( H ) BMDMs were treated as in ( A - D ) . Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate . Infected cells are incubated with different concentration of BTP15 ( 0 , 2 , 10 , 50 μM ) during the course of the experiment . Each symbol represents the percentage of dead macrophages for a single biological replicate ( n ≥ 3 replicates with ≥ 70 cells per replicate ) . Bars represent means and standard deviations . P - values were calculated using a one - way ANOVA test comparing the treated vs . untreated samples .

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

(H) BMDMs were treated as in (A-D). Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate. Infected cells are incubated with different concentration of BTP15 (0, 2, 10, 50 μM) during the course of the experiment. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test comparing the treated vs. untreated samples.


( A ) Hybridization Chain Reaction ( HCR ) detection of CGG repeat RNA along with immunocytochemistry ( ICC ) to detect SRSF1 - FLAG in U2OS cells . Expanded view of merged channels shows the CGG RNA foci ( arrowhead ) and an example of SRSF1 - CGG RNA foci co - localization ( arrow ) . DAPI marks the nucleus . Scale bars are 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.


( B ) Co - localization analysis of CGG RNA ( red traces ) and SRSF1 ( green traces ) showing overlaps of CGG RNA and SRSF1 . ( a . u . = arbitrary unit )

O O O O O O O O O O O O O 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) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)


( C ) Co - immunoprecipitation of indicated SRSF proteins with PP7 - tagged control sequence ( partial nLUC sequence ) to correct for non - specific binding , AUG and CGG reporter RNAs . SRSF1 and 2 specifically immunoprecipitated with PP7 - tagged CGG reporter .

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

(C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.


( D ) Representative photographs of fly eyes and quantitation ( below ) of rough eye scores with fly SRSF1 overexpression ( dSF2 OE ) ; n = 20 - 32 / genotype . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( F ) Quantitation of ( GGGGCC ) 28 - EGFP rough eye phenotype with SRSF modifiers ( n > 30 flies / genotype ) . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

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

(F) Quantitation of (GGGGCC)28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( G ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP at 29 ˚ C along with the quantifications of necrosis and eye width . n = 28 - 30 / genotype . Error bars represent mean + / - SD .

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(G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD.


( H - I ) Survival assays of flies expressing ( CGG ) 90 - EGFP under Tub5 - GS ( H ) and ELAV - GS ( I ) drivers with control or SRSF1 siRNAs . Expression of ( CGG ) 90 - EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment ( Log - rank Mantel - Cox test ; n = 98 - 101 / genotype for Tub - GS and n = 120 - 141 / genotype for ELAV - GS flies ) ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 . ( J ) Survival assays of ( GGGGCC ) 28 - EGFP expressing fly under Tub5 - GS driver ( Log - rank Mantel - Cox test ; n = 71 - 93 / genotype ) with control or SRSF1 siRNAs . ∗ ∗ p < 0 . 01 .

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(H-I) Survival assays of flies expressing (CGG)90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01. (J) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.


( A , B ) Representative ( A ) photographs of fly eyes and ( B ) quantitation with siRNA mediated knockdown of SRPK1 ( dSRPK1 ) ; n = 30 - 34 / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( C ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion . ( D ) Quantitation of rough eye phenotypes . t - test with Welch corrections for comparisons with the control ; n = 31 - 34 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

(C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( E ) Representative external eye imaging for detection of GFP aggregates caused by ( CGG ) 90 - EGFP transgene expression ( top ) . Converted images used to quantify total intensity of GFP puncta ( bottom ) . ( F ) Depletion of SRSF1 or SRPK1 by RNAi results in reduced ( CGG ) 90 - EGFP puncta compared to control siRNA as quantified by total intensity ( a . u . = arbitrary unit ) . n = 13 - 15 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( B ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . β - Actin is used as a loading control . To prevent signal saturation , AUG - nLuc lysate was diluted 1 : 3 in sample buffer prior to loading ( n = 3 biological replicates ) . Schematics of the AUG - nLUC - 3xFLAG and + 1CGG ( 100 ) - nLuc - 3xFLAG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 . To prevent over - exposure , the AUG - nLuc lysate was diluted 1 : 3 in sample buffer .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.


( D ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Schematics of the GA70 ( GGGGCCx70 ) and + 2CGG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) Immunoblot of DMSO and SPHINX31 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . ( n = 3 biological replicates ) . ( F ) Anti - FLAG of DMSO and SPHINX31 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(E) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( C , D ) Expression of GGGGCC - nLuc - 3xFLAG ( C ) and + 2CGG - nLuc - 3xFLAG ( D ) RAN translation reporters in HEK293T cells treated with 2 μM TG ( for stress induction ) analyzed by immunoblot ( n = 3 biological replicates ) . To evaluate effects of SRPK1 inhibition cells were pre - treated with DMSO or SRPIN340 before reporter transfection . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(C, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection. Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG ( for stress induction ) in HEK293T cells by immunoblot ( n = 3 biological replicates ) . ( F ) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG ( for stress induction ) in the presence of the + 1CGG reporter . Cells were pre - treated with DMSO or SRPIN340 before reporter transfection ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(E) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates). (F) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( B ) ICC images of FLAG - tagged SRSF1 after SRPIN340 treatment compared to vehicle ( DMSO ) . Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean + / - 95 % CI ( n = 85 - 126 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(B) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( C ) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle ( DMSO ) as detected by HCR . Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole . Error bars indicate mean + / - 95 % CI ( n = 124 - 151 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(C) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( D ) Anti - FLAG immunoblot blot of DMSO and SRPIN340 pre - treated HEK293T cells transfected with in vitro transcribed CGG - nLuc - 3xFLAG reporter RNA . β - Actin is used as a loading control . Error bars represent mean + / - SD ( n = 6 biological replicates ) . Statistical analysis was performed using Student ' s t test with Welch ' s correction . ∗ p < 0 .

