A , Example of hippocampal neurons co - transfected with Mito - mKate2 VGlut1pHluorin before during field stimulation . arrow indicates a terminal occupied with a mitochondrion , while empty arrow indicates a terminal without a mitochondrion . B , Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin Mito - mKate2 stimulated using field stimulation at 10 Hz for 20s ( 40 - 60s ) . Terminals with a mitochondrion are represented by red trace terminals without are represented by black trace . C , Summary graph corresponds to traces shown in panel B ) . Mean stimulation ( time points 60 - 70s ) comparing terminals of same axon which contain mitochondria ( right , ΔF / F0 = 2 . 3 0 . 9 ) compared to terminals without mitochondria ( left , ΔF / F0 = 3 . 2 0 . 7 ) ( n = 9 neurons , 66 terminals , * p < 0 . 05 , paired t - ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

A, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , C E , Confocal of neuronal processes of fixed neurons transfected with MtDsRed SYN - GFP C , myc - Miro1 ( Miro OE ) or E , myc - Miro1ΔEF ( Miro ΔEF ) . Neurons are either non - treated ( NT ) , TTX treated ( 1 M , 48h ) , PTX treated ( 100 M , 48h ) , or DMSO treated ( 1 : 2000 , as PTX ) . Scale bar , 10 m . B , D F , Fraction of SYN - GFP clusters co - localising with mitochondria in cultures in B , control conditions , DMSO 28 . 7 % 3 . 9 , TTX 16 . 6 % 3 . 1 , PTX 50 . 0 % 7 . 2 , ANOVA * * p0 . 001 , n = 8 - 9 neurons , D , with expression of myc - Miro1 , DMSO 33 . 9 % 4 . 7 , TTX 35 . 9 % 5 . 4 , PTX 46 . 7 % 4 . 0 , ANOVA , * p0 . 05 , n = 8 - 9 , F , with expression of myc - Miro1ΔEF , ANOVA p0 . 05 , n = 9 - 12 . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

A, C E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Live of neurons co - transfected with SyGCaMP5 MtDsRed or myc - ΔEF - Miro1 - IRES - MtDsRed ( ΔEF Miro ) before during 10 Hz field stimulation with without TTX treatments . Scale bar , 10 m . B , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 2 . 5 0 . 5 ) , n = 10 neurons ( 62 terminals ) non - treated terminals ( black trace , ΔF / F0 = 1 . 3 0 . 6 ) , n = 11 neurons ( 69 terminals ) co - transfected with MtDsRed , * p < 0 . 05 , t - . C , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 1 . 8 0 . 3 ) in n = 7 neurons ( 33 terminals ) non - treated terminals ( black trace , ΔF / F0 = 2 . 3 0 . 6 ) in n = 7 neurons ( 32 terminals ) co - transfected with myc - ΔEF - Miro1 - IRES - MtDsRed ; p = 0 . 5 , t - . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed or myc-ΔEF-Miro1-IRES-MtDsRed (ΔEF Miro) before and during 10 Hz field stimulation with and without TTX treatments. Scale bar, 10 m. B, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 2.5 0.5), n=10 neurons (62 terminals) and non-treated terminals (black trace, ΔF/F0 = 1.3 0.6), n=11 neurons (69 terminals) co-transfected with MtDsRed, *p<0.05, t-test. C, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 1.8 0.3) in n=7 neurons (33 terminals) and non-treated terminals (black trace, ΔF/F0 = 2.3 0.6) in n=7 neurons (32 terminals) co-transfected with myc-ΔEF-Miro1-IRES-MtDsRed; p=0.5, t-test. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Luciferase sequestration as an for in vitro uptake . ( A ) Luciferase cofractionating with vacuoles is proteinase K protected . In vitro uptake reactions of three times standard volume were performed ( 60 min , 27 ° C ) in presence or absence of ATP - regenerating system . vacuoles were reisolated , washed , resuspended in 150 mM KCl in PS buffer to a of 0 . 23 mg / ml , split into aliquots . After addition of 1 % Triton X - 100 or buffer , mixture was digested with indicated concentrations of proteinase K ( 15 min , 0 ° C ) . Proteinase treatment was stopped by adding one volume of 0 . 5 mM PMSF in 150 mM KCl in PS buffer . Then , luciferase activity was assayed .

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

Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.


( B ) Luciferase can be released from vacuolar lumen by freeze - thaw treatment . An uptake reaction was performed ( 60 min , 27 ° C ) . vacuoles were reisolated washed as described in Materials Methods section . final pellet was resuspended in 90 μl PS buffer with 150 mM KCl split into two aliquots . One was frozen at − 80 ° C thawed again slowly , whereas other remained on ice . samples were centrifuged ( 15 min , 145 , 000 g , 4 ° C ) . supernatants ( Sup ) were recovered pellets were resuspended in an equal volume of PS with 150 mM KCl . Luciferase activity in both fractions was determined . reaction tubes for this experiment had been coated with BSA ( 0 . 5 mg / ml , 15 min at room ) to reduce unspecific binding of luciferase to surface .

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

(B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.


time course dependence of uptake . ( A ) Standard uptake reactions were performed in presence or absence of ATP - regenerating system . After indicated times at 27 ° C , vacuoles were reisolated , washed , treated with protease , luciferaseuptake activity was assayed .

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

time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.


( B ) A standard uptake reaction was performed . After 70 min of incubation , vacuoles were reisolated , washed , treated with protease . vacuoles were reisolated again , resuspended in reaction buffer without luciferase , incubated for indicated times at 27 ° C . Aliquots were withdrawn to total luciferase remaining . As a control , luciferase was incubated in reaction buffer for same periods of time assayed for activity .

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

(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.


Requirements for in vitro uptake . Standard uptake reactions were performed ( 60 min ) ( A ) at different temperatures , or in presence of different concentrations of ( B ) cytosol , ( C ) ATP , or ( D ) MgCl2 . Luciferase uptake was determined as described in legend to Fig . 3 A . In B , titration curves for four independent cytosol preparations are shown . maximal uptake signal of each titration curve was set at 100 % . In D , sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in reaction .

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

Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.


In vitro uptake is independent of components involved in homotypic vacuolar fusion . ( A ) Standard uptake reactions were performed ( 60 min , 27 ° C ) in presence of indicated reagents using a mixture of vacuoles from strains DBY5734 - 16 ( Δpep4 ) DBY5734 - 19 ( Δpho8 ) . Therefore , homotypic vacuolar fusion luciferase uptake could be assayed simultaneously from same sample . Four independent experiments were averaged . Error bars indicate SD . For averaging , uptake fusion activities of control sample without inhibitor were set at 100 % . Inhibitors were used at following concentrations : anti - Sec17p , 1 μM ; anti - Sec18p , 1 μM ; Gdi1p , 2 . 5 μM ; anti - Vam3p , 1 μM ; anti - Vam7p , 1 μM ; anti - Nyv1p , 1 μM .

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

In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM.


( B ) Vacuoles cytosols were prepared from sec18 - 1 from corresponding wild - type ( Wt ) grown spheroplasted at 23 ° C . isolated vacuoles cytosols were exposed to heat treatment ( 10 min , 37 ° C ) . Standard uptake reactions were performed with vacuoles cytosols in indicated combinations . Control reactions were performed in absence of ATP . Five independent experiments were averaged ; sample with vacuoles cytosol from wild - type was used as 100 % reference . Error bars indicate SD . Mut , mutant .

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

(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.


In vitro uptake depends on GTP hydrolysis on V - ATPase . ( A ) Standard uptake reactions were performed in presence of different concentrations of GTPγS . Luciferase uptake was assayed as in legend to Fig . 3 A . Four independent experiments were averaged , using sample without inhibitor as 100 % reference . Error bars indicate SD . GTPγS did not influence luciferase activity by itself ( not shown ) .

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In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).


( B ) Standard uptake reactions were performed in presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in absence of ATP .

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(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.


( H ) bur1 - ΔC mutant exhibits a synthetic growth defect with vac17Δ mutant . Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [ URA3 ] BUR1 . Plasmids tested were pRS415 [ LEU2 ] ( mock ) , pRS415 BUR1 , or pRS415 bur1 - ΔC . Transformed colonies were cultured in liquid medium serial dilutions spotted onto SC + 5 - FOA or SC - Leu - Ura plates . Plates were incubated at 24 ̊ C for 3 days .

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

(H) The bur1-ΔC mutant exhibits a synthetic growth defect with the vac17Δ mutant. Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [URA3] BUR1. Plasmids tested were pRS415 [LEU2] (mock), pRS415 BUR1, or pRS415 bur1-ΔC. Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura plates. Plates were incubated at 24 ̊C for 3 days.


( A , B ) bur1 - ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of cycle . Yeast were grown in YPD medium collected in log phase growth . content was measured using propidium iodide ( PI ) assessed by flow cytometry . p - value determined with a one - way ANOVA Tukey post hoc . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . ( C , D ) A bur1 - 267 mutant also exhibits a delay in progression through G1 phase of cycle . content was measured using PI assessed by flow cytometry . p - value determined with a one - way ANOVA Tukey post hoc . * * * * ( p - value < 1 x 10 - 4 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment .

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

(A, B) The bur1-ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of the cell cycle. Yeast were grown in YPD medium and collected in log phase growth. DNA content was measured using propidium iodide (PI) staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. (C, D) A bur1-267 mutant also exhibits a delay in progression through G1 phase of the cell cycle. DNA content was measured using PI staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. **** (p-value < 1 x 10-4). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.


( E , F ) bur1 - ΔC vac17Δ double mutant shows accumulation of Whi5 in nucleus . WT , vac17Δ , bur1 - ΔC , bur1 - ΔC vac17Δ , which express Whi5 - 3xGFP Nup188 - mCherry from endogenous loci , were incubated at 24 ̊ C . were scored for presence of Whi5 - 3xGFP in within nucleus ( Nup188 - mCherry ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . p - value determined with a one - way ANOVA Tukey post hoc . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . Scale bar , 2 μm .

O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) The bur1-ΔC vac17Δ double mutant shows accumulation of Whi5 in the nucleus. WT, vac17Δ, bur1-ΔC, and bur1-ΔC vac17Δ cells, which express Whi5-3xGFP and Nup188-mCherry from endogenous loci, were incubated at 24 ̊C. Cells were scored for the presence of Whi5-3xGFP in within the nucleus (Nup188-mCherry). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. Scale bar, 2 μm.


( B ) bur1 - ∆ C vac17 ∆ double mutant have ability to generate a vacuole de novo , which suggests that - cycle delay is due to a defect in Bur1 - dependent signaling . WT , vac17Δ , bur1 - ∆ C , bur1 - ∆ C vac17 ∆ which express Vph1 - GFP from its endogenous locus , were labeled with vacuole specific dye FM4 - 64 . WT bur1 - ∆ C have both FM4 - 64 Vph1 - GFP signals in both mother daughter . vac17Δ bur1 - ∆ C vac17 ∆ have both Vph1 - GFP FM4 - 64 on vacuole in mother , however daughter solely have a Vph1 - GFP labeled vacuole , indicating that vacuole was generated de novo , rather than inherited from mother vacuole . Arrowheads ; new vacuoles in daughter . Scale bar , 2 μm .

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

(B) The bur1-∆C vac17∆ double mutant cells have the ability to generate a vacuole de novo, which suggests that the cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with the vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both Vph1-GFP and FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.


( A ) In vivo phosphorylation of Sch9 is reduced in bur1 - ∆ C vac17 ∆ . WT , bur1 - ∆ C , vac17 ∆ , or bur1 - ∆ C vac17 ∆ expressing SCH9 - HA from URA3 locus were grown in YPD . lysates were analyzed by immunoblot with antibodies directed against HA ( top ) Pgk1 ( loading control ) . ( B ) In vivo phosphorylation of Sch9 is reduced in bur1 - 267 . WT , vac17 ∆ , bur1 - 267 , or bur1 - 267 vac17 ∆ expressing SCH9 - HA from URA3 locus were grown in YPD . lysates were analyzed by immunoblot with antibodies directed against HA ( top ) Pgk1 ( loading control ) .

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

(A) In vivo phosphorylation of Sch9 is reduced in bur1-∆C vac17∆ cells. WT, bur1-∆C, vac17∆, or bur1-∆C vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control). (B) In vivo phosphorylation of Sch9 is reduced in bur1-267 cells. WT, vac17∆, bur1-267, or bur1-267 vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control).


( C ) bur1 - ∆ C shows high rapamycin sensitivity . WT , tor1 ∆ , bur1 - ∆ C , tor1 ∆ bur1 - ∆ C or vac17 ∆ were cultured in liquid YPD medium serial dilutions were spotted onto YPD plates with 0 , 2 , 4 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(C) bur1-∆C shows high rapamycin sensitivity. WT, tor1∆, bur1-∆C, tor1∆ bur1-∆C or vac17∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0, 2, 4 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) In vitro , Bur1 directly phosphorylates kinase domain of Sch9 as well as Bur1 itself . pRS425 BUR1 - HA pVT102 - Ura BUR2 , essential cyclin for BUR1 , were co - expressed in a bur1Δ mutant , immunoprecipitated using anti - HA antibody A beads , then incubated with [ γ - 32P ] ATP GST , GST - N - Sch9 ( 1 - 390 ) or GST - C - Sch9 ( 391 - 824 ) . were eluted with SDS sample buffer , separated by SDS - PAGE , transferred to nitrocellulose membranes . Membranes exposed to x - ray film ( top ) or stained with Ponceau S ( bottom ) . Red arrowhead ; phosphorylated GST - C - Sch9 . Gray arrowhead ; auto - phosphorylated Bur1 .

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

(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.


( D ) Sch9 - 2D3E , but not Sch9 - 3A - 2D3E , partially suppressed rapamycin sensitivity of tor1 ∆ . tor1 ∆ which expresses pRS426 ( mock ) , pRS425 TOR1 , pVT102 - Ura ( mock ) , pVT102 - Ura Sch9 , pVT102 - Ura Sch9 - 2D3E , or pVT102 - Ura Sch9 - 3A - 2D3E , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 - 3 days .

O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(D) Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells. tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days.


( E ) Sch9 - 2D3E mutant did not suppress rapamycin sensitivity of bur1 - ∆ C . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pVT102 - Ura ( mock ) , pVT102 - Ura SCH9 , pVT102 - Ura sch9 - 2D3E , pVT102 - Ura sch9 - 3A - 2D3E , pVT102 - Ura sch9 - 3D / E , or pVT102 - Ura sch9 - 3D / E - 2D3E were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 3 days .

O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.


( A ) Bur1 fused SV40 - NLS does not rescue rapamycin sensitivity of bur1 - ∆ C compared to Bur1 without NLS . bur1 - ∆ C which expresses pRS416 ( mock ) , pRS416 BUR1 , pRS416 BUR1 - mNG , pRS416 BUR1 - mNG - NLS , pRS416 bur1 - ∆ C , pRS416 bur1 - ∆ C - mNG - NLS or pRS416 bur1 - ∆ N , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

O O O B-GENEPROD O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(A) Bur1 fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416 bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) Adding - export signal ( NES ) of PKI to Bur1 / or deletion of importin α - dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pRS426 bur1 - ∆ N , pRS426 BUR1 - mNG , pRS426 BUR1 - mNG - NES , or pRS426 bur1 - ∆ N - mNG - NES , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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

(B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the importin α-dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426 BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


C , Immunoblot of lysates from MEF treated with EBSS , 250 nM Torin - 1 or 100 μM NaAsO2 for 1h run under non - reducing conditions . For refeed condition , starved were incubated in complete medium for indicated times .

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

C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1h and run under non-reducing conditions. For refeed condition, starved cells were incubated in complete medium for the indicated times.


C , Immunoblot of lysates from ARPE - 19 expressing TFEB - FLAG - WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h run under non - reducing condition .

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

C, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-FLAG-WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing condition.


D , Immunoblot of lysates from ARPE - 19 overexpressing TFEB - FLAG or TFE3 - MYC , treated with 250 nM Torin - 1 for 1 h run under non - reducing condition . E , of immunoblot data shown in ( D ) . Data are presented as mean ± SD using two - t - ( * ) p < 0 . 05 from three independent experiments .

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

D, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG or TFE3-MYC, treated with 250 nM Torin-1 for 1 h and run under non-reducing condition. E, Quantification of immunoblot data shown in (D). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 from three independent experiments.


D , Relative quantitative RT - PCR of mRNA expression of autophagy - ( UVRAG ) , mitochondria - ( PGC - 1α ) lysosome ( MCOLN1 , ATPV1C1 HEXA ) - related genes in ARPE - 19 overexpressing TFEB - FLAG - WT or TFEB - FLAG - C212A . Data are presented as mean ± SD using two - way ANOVA , ( ns ) not significant as compared to same gene in TFEB - WT overexpressing , from three independent experiments .

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

D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), mitochondria- (PGC-1α) and lysosome (MCOLN1, ATPV1C1 and HEXA)-related genes in ARPE-19 cells overexpressing TFEB-FLAG-WT or TFEB-FLAG-C212A. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same gene in TFEB-WT overexpressing cells, from three independent experiments.


G , Relative quantitative RT - PCR of mRNA expression of lysosome - related genes ( MCOLN1 ATPV61C1 ) in ARPE - 19 HeLa treated with 100 μM 50 μM NaAsO2 respectively for indicated times . Data are presented as mean ± SD using one - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 from two independent experiments .

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

G, Relative quantitative RT-PCR analysis of the mRNA expression of lysosome-related genes (MCOLN1 and ATPV61C1) in ARPE-19 and HeLa cells treated with 100 μM and 50 μM NaAsO2 respectively for the indicated times. Data are presented as mean ± SD using one-way ANOVA (*)p<0.05, (**)p< 0.01, (***)p<0.001 from two independent experiments.


D , Relative quantitative RT - PCR of mRNA expression of autophagy - ( UVRAG ) , lysosome - ( ATP6V1C1 ) , oxidative stress response - ( TXNRD1 HMOX1 ) , cycle control - ( CDKN1a ) cytokine production ( IL6 ) - related genes in TFEB / TFE3 - double knockout MEFs stably expressing TFEB - FLAG - WT or TFEB - FLAG - C212A treated with 5 μM arsenic trioxide ( ATO ) or 12 . 5 μM NaAsO2 for 12 h . Data are presented as mean ± SD using two - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 , ( * * * * ) p < 0 . 0001 from three independent experiments .

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

D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), lysosome- (ATP6V1C1), oxidative stress response- (TXNRD1 and HMOX1), cell cycle control- (CDKN1a) and cytokine production (IL6) -related genes in TFEB/TFE3-double knockout MEFs cells stably expressing TFEB-FLAG-WT or TFEB-FLAG-C212A and treated with 5 μM arsenic trioxide (ATO) or 12.5 μM NaAsO2 for 12 h. Data are presented as mean ± SD using two-way ANOVA (*)p<0.05, (**)p<0.01, (***)p<0.001, and (****)p<0.0001 from three independent experiments.


C , Representative examples of HLH - 30 : : 3xFLAG : : eGFP subcellular localization : , diffuse GFP distribution along whole of worm ; Weak , some in posterior part of worm display GFP labeling but no labeling is found in head ; Strong , distinct GFP labeling in along worm , including neurons in head . Arrowheads denote localization of HLH - 30 : : 3xFLAG : : eGFP . Scale bar 200 µm 50 µm . D , of subcellular distribution of HLH - 30 : : 3xFLAG : : eGFP HLH - 30 ( C284A ) : : 3xFLAG : : eGFP upon treatment with different stimuli . Data are from three independent experiments with at least 50 per experiment .

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

C, Representative examples of HLH-30::3xFLAG::eGFP subcellular localization: Cytoplasmic, diffuse GFP distribution along the whole body of the worm; Weak nuclear, some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells; Strong nuclear, distinct nuclear GFP labeling in cells along the worm body, including neurons in the head area. Arrowheads denote nuclear localization of HLH-30::3xFLAG::eGFP. Scale bar 200 µm and 50 µm. D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP upon treatment with different stimuli. Data are from three independent experiments with at least 50 animals per experiment.


A , Lifespan of hlh - 30 mutants at 25 ° C . Graph represents Kaplan - Meier survival plots of two independent experiments initiated with 100 per . ( * * ) p < 0 . 01 ( * * * * ) p < 0 . 0001 by Log - rank ( Mantel - Cox ) compared to wild type control . B , Survival of hlh - 30 mutants on 7 . 5 mM NaAsO2 . Data are mean ± SD of three independent experiments initiated with 120 per . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control . C , Survival of hlh - 30 mutants to infection by Staphylococcus aureus . Data are mean ± SD of three independent experiments initiated with 100 per . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control .

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

A, Lifespan assay of hlh-30 mutants at 25°C. Graph represents Kaplan-Meier survival plots of two independent experiments initiated with 100 animals per strain. (**)p<0.01 and (****)p<0.0001 by Log-rank (Mantel-Cox) test compared to wild type control. B, Survival of hlh-30 mutants on 7.5 mM NaAsO2. Data are the mean ± SD of three independent experiments initiated with 120 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control. C, Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± SD of three independent experiments initiated with 100 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control.


E , Synthetic developmental delay phenotype of eat - 2 ( ad1116 ) worms carrying mutations in hlh - 30 gene . Data are mean ± SD of three independent experiments with at least 20 per . ( * * * * ) p < 0 . 0001 by One - way ANOVA with multiple comparison . F , Representative of worms quantified in ( E ) . Scale bars 200 µm .

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E, Synthetic developmental delay phenotype of eat-2(ad1116) worms carrying mutations in the hlh-30 gene. Data are the mean ± SD of three independent experiments with at least 20 animals per assay. (****)p<0.0001 by One-way ANOVA with multiple comparison test. F, Representative images of worms quantified in (E). Scale bars 200 µm.


ISCts midguts expressing YFP alone ( control ) , or expressing two non - overlapping EcR - RNAi , of number of YFP - positive ( yellow ) . graph also plots number of GFP - positive in esgts midgut expressing GFP alone ( ctrl ) , or expressing EcR - RNAi # 1 , EcR - RNAi # 2 , mcherry - RNAi as an additional negative control ( see EV2B ) .

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ISCts midguts expressing YFP alone (control), or expressing two non-overlapping EcR-RNAi, and quantification of the number of YFP-positive cells (yellow). The graph also plots the number of GFP-positive cells in esgts midgut expressing GFP alone (ctrl), or expressing EcR-RNAi#1, EcR-RNAi#2, and mcherry-RNAi as an additional negative control (see EV2B).


esgts midguts expressing GFP alone ( control ) , or expressing EcR - DN , EcR - DN + pri . Samples were stained for GFP ( green ) . graph shows of number of GFP - positive in different genotypes .

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esgts midguts expressing GFP alone (control), or expressing EcR-DN, and EcR-DN + pri. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in the different genotypes.


Posterior midguts containing control Ubr3 null MARCM clones ( GFP , green ) .

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Posterior midguts containing control and Ubr3 null MARCM clones (GFP, green).


esgts midguts expressing GFP alone ( control ) , or expressing Ubr3 - RNAi , Ubr3 - RNAi + OvoB . Samples were stained for GFP ( green ) . H ' of GFP - positive from H .

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esgts midguts expressing GFP alone (control), or expressing Ubr3-RNAi, and Ubr3-RNAi + OvoB. Samples were stained for GFP (green). H' quantification of GFP-positive cells from H.


esgts midguts expressing GFP alone ( control ) or expressing SvbACT . Samples were stained for GFP ( green ) , ß - catenin ( purple ) , DE - Cadherin ( ) or Scribble ( yellow ) . Top bottom pictures show separate channels for a same region .

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esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region.


esgts midguts expressing GFP alone ( control ) , or expressing miR8 , OvoB , mir8 + OvoB . Samples were stained for GFP ( green ) ß - catenin ( purple ) . graph shows of GFP - positive for each genotype . Y axis is drawn using a log ( 10 ) scale .

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esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale.


esgts midguts expressing GFP alone ( control ) , or expressing Notch Intra Domain ( NICD ) , NICD + OvoB . In closeups ( right ) , DAPI is shown in purple for improved contrast ; arrow highlights a with intermediate phenotype .

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esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.


esgtsF / O midguts expressing GFP alone ( control ) , or expressing RasV12 , RasV12 + svb - RNAi ( top panels ) . Bottom panels show esgts midguts expressing GFP alone ( control ) , or expressing EGFR - DN , EGFR - DN + OvoB . Samples were stained for GFP ( green ) . graph shows of number of GFP - positive in esgts midguts expressing GFP alone ( ctrl ) , or expressing EGFR - DN , EGFR - DN + OvoB , EGFR - DN + svb - RNAi . Y axis is plotted as log ( 10 ) .

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esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing ArmS10 , ArmS10 + svb - RNAi , ArmS10 + OvoB , TCF - DN , TCF - DN + OvoB . Samples were stained for GFP ( green ) . graph shows of number of GFP - positive in esgts midguts expressing GFP alone ( ctrl ) , or expressing TCF - DN , TCF - DN + OvoB , TCF - DN + svb - RNAi . Y axis is plotted as log ( 10 ) .

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esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) Prospero ( red ) . Closeups correspond to boxed regions , with DAPI shown in purple GFP - positive outlined in yellow .

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esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.


esgts - F / O midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) DCP1 ( red ) . E ' shows of number of GFP - positive in esgts guts expressing GFP alone ( ctrl ) , or DIAP , SvbREP , SvbREP + DIAP .

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esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP.


esgts midguts expressing GFP alone ( control ) , or expressing Notch - RNAi , Notch - RNAi + svb - RNAi , Notch - RNAi + SvbREP . Samples were stained for GFP ( green ) Prospero ( red ) . graph shows of number of GFP - positive .

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esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


esgts midguts expressing GFP alone ( control ) , or expressing STAT - RNAi , STAT - RNAi + svb - RNAi STAT - RNAi + SvbREP . Samples were stained for GFP ( green ) Prospero ( red ) . graph shows of number of GFP - positive .

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esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


Cross sections of control MyoIAts midguts ( expressing GFP mCherry - RNAi ) , or expressing svb - RNAi , SvbACT , Pri . Samples were stained for F - actin ( ) , GFP ( green ) Tsp2a ( yellow ) .

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Cross sections of control MyoIAts midguts (expressing GFP and mCherry-RNAi), or expressing svb-RNAi, SvbACT, and Pri. Samples were stained for F-actin (white), GFP (green) and Tsp2a (yellow).


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) , Scribble ( yellow ) , ß - catenin ( purple ) DAPI ( Blue ) . A ' pictures display cross sections of regions shown in A .

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MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) PH3 ( red ) . graph plots number of mitotic PH3 - positive per midgut of MyoIAts guts expressing GFP alone ( ctrl ) , or expressing SvbACT , SvbREP + pri .

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MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.


A . qPCR shows loss of Setd1b in forebrain regions while in cerebellum are not affected ( CA : Control , n = 6 ; cKO , n = 6 . DG : Control , n = 6 ; cKO , n = 6 . Cortex : Control , n = 7 ; cKO , n = 7 . Cerebellum : Control , n = 4 ; cKO , n = 4 ) . * p - value < 0 . 05 ( Student t - ) .

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A. qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected (CA: Control, n = 6; cKO, n = 6. DG: Control, n = 6; cKO, n = 6. Cortex: Control, n = 7; cKO, n = 7. Cerebellum: Control, n = 4; cKO, n = 4). * p-value < 0.05 (Student t-test).


B . Immunoblot shows loss of Setd1b in hippocampus of Setd1b cKO mice ( Control , n = 4 ; cKO , n = 4 ) . * * p - value < 0 . 01 ( Student t - ) .

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B. Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice (Control, n = 4; cKO, n = 4). ** p-value < 0.01 (Student t-test).


C . Immunohistochemical ( upper level ) for marker of neuronal integrity ( lower panel ) show no difference in control Setd1b cKO mice ( NeuN : Control , n = 6 ; cKO , n = 6 ; Student t - p - value = 0 . 57 . MAP2 : Control , n = 4 ; cKO , n = 4 ; Student t - p - value = 0 . 72 . Iba1 : Control , n = 5 ; cKO , n = 5 ; Student t - p - value = 0 . 8 . Gfap : Control , n = 5 ; cKO , n = 5 ; Student t - p - value = 0 . 09 . ) . Scale bar : 100 µm .

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C. Immunohistochemical staining (upper level) for marker proteins of neuronal integrity and quantification (lower panel) show no difference in control and Setd1b cKO mice (NeuN: Control, n = 6; cKO, n = 6; Student t-test p-value = 0.57. MAP2: Control, n = 4; cKO, n = 4; Student t-test p-value = 0.72. Iba1: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.8. Gfap: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.09.). Scale bar: 100 µm.


D . Average speed ( left panel ) time spent in center ( right panel ) during exposure to open field was similar in control Setd1b cKO mice ( Average speed : Control , n = 15 ; cKO , n = 15 ; Student t - p - value = 0 . 075 . Time spent in center : Control , n = 15 ; cKO , n = 15 ; Student t - p - value = 0 . 96 ) .

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D. Average speed (left panel) and percent time spent in the center (right panel) during exposure to the open field test was similar in control and Setd1b cKO mice (Average speed: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.075. Time spent in center: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.96).


E . Short term memory was not different between control Setd1b cKO mice as indicated by similar of alternations in Y - maze ( Control , n = 15 ; cKO , n = 15 ; Student t - p - value = 0 . 3 ) .

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E. Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y-maze test (Control, n = 15; cKO, n = 15; Student t-test p-value = 0.3).


F . Depressive - like behavior was measured in Porsolt forced swim . There was no significant difference in % time spent immobile between groups ( Control , n = 8 ; cKO , n = 8