O O O O O O

(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm.


( B ) Representative images of double - immunofluorescent stained for 53BP - 1 and dsDNA ( upper panel ) , Iba - 1 and dsDNA ( middle panel ) , and for GFAP and dsDNA ( lower panel ) . Scale bars , 5 μm .

O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O

(B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm.


( C ) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO . ( D ) Quantitative analysis for Western blot analysis .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O B-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis.


( E ) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri - ischemic area as well as in the corresponding regions of sham control brains . Scale bars , 50 μm , 20 μm in insets . ( F ) Quantitative analysis of cGAS immunohistochemical staining . n = 6 mice per group . * * P < 0 . 01 , two - tailed unpaired Student ' s t test .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-DISEASE O B-DISEASE O O O B-DISEASE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test.


( G ) Representative double immunofluorescent stainings for cGAS ( red ) and 53BP1 ( green ) . n = 6 mice per group .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O

(G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group.


Mice received daily intraperitoneal ( IP ) injections of A151 ( 300 μg ) or an equal volume of vehicle for 3 consecutive days after MCAO induction . ( A ) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT - PCR . n = 6 mice per group . * *

B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-DISEASE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O

Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **


( B ) The expression levels of cGAS , STING , and NF - κB proteins in the brains of indicated groups were measured using Western blot analysis . ( C ) Bar graph shows the relative protein expression levels of cGAS , STING , and NF - κB in the brains of indicated groups . n = 6 mice per group .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-ORGANISM O O O

(B) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group.


( D ) The mRNA expression levels of the AIM2 inflammasome components ( AIM2 , ASC , and caspase - 1 ) , effector of pyroptosis GSDMD , IL - 1β , and IL - 18 after MCAO were determined using qRT - PCR . n = 6 mice per group .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O

(D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group.


( E ) The expression of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups was measured using Western blot analysis . ( F ) Bar graph shows the relative protein expression levels of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups . n = 6 mice per group .

O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-ORGANISM O O O

(E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group.


( A ) Representative images of immunohistochemical staining of IL - 1β , caspase - 1 , and GSDMD in the brains of indicated group . Scale bars , 50 μm . ( B ) Quantitative analysis for immunohistochemical staining . n = 6 mice per group .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O

(A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group.


( C ) Representative images of double - immunofluorescent stained for GFAP and GSDMD , for Iba - 1 and GSDMD , and for NeuN and GSDMD in post - ischemic brains after tMCAO .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE B-TISSUE O B-DISEASE O

(C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO.


( D ) Representative images of double - immunofluorescent stained for Iba - 1 and GSDMD , for Iba - 1 and caspase - 1 , and for Iba - 1 and IL - 1β of sham + vehicle , MCAO + vehicle , and MCAO + A151 groups . Scale bars , 50 μm . ( E ) Bar graph shows the relative immunofluorescence intensity of GSDMD , caspase - 1 , and IL - 1β in the brains of indicated groups . n = 6 mice per group . DAPI staining : nuclei .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-DISEASE O O O O B-DISEASE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O

(D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.


( A ) Expression of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 ( FL ) , and GSDMD in primary microglia lysates was detected using western blot . ( B ) Quantitative analysis for Western blot analysis of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 and GSDMD .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O

(A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD.


( C ) The protein levels of caspase - 1 p20 and IL - 1β ( p17 ) in the supernatant of cultured primary microglia were determined by western blot . ( D ) Quantitative analysis for Western blot analysis of caspapse - 1 and IL - 1β . * P < 0 . 05 , * * P < 0 . 01 compared with the untreated group , # P < 0 . 05 , # # P < 0 . 01 compared with the LPS + poly ( dA : dT ) group .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

(C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group.


( E - F ) Immunofluorescence was conducted to detect the expression levels of cGAS , NF - κB , and pyroptosis associated molecules ( GSDMD , caspase - 1 , and IL - 1β ) in BV2 cells . n = 3 in each group . Scale bars , 50 μm .

O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O

(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.


( A ) Gating strategy of brain - infiltrating immune cells including CD4 + T cells ( CD3 + CD4 + ) , CD8 + T cells ( CD3 + CD8 + ) , B cells ( CD3 - CD19 + ) , NK cells ( CD3 - NK1 . 1 + ) , monocyte / macrophages ( CD11b + CD45highF4 / 80 + ) , neutrophils ( CD11b + CD45highLy6G + ) , and microglia ( CD11b + CD45int ) and their expression of IL ( interleukin ) - 6 , TNF - α ( tumor necrosis factor - α ) , TGF - β ( transforming growth factor - β ) , and IL - 4 . FMO , fluorescence minus one . ( B ) Quantitative analysis of the counts of brain - infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment . ( C ) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment . n = 6 mice per group . ( D ) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group . *

O O O O O O B-TISSUE O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O B-CELL_TYPE O O B-TISSUE O B-DISEASE B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O O B-TISSUE O B-DISEASE B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O B-DISEASE B-ORGANISM O O O O O O O B-ORGANISM O O O O

(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *


( E ) Bar graph shows percentages of microglia expressing IL - 6 , TNF - α , TGF - β , and IL - 10 in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group .

O O O O O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-DISEASE B-ORGANISM O O O O O O O B-ORGANISM O O O

(E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group.


( F ) Representative images of immunofluorescent staining for ly6G in peri - infarct areas of MCAO mice receiving indicated treatment . ( G ) Bar graph shows the number of ly6G + neutrophils . n = 6 mice per group .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-DISEASE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-ORGANISM O O O

(F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group.


( H ) Representative images of immunofluorescent staining for Iba - 1 in peri - infarct areas of MCAO mice receiving indicated treatment . ( I ) Bar graph shows the number of Iba - 1 + microglia . n = 6 mice per group .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-ORGANISM O O O

(H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.


( B ) Neurological tests were performed to evaluate the motor , sensory , and balance functions in mice receiving A151 , or vehicle at days 1 , 3 , 7 , and 14 after MCAO . n = 10 mice per group .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-DISEASE O O O O B-ORGANISM O O O

(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.


( C ) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO , the infarct area is shown in white .

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(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.


( D ) Bar graph shows percentages of infarct volume of indicated groups . n = 10 mice per group . *

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(D) Bar graph shows percentages of infarct volume of indicated groups. n = 10 mice per group. *


( E ) Flow cytometry plots and ( F ) summarized results show percentages of Annexin V + PI + cells in the brains of MCAO mice receiving A151 or vehicle . n = 6 mice per group .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-TISSUE O B-DISEASE B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O

(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.


( G ) Representative histology images for TUNEL staining . ( H ) Bar gragh shows percentages of TUNEL - positive cells . n = 6 mice per group .

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(G) Representative histology images for TUNEL staining. (H) Bar gragh shows percentages of TUNEL-positive cells. n = 6 mice per group.


( A ) Representative double immunofluorescent stainings for Iba - 1 and cGAS , and for GFAP and cGAS . Scale bars , 10 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O

(A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm.


( D ) Representative genotyping analysis for WT , cGAS - KD , and cGAS - KO mice .

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(D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice.


( F ) Representative images of TTC staining of brain sections from vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice on day 3 after tMCAO . Three representative rostrocaudal levels of TTC staining are shown . ( G ) Bar graph shows percentages of infarct volume of indicated group . n = 6 mice per group . ( H ) Bar graph shows modified Neurological Severity Score ( mNSS ) in vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice n = 10 mice per group .

O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O O O O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O B-DISEASE O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O B-DISEASE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O B-ORGANISM O O O

(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.