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(D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05


( A , B ) Pharmacological targeting of SPRK1 with ( A ) 40 μM SRPIN340 or ( B ) 8 μM SPHINX31 reduced the cumulative risk of death in + 1 ( CGG ) 100 - EGFP ( encoding FMRpolyG ) expressing neurons . n = # of neurons quantified for each condition ; Cox proportional hazard analysis , ∗ ∗ ∗ p < 0 . 001 .

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

(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.


a , Sections of the thymus and muscle of fed and starved GFP - LC3 mice were stained with anti - GFP Alexa 488 antibody ( green ) and 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Images are representative of n = 6 .

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a, Sections of the thymus and muscle of fed and starved GFP-LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6.


b , Thymic stromal cells from GFP - LC3 mice were fixed onto glass slides and stained with anti - GFP Alexa 488 and DAPI . Typical autophagy - positive cTECs and mTEChi are shown . The average frequencies ± s . d . of cTECs and mTEChi scoring autophagy positive ( that is , ≥ 5 autophagosomes ) in two independent preparations are indicated . Less than 1 % of dendritic cells ( DCs ) and mTEClo were positive .

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b, Thymic stromal cells from GFP-LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive.


c , Relative distribution of the number of autophagosomes in autophagy - positive cTECs and mTEChi ; a . u . , arbitrary units .

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c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.


a , Thymi from wild - type ( WT ) or Atg5 - / - embryos were transplanted under the kidney capsule of wild - type recipients and analysed 6 weeks later . The average cellularities ± s . d . are shown ; n = 5 .

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a, Thymi from wild-type (WT) or Atg5-/- embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5.


b , Normal compartmentalization of Atg5 - / - grafts in cytokeratin - 8 - positive ( red ; K8 ) cortical and cytokeratin - 5 - positive ( green ; K5 ) medullary regions ; n = 8 .

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b, Normal compartmentalization of Atg5-/- grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8.


c , Expression of MHC class I and II on cTECs or mTECs from Atg5 - / - to wild - type ( WT ) grafts ( red ) , or wild - type to wild - type grafts ( blue ) . Staining controls are depicted in grey ; data are representative of three experiments .

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c, Expression of MHC class I and II on cTECs or mTECs from Atg5-/- to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. d


d , RT - PCR analysis of autoimmune regulator ( Aire ) and TRA expression in mTECs isolated from Atg5 - / - to wild - type , or wild - type to wild - type grafts . Expression of β - actin ( Actb ) , intestinal trefoil factor ( Tff3 ) , casein 2 ( Csn2 ) and salivary protein 1 ( Spt1 ) are also shown ( 5 - fold serial dilutions ) . Data are representative of two experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RT-PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5-/- to wild-type, or wild-type to wild-type grafts. Expression of β-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.


a , Polyclonal T - cell development in Atg5 - / - to wild - type ( WT ) , or wild - type to wild - type grafts . The top dot plots show CD4 and CD8 staining ; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes ; n ≥ 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Polyclonal T-cell development in Atg5-/- to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5.


b , Atg5 - / - or wild - type lobes were grafted into TCR - HA transgenic recipients . Graft cellularity ± s . d . and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of TCR - HA + cells among CD4 SP cells was 32 . 1 % ± 3 . 2 % in wild - type to TCR - HA , and 19 . 6 % ± 4 . 3 % in Atg5 - / - to TCR - HAlobes ; P = 1 . 8 × 10 - 8 ( n ≥ 14 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Atg5-/- or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5-/- to TCR-HAlobes; P = 1.8 × 10-8 (n ≥ 14).


c , Atg5 - / - or wild - type lobes were grafted into AND transgenic mice . Graft cellularity ± s . d . and the expression of the transgenic TCR ( Vb3 - Va11 ) on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of Vb3 - Va11 + CD4 SP cells was 93 . 5 % ± 0 . 2 % in wild - type to ANDlobes ( n = 3 ) and 97 . 9 % ± 1 . 1 % ( n = 6 ) in Atg5 - / - to ANDlobes ( P = 9 × 10 - 4 ) .

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

c, Atg5-/- or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (Vb3-Va11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of Vb3-Va11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to ANDlobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5-/- to ANDlobes (P = 9 × 10-4).


d , Thymi from wild - type or Atg5 - / - embryos were grafted into P14 , OT - I or HY TCR - transgenic recipients . The dot plots show the expression of the transgenic TCR V - segments on CD8 SP cells . Thymus cellularities ± s . d . are shown in the respective bar diagrams . Data are representative of n = 4 ( P14 ) , n ≥ 4 ( OT - I ) and n ≥ 2 ( HY ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Thymi from wild-type or Atg5-/- embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY).


e , Thymi from wild - type or ( BALB / c3C57BL / 6 ) F1 Atg5 - / - embryos were transplanted into ( BALB / c3C57BL / 6 ) F1 recipients . Six weeks after grafting , expression of the I - Ea52 - 68 - I - Ab complex on cTECs was analysed using the monoclonal antibody Y - Ae . Atg5 - / - cTECs are shown in red ; wild - type cTECs are shown in blue . Control stainings are shown in orange ( Atg5 - / - ) and green ( wild type ) . Data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Thymi from wild-type or (BALB/c3C57BL/6)F1 Atg5-/- embryos were transplanted into (BALB/c3C57BL/6)F1 recipients. Six weeks after grafting, expression of the I-Ea52-68-I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5-/- cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5-/-) and green (wild type). Data are representative of two independent experiments.


Atg5 - / - or wild - type ( WT ) lobes were grafted into athymic ( nu / nu ) mice ( nu / nuAtg5 - / - or nu / nuWT , respectively ) . Data are representative of three experiments with n ≥ 4 . a , Expression of activation markers on lymph node CD4 T cells in nu / nuAtg5 - / - or nu / nuWT chimaeras .

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

Atg5-/- or wild-type (WT) lobes were grafted into athymic (nu/nu) mice (nu/nuAtg5-/- or nu/nuWT, respectively). Data are representative of three experiments with n ≥ 4. a, Expression of activation markers on lymph node CD4 T cells in nu/nuAtg5-/- or nu/nuWT chimaeras.


b , The changes in body weight over time are shown .

b, The changes in body weight over time are shown.


c , The appearance of nu / nuAtg5 - / - or nu / nuWT chimaeras . The colon ( arrow ) and uterus ( arrowhead ) are highlighted ; note the absence of fat pads ( asterisk ) in nu / nuAtg5 - / - chimaeras .

c, The appearance of nu/nuAtg5-/- or nu/nuWT chimaeras. The colon (arrow) and uterus (arrowhead) are highlighted; note the absence of fat pads (asterisk) in nu/nuAtg5-/- chimaeras.


d , Haematoxylin and eosin staining of organs from nu / nuAtg5 - / - or nu / nuWT chimaeras . Arrows indicate infiltrates .

d, Haematoxylin and eosin staining of organs from nu/nuAtg5-/- or nu/nuWT chimaeras. Arrows indicate infiltrates.


Heat map showing absolute counts of unique peptides for S . indica apoplastic proteins present at all three time points and in culture filtrate . S1 / S2 / S3 = biologically independent sample 1 , 2 and 3 ; d = deglycosylated .

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

Heat map showing absolute counts of unique peptides for S. indica apoplastic proteins present at all three time points and in culture filtrate. S1/S2/S3 = biologically independent sample 1, 2 and 3; d = deglycosylated.


eATP levels in APF from S . indica inoculated and mock - treated barley root samples collected at the predominantly biotrophic phase ( 3 and 5 dpi ) as well as the predominantly cell death associated phase ( 7 and 10 dpi ) . Error bars show the standard error of the mean obtained from three independent biological replicates at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - test .

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

eATP levels in APF from S. indica inoculated and mock-treated barley root samples collected at the predominantly biotrophic phase (3 and 5 dpi) as well as the predominantly cell death associated phase (7 and 10 dpi). Error bars show the standard error of the mean obtained from three independent biological replicates at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


eATP levels measured from culture medium collected from mock treated and S . indica colonized Arabidopsis seedlings at 3 , 5 , 7 and 10 dpi . Error bars represent ± SE of the mean from three independent biological replicates . RLU : relative light units . Asterisks indicate significance at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - test .

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

eATP levels measured from culture medium collected from mock treated and S. indica colonized Arabidopsis seedlings at 3, 5, 7 and 10 dpi. Error bars represent ±SE of the mean from three independent biological replicates. RLU: relative light units. Asterisks indicate significance at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


S . indica colonization of Arabidopsis dorn1 - 3 mutant and the parental Col - 0aq lines quantified by qPCR at 3 , 5 , 7 and 10 dpi . The ratio of fungal ( SiTEF ) to plant ( AtUBI ) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2 - ΔCT method . Error bars represent standard error of the mean of three technical replicates . The experiment was repeated 3 times for 3 , 5 and 7 dpi with similar outcomes .

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

S. indica colonization of Arabidopsis dorn1-3 mutant and the parental Col-0aq lines quantified by qPCR at 3, 5, 7 and 10 dpi. The ratio of fungal (SiTEF) to plant (AtUBI) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2-ΔCT method. Error bars represent standard error of the mean of three technical replicates. The experiment was repeated 3 times for 3, 5 and 7 dpi with similar outcomes.


Transcript levels of S . indica E5 ′ NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture . Error bars represent standard error of the mean of three independent biological replicates . CM = complex medium .

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

Transcript levels of S. indica E5′NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture. Error bars represent standard error of the mean of three independent biological replicates. CM = complex medium.


Ecto - 5 ′ - nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S : : E5 ′ NT ( # 303 ) , Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) or Pro35S : : mCherry ( # 305 ) . E5 ′ NT activity was measured after incubation with 100 µM of either ATP , ADP or AMP . In the membrane protein preparations from Pro35S : : E5 ′ NT ( # 303 ) lines phosphate release was specifically increased upon incubation with purines . Error bars represent the standard error of the mean from three technical repetitions . The coomassie stained SDS - PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines . Equal volumes were loaded . The experiment was repeated two times with similar results .

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

Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results.


Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry ( # 305 ) or Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) showing secretion of the E5 ′ NT fusion protein . mCherry images show z - stacks of 14 image planes of 1 µm each . Scale bar = 20 µm .

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

Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry (#305) or Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) showing secretion of the E5′NT fusion protein. mCherry images show z-stacks of 14 image planes of 1 µm each. Scale bar=20 µm.


The transgenic Arabidopsis line Pro35S : : E5 ′ NT ( # 303 ) expressing untagged full length SiE5 ′ NT was better colonized by S . indica . Error bars of the qPCR data represent ± SE of the mean from three independent biological replicates . Asterisks indicate significance ( Student ' s t - test , * p < 0 . 05 ) .

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

The transgenic Arabidopsis line Pro35S::E5′NT (#303) expressing untagged full length SiE5′NT was better colonized by S. indica. Error bars of the qPCR data represent ±SE of the mean from three independent biological replicates. Asterisks indicate significance (Student's t-test, * p< 0.05).


S . indica induced eATP release in different Arabidopsis transgenic lines . Culture medium was collected from mock treated or S . indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured . RLU : relative light units . Error bars represent ± SE of the mean from three biological replicates . Asterisks indicate significance to all other samples within the same treatment group ( ANOVA , p < 0 . 05 ) .

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

S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


S . indica colonization of transgenic lines at 5 dpi . Error bars represent ± SE of the mean from three biological replicates . Asterisks indicate significance to all other samples within the same treatment group ( ANOVA , p < 0 . 05 ) .

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

S. indica colonization of transgenic lines at 5 dpi. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


Expression analysis of the eATP responsive gene At1g58420 measured by qRT - PCR . Error bars represent ± SE of the mean from three independent biological replicates ( independent from those shown in Fig . 2C ) . Letters indicate significant groups ( ANOVA , p < 0 . 01 , for the line 305 p < 0 . 05 ) .

Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).


Relative fluxes of phosphates ( Pi ) and sugars ( C ) across the fungal plasma membrane . Without additional Pi - source in the apoplast ( value 0 at the x - axis , black dots ) there is a constant flux of phosphate via the proton - coupled phosphate ( H / P ) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus . For better comparison these fluxes were normalized to 1 and - 1 , respectively , and all other fluxes were calculated relative to these control values . With increasing ATP - release and Pi - production the Pi - efflux gets smaller and is zero at a relative Pi - production rate of 1 ( grey triangles ) . At higher ATP - release and Pi - production rates the fungus imports Pi , i . e . the transport direction of phosphate has been inverted in comparison to the control condition ( white square ) . The C - flux via the proton - coupled sugar ( H / C ) transporter is not affected by the additional Pi - source ( horizontal grey line ) .

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

Relative fluxes of phosphates (Pi) and sugars (C) across the fungal plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of phosphate via the proton-coupled phosphate (H/P) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus. For better comparison these fluxes were normalized to 1 and -1, respectively, and all other fluxes were calculated relative to these control values. With increasing ATP-release and Pi-production the Pi-efflux gets smaller and is zero at a relative Pi-production rate of 1 (grey triangles). At higher ATP-release and Pi-production rates the fungus imports Pi, i.e. the transport direction of phosphate has been inverted in comparison to the control condition (white square). The C-flux via the proton-coupled sugar (H/C) transporter is not affected by the additional Pi-source (horizontal grey line).


Relative fluxes of Pi and C across the plant plasma membrane . Without additional Pi - source in the apoplast ( value 0 at the x - axis , black dots ) there is a constant flux of sugar via the H / C transporter from the plant to the apoplast ( and thereafter to the fungus ) and a constant flux of phosphate ( coming from the fungus ) via the H / P transporter from the apoplast to the plant . With increasing ATP - release and Pi - production the Pi - influx via the H / P transporter increases while the C - flux via the H / C transporter is unaffected by the additional Pi - source ( horizontal grey line ) .

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

Relative fluxes of Pi and C across the plant plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of sugar via the H/C transporter from the plant to the apoplast (and thereafter to the fungus) and a constant flux of phosphate (coming from the fungus) via the H/P transporter from the apoplast to the plant. With increasing ATP-release and Pi-production the Pi-influx via the H/P transporter increases while the C-flux via the H/C transporter is unaffected by the additional Pi-source (horizontal grey line).


Phosphate fluxes across the plant plasma membrane . Besides the Pi - uptake via the H / P transporter ( B , black line , for clarity not shown in C ) , the plant loses Pi due to the ATP - release ( light grey line ) . The difference between Pi - uptake and Pi - loss is the net Pi - balance of the plant ( dark grey line ) . Without ATP - release ( value 0 at the x - axis , black dots ) the plant gains Pi . At a relative ATP - release and Pi - production of 1 ( grey triangles ) the plant release as much phosphate as it takes up , while at higher ATP - release values , the plant loses Pi .

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

Phosphate fluxes across the plant plasma membrane. Besides the Pi-uptake via the H/P transporter (B, black line, for clarity not shown in C), the plant loses Pi due to the ATP-release (light grey line). The difference between Pi-uptake and Pi-loss is the net Pi-balance of the plant (dark grey line). Without ATP-release (value 0 at the x-axis, black dots) the plant gains Pi. At a relative ATP-release and Pi-production of 1 (grey triangles) the plant release as much phosphate as it takes up, while at higher ATP-release values, the plant loses Pi.


( A ) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ .

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(A) Dot plot depicting the expression of mRNA for neurogenic transcription factor MEIS2 in prospectively isolated cellular populations from the SEZ.


( B ) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells . Note that Meis2 mRNA positive LRCs have low ( no ) MEIS2 protein .

O O O O O O O O O O O O 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) Micrographs depicting the expression of neurogenic fate determinant Meis2 mRNA and MEIS2 protein in acutely dissociated SEZ cells. Note that Meis2 mRNA positive LRCs have low (no) MEIS2 protein.


( C IHC labeling for MEIS2 ( C ) of LRCs positive for BrdU only and neuroblasts marked by DCX .

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

(C IHC labeling for MEIS2 (C) of LRCs positive for BrdU only and neuroblasts marked by DCX.


D ) IHC labeling for MCM6 ( D ) of LRCs positive for BrdU only and neuroblasts marked by DCX .

D) IHC labeling for MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.


( E ) Dot plot showing the proportion of BrdU + LRCs negative for MEIS2 or MCM6 protein .

(E) Dot plot showing the proportion of BrdU+ LRCs negative for MEIS2 or MCM6 protein.


( F ) Dot plot depicting the expression of miR - 204 in prospectively isolated cells of neural lineage .

(F) Dot plot depicting the expression of miR-204 in prospectively isolated cells of neural lineage.


( B ) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi .

(B) Dot plot depicting the immunoreactivity of LRCs for MEIS2 in control and antagomirs treated animals 7 dpi.


( C , D ) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF ( control , C ) or AntimiR204 ( D ) . White arrows indicate LRCs without detectable MEIS2 protein . The white arrowheads are pointing out the loss of neurogenic priming ( LRCs positive for MEIS2 protein ) upon the antagomir treatments .

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(C, D) Micrographs showing IHC for the neurogenic priming factor MEIS2 in the SEZ 7 days after injection of artificial CSF (control, C) or AntimiR204 (D). White arrows indicate LRCs without detectable MEIS2 protein. The white arrowheads are pointing out the loss of neurogenic priming (LRCs positive for MEIS2 protein) upon the antagomir treatments.


( A , B ) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF ( control , A ) or AntimiR204 ( B ) . Boxed areas correspond to higher magnifications in adjacent panels ( A ' , B ' ) .

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(A, B) Micrographs depicting the cellular composition in the SEZ 7 days after injection of aCSF (control, A) or AntimiR204 (B). Boxed areas correspond to higher magnifications in adjacent panels (A', B').


Dot plots depicting the abundance of LRCs ( BrdU + , Ki67 - , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting the abundance of aNSCs and TAPs ( BrdU - , Ki67 + , DCX - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.


Dot plots depicting the abundance of neuroblasts ( DCX + Ki67 + or DCX + Ki67 - ) in the SEZ 7 days after aCSF or AntimiR204 injection .

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Dot plots depicting the abundance of neuroblasts (DCX+ Ki67+ or DCX+ Ki67-) in the SEZ 7 days after aCSF or AntimiR204 injection.


( F , G ) Micrographs depicting the elbow - the most ventral region of the RMS ( F ) and OB neuronal layers ( G ) upon injection of artificial CSF ( control ) or AntimiR204 . ( H ) Dot plot showing the density of BrdU + cells ( progeny of NSCs ) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection .

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(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204. (H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.


( A ) Micrographs of in situ hybridization for miR - 204 in an adult mouse brain section . ( A ' and A ' ' ) are magnifications of boxed areas in ( A or A ' ) , respectively .

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(A) Micrographs of in situ hybridization for miR-204 in an adult mouse brain section. (A' and A'') are magnifications of boxed areas in (A or A'), respectively.


( B ) Dot plot showing miR - 204 levels in ChP and SEZ in the adult mouse brain measured by RT - qPCR . ( C ) Agarose gel of RT - qPCR product loaded after the saturation phase showing presence of miR - 204 and U6 in mouse ChP , SEZ and CSF .

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(B) Dot plot showing miR-204 levels in ChP and SEZ in the adult mouse brain measured by RT-qPCR. (C) Agarose gel of RT-qPCR product loaded after the saturation phase showing presence of miR-204 and U6 in mouse ChP, SEZ and CSF.


Plots depicting the number and size of EVs isolated from human CSF ( D )

Plots depicting the number and size of EVs isolated from human CSF (D)


Plots depicting the miR - 204 levels ( E ) in EVs and EV - free CSF ( n = 2 ) . ( F ) Agarose gel of RT - qPCR analysis depicting levels of miR - 204 in the EV - free supernatant and EV containing fraction of human CSF loaded at the saturation phase .

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Plots depicting the miR-204 levels (E) in EVs and EV-free CSF (n=2). (F) Agarose gel of RT-qPCR analysis depicting levels of miR-204 in the EV-free supernatant and EV containing fraction of human CSF loaded at the saturation phase.


( G ) Micrographs of transmission electron microscopy imaged extracellular vesicles ( EVs ) isolated from sham treated ( upper row ) and GW4869 treated animals ( lower row ) . ( G ' ' and G ' ' ' ) are magnifications of boxed areas in overview images to the left . ( H ) Dot plot depicting miR - 204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor .

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(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.


( A ) Gel pictures of the saturation phase of RT - qPCR analysis for miR - 204 in SEZ primary culture cells , ChP co - culture and ChP explants 7 day post preparation .

(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation.


( B ) Gel electrophoresis of RT - qPCR analysis loaded at the saturation phase for miR - 204 in SEZ culture medium and SEZ medium from the ChP co - culture .

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(B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture.


( C ) Dot plot showing the proportion of neural stem cells ( GFAP + ) out of DAPI + cells in primary SEZ / ChP co - culture with or without treatment with GW4869 inhibitor or AntimiR204 .

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(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.


( D - G ) Micrographs depicting the cellular compositions of SEZ cultures after co - culturing with control treated , GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating . Boxed areas correspond to the higher magnification images in adjacent panels ( D ' , E ' , F ' , G ' ) .

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(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').


( B , C ) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle . ( B ' and C ' ) are magnifications of the SEZ and ( B ' ' and C ' ' ) of the ChP . ( D ) Dot plot depicting expression of miR - 204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR - 204 - specific TuD compared to scrambled control .

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(B, C) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle. (B' and C') are magnifications of the SEZ and (B'' and C'') of the ChP. (D) Dot plot depicting expression of miR-204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR-204-specific TuD compared to scrambled control.


Micrographs depicting immunoreactivity for MEIS2 ( E , F ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR - 204 interference .

Micrographs depicting immunoreactivity for MEIS2 (E, F) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR-204 interference.


Micrographs depicting immunoreactivity for MCM6 ( H , I ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MCM6 + LRCs . Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 ( J ) after ChP specific miR - 204 interference .

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Micrographs depicting immunoreactivity for MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MCM6+ LRCs. Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 (J) after ChP specific miR-204 interference.


( K , L ) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control ( K ) and miR - 204 specific TuD ( L ) . Note the reduction of primed LRCs ( BrdU + only , white arrows in K and L ) upon TuD - 204 injection . ( M ) Dot plot depicting the number of LRCs ( BrdU + only ) in the SEZ 7 days after miR - 204 inhibition .

(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection. (M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition.


( A ) TNF concentrations in supernatants of WT and Tnf - / - BMDMs unstimulated or stimulated with 10 ng / ml Pam3CSK4 , quantified by ELISA . ( Left ) 0 h , 4 h and 8 h time points . ( Right ) Close - up of 0 h time point . Dashed line indicates mean of blank measurements . Data are represented as mean ± SD of 3 ( 4 h , 8 h ) or 5 ( 0 h ) biological replicates using cells from 2 ( 4 h , 8 h ) or 3 ( 0 h ) mice . Statistical significance was determined by two - tailed paired - sample t - test .

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(A) TNF concentrations in supernatants of WT and Tnf-/- BMDMs unstimulated or stimulated with 10 ng/ml Pam3CSK4, quantified by ELISA. (Left) 0 h, 4 h and 8 h time points. (Right) Close-up of 0 h time point. Dashed line indicates mean of blank measurements. Data are represented as mean ± SD of 3 (4 h, 8 h) or 5 (0 h) biological replicates using cells from 2 (4 h, 8 h) or 3 (0 h) mice. Statistical significance was determined by two-tailed paired-sample t-test.


( E - J ) Nuclear localization of mVenus - RelA upon stimulation with 10 ng / ml TNF ( E - G ) or 10 ng / ml Pam3CSK4 ( H - J ) , measured over time in WT and Tnf - / - BMDMs by fluorescence microscopy and quantified by automated image analysis . ( E , H ) Each row of the heatmap represents the NFκB signaling trajectory of one cell . Trajectories are sorted by maximum amplitude . Example trajectories are shown below . Data from a single representative experiment is depicted . ( F , I ) Indicated dynamic features were calculated from signaling trajectories . Violin plots depict pooled data from 8 ( F ) or 6 ( I ) biological replicates ( total # of cells : 5769 / 5238 ( F ) , 3843 / 3899 ( I ) ) . Marker indicates median of distribution . Statistical significance was determined by Wilcoxon rank sum test . Number indicates log10 ( p - value ) . ( G , J ) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment . Data are represented as mean ± SD of 8 ( G ) or 6 ( J ) biological replicates . Statistical significance was determined by two - tailed paired - sample t - test . ( F , G ) Some replicates for Tnf - / - BMDMs overlap with

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(E - J) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF (E - G) or 10 ng/ml Pam3CSK4 (H - J), measured over time in WT and Tnf-/- BMDMs by fluorescence microscopy and quantified by automated image analysis. (E, H) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment is depicted. (F, I) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 8 (F) or 6 (I) biological replicates (total # of cells: 5769/5238 (F), 3843/3899 (I)). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (G, J) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 8 (G) or 6 (J) biological replicates. Statistical significance was determined by two-tailed paired-sample t-test. (F, G) Some replicates for Tnf-/- BMDMs overlap with data


( B ) IκBα mRNA expression in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by RT - qPCR . IκBα expression was normalized to GAPDH expression and then to maximum WT value in each time course replicate . Data are represented as mean ± SD of 5 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Some replicates for Tnf - / - BMDMs overlap with

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(B) IκBα mRNA expression in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value in each time course replicate. Data are represented as mean ± SD of 5 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Some replicates for Tnf-/- BMDMs overlap with data


Phosphorylation of IKK , JNK , ERK , and p38 and total IKK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by SDS - PAGE followed by Western Blotting . ( C ) One of three representative experiments is shown . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point .

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Phosphorylation of IKK, JNK, ERK, and p38 and total IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by SDS-PAGE followed by Western Blotting. (C) One of three representative experiments is shown. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.


Phosphorylation of IKK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h For quantification of p - IKK ( D ) levels , basal levels ( 0 min ) were deducted per genotype before normalizing to maximum WT value in each experiment . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point .

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Phosphorylation of IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h For quantification of p-IKK (D) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.


Phosphorylation of JNK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , p - JNK ( E ) levels , basal levels ( 0 min ) were deducted per genotype before normalizing to maximum WT value in each experiment . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point .

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Phosphorylation of JNK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, p-JNK (E) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.


( E ) TNF receptor 1 ( TNFR1 , left ) , TNF receptor 2 ( TNFR2 , center ) , and CD11b ( right ) cell surface expression in Tnf - / - compared to WT BMDMs unstimulated or stimulated for 30 min with 10 ng / ml TNF , measured by flow cytometry . Per experiment , median fluorescence intensity average of 3 culture replicates in Tnf - / - cells is expressed as percent of expression in WT cells . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed one - sample t - test using a hypothesis mean of 100 .

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(E) TNF receptor 1 (TNFR1, left), TNF receptor 2 (TNFR2, center), and CD11b (right) cell surface expression in Tnf-/- compared to WT BMDMs unstimulated or stimulated for 30 min with 10 ng/ml TNF, measured by flow cytometry. Per experiment, median fluorescence intensity average of 3 culture replicates in Tnf-/- cells is expressed as percent of expression in WT cells. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed one-sample t-test using a hypothesis mean of 100.


( F ) ( Left ) TNF receptor 1 ( TNFR1 ) cell surface expression at baseline in BMDMs pre - treated with TNFR1 siRNA , control ( ctrl ) siRNA , or untreated ( - ) . Median fluorescence intensity is expressed as percent of expression in untreated WT cells . ( Right ) Percentage of cells with oscillatory NFκB activity ( measured by fluorescence microscopy ) among all responder cells upon indicated siRNA pre - treatment and stimulation with 10 ng / ml TNF . Per condition , 1032 - 1484 responding cells were analyzed . ( Left , right ) Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by one - tailed paired - sample t - test .

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(F) (Left) TNF receptor 1 (TNFR1) cell surface expression at baseline in BMDMs pre-treated with TNFR1 siRNA, control (ctrl) siRNA, or untreated (-). Median fluorescence intensity is expressed as percent of expression in untreated WT cells. (Right) Percentage of cells with oscillatory NFκB activity (measured by fluorescence microscopy) among all responder cells upon indicated siRNA pre-treatment and stimulation with 10 ng/ml TNF. Per condition, 1032 - 1484 responding cells were analyzed. (Left, right) Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test.


( D ) IκBα mRNA expression in Tnf - / - and Tnf - / - p100 - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by RT - qPCR . IκBα expression was normalized to GAPDH expression and then to maximum WT value also obtained with each replicate . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Data for Tnf - / - BMDMs is also

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(D) IκBα mRNA expression in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also included


( E ) Levels of nuclear NFκB DNA binding activity in Tnf - / - and Tnf - / - p100 - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by EMSA . Quantification of the NFκB - DNA complex was normalized to the quantified NFY - DNA complex , and then to maximum WT value also obtained with each replicate . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Data for Tnf - / - BMDMs is also

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(E) Levels of nuclear NFκB DNA binding activity in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by EMSA. Quantification of the NFκB-DNA complex was normalized to the quantified NFY-DNA complex, and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also shown


( F - H ) Nuclear localization of mVenus - RelA upon stimulation with 10 ng / ml TNF was measured over time by fluorescence microscopy in Tnf - / - and Tnf - / - p100 - / - BMDMs and quantified by automated image analysis . ( F ) Each row of the heatmap represents the NFκB signaling trajectory of one cell . Trajectories are sorted by maximum amplitude . Example trajectories are shown below . Data from a single representative experiment are depicted . ( G ) Indicated dynamic features were calculated from signaling trajectories . Violin plots depict pooled data from 6 biological replicates ( total # of cells : 4030 / 4133 ) . Marker indicates median of distribution . Statistical significance was determined by Wilcoxon rank sum test . Number indicates log10 ( p - value ) . ( H ) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment . Data are represented as mean ± SD of 6 biological replicates . Statistical significance was determined by one - tailed paired - sample t - test . ( G , H ) Data for Tnf - / - BMDMs is also

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(F - H) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF was measured over time by fluorescence microscopy in Tnf-/- and Tnf-/-p100-/- BMDMs and quantified by automated image analysis. (F) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment are depicted. (G) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 6 biological replicates (total # of cells: 4030/4133). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (H) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 6 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test. (G, H) Data for Tnf-/- BMDMs is also included


( E ) Volcano plot of single - cell gene expression in Tnf - / - vs . WT BMDMs upon TNF stimulation . Dashed lines indicate p - value threshold of 0 . 01 and absolute log2 ( FC ) threshold of 0 . 5 . Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included ( p - value threshold of 0 . 05 and log2 ( FC ) threshold of 0 . 25 ) .

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(E) Volcano plot of single-cell gene expression in Tnf-/- vs. WT BMDMs upon TNF stimulation. Dashed lines indicate p-value threshold of 0.01 and absolute log2(FC) threshold of 0.5. Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included (p-value threshold of 0.05 and log2(FC) threshold of 0.25).


( F ) Violin plots of single - cell gene expression in WT and Tnf - / - BMDMs upon mock , TNF , or Pam3CSK4 stimulation . The 10 genes shown are significantly upregulated in Tnf - / - vs . WT cells upon TNF stimulation ( see ( E ) ) .

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(F) Violin plots of single-cell gene expression in WT and Tnf-/- BMDMs upon mock, TNF, or Pam3CSK4 stimulation. The 10 genes shown are significantly upregulated in Tnf-/- vs. WT cells upon TNF stimulation (see (E)).


( D ) Violin plot of log 2 ( FC ) of normalized H3K4me1 ChIP - seq signal in TNF - or Pam3CSK4 - stimulated over unstimulated WT or Tnf - / - BMDMs within Cluster 1 regions ( 533 regions ) . Statistical significance was determined by Kolmogorov - Smirnov test . Data are represented as mean of two biological replicates for each genomic region . In the boxplot , central band indicates the median , boxes the interquartile range ( IQR ) , whiskers values within 1 . 5 IQR from the median .

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(D) Violin plot of log2(FC) of normalized H3K4me1 ChIP-seq signal in TNF- or Pam3CSK4-stimulated over unstimulated WT or Tnf-/- BMDMs within Cluster 1 regions (533 regions). Statistical significance was determined by Kolmogorov-Smirnov test. Data are represented as mean of two biological replicates for each genomic region. In the boxplot, central band indicates the median, boxes the interquartile range (IQR), whiskers values within 1.5 IQR from the median.


( E ) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1 . Tracks for WT and Tnf - / - BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates ( R1 , R2 ) are shown .

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(E) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1. Tracks for WT and Tnf-/- BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates (R1, R2) are shown.


( B ) Violin plots showing the expression of prostaglandin ( PG ) receptors in pre - adipocytes and mature adipocytes from human deep - neck BAT ( E - MTAB - 8564 ) .

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(B) Violin plots showing the expression of prostaglandin (PG) receptors in pre-adipocytes and mature adipocytes from human deep-neck BAT(E-MTAB-8564).


( A ) Oil Red O staining of mouse BAs differentiated from normal or EP3 - deficient brown pre - adipocytes . Scale bar : 50 μm . ( B ) Mean lipid droplet area of differentiated BAs from normal or EP3 - deficient brown pre - adipocytes . * P < 0 . 05 vs WT ( n = 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests ( B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 .

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(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05


( D ) Representative image of hematoxylin and eosin ( H & E ) staining for iBAT from EP3F / F and EP3F / FMyf5Cre mice . Scale bar : 50 μm . ( E ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(D) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( K ) Representative image of H & E staining of iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD . Scale bar : 50 μm . ( L ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates L , Statistical significance was evaluated by Mann - Whitney U tests L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm. (L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates L, Statistical significance was evaluated by Mann-Whitney U tests L, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) N6 - methyladenosine ( m6A ) modification levels of mRNAs in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction , detected by m6A RNA Methylation Quantification Kit ( Colorimetric ) ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test ( A * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction, detected by m6A RNA Methylation Quantification Kit (Colorimetric) (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( B ) qRT - PCR analysis of the relative mRNA levels of m6A writers ( METTL3 , METTL4 , METTL14 and WTAP ) and erasers ( FTO and ALKBH5 ) in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction ( n = 6 - 9 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

(B) qRT-PCR analysis of the relative mRNA levels of m6A writers (METTL3, METTL4, METTL14 and WTAP) and erasers (FTO and ALKBH5) in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction (n = 6-9). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( C ) Western blotting analysis of m6A writers and erasers in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction .

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(C) Western blotting analysis of m6A writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.


( D ) Quantification of protein levels in C ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates , D , Statistical significance was evaluated by unpaired two - tailed student ' s t test D , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(D) Quantification of protein levels in C (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates , D, Statistical significance was evaluated by unpaired two-tailed student's t test D, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( E ) Western blotting analysis of WTAP in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD for 1 week .

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(E) Western blotting analysis of WTAP in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD for 1 week.


( F ) Western blotting analysis of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes .

(F) Western blotting analysis of WTAP in BAs differentiated from EP3γ-expressed brown pre-adipocytes.


( G ) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed brown pre - adipocytes detected by RT - qPCR ( n = 6 - 7 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates G , Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons ( G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(G) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed brown pre-adipocytes detected by RT-qPCR (n = 6-7). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates G, Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons (G, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( H ) qRT - PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . ( I ) qRT - PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test H ) , two - way ANOVA followed by Tukey ' s test for post - hoc comparisons I , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(H) qRT-PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). (I) qRT-PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test H), two-way ANOVA followed by Tukey's test for post-hoc comparisons I, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( J ) Representative picture of oil red O - stained BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes with WTAP siRNA treatment . Scale bar : 50 μm .

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(J) Representative picture of oil red O-stained BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP siRNA treatment. Scale bar: 50 μm.


( K - Q ) qRT - PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

(K-Q) qRT-PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons K-Q). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( A ) Western blotting analysis of ubiquitination of WTAP in the control and EP3 - deficient brown pre - adipocytes with or without sulprostone treatment . Data information : Data are representative of two independent experiments with biological

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(A) Western blotting analysis of ubiquitination of WTAP in the control and EP3-deficient brown pre-adipocytes with or without sulprostone treatment. Data information: Data are representative of two independent experiments with biological replicates


( B ) Western blotting analysis of ERK1 / 2 phosphorylation in the control and EP3 - deficient brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

(B) Western blotting analysis of ERK1/2 phosphorylation in the control and EP3-deficient brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( C ) Effect of ERK1 / 2 inhibitor , ravoxertinib , on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( D ) Effect of wortmannin ( Wor ) , pertussis toxin ( PTX ) , U73122 , barbadin , and melittin on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(D) Effect of wortmannin (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( E ) Analysis of PKA activity in the control and EP3 - deficient brown pre - adipocytes ( n = 4 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological

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(E) Analysis of PKA activity in the control and EP3-deficient brown pre-adipocytes (n = 4). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates


( F ) Effect of PKA inhibitor , H89 , on ubiquitin - dependent degradation of WTAP in EP3 - / - brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(F) Effect of PKA inhibitor, H89, on ubiquitin-dependent degradation of WTAP in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( G ) Effect of ERK1 / 2 phosphorylation site mutation ( HA - WTAP - 2A ) on EP3 - mediated ubiquitin - dependent degradation of WTAP in brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological

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(G) Effect of ERK1/2 phosphorylation site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation of WTAP in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates


( H ) Representative picture of oil red O - stained BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(H) Representative picture of oil red O-stained BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( I ) Western blotting assay of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , and UCP1 in BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Data information : Data are representative of two independent experiments with biological

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(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1 in BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates


( B ) Representative pictures of nile red - stained human BAs differentiated from hESC - derived brown progenitors . Scale bar : 50 μm .

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(B)Representative pictures of nile red-stained human BAs differentiated from hESC-derived brown progenitors. Scale bar: 50 μm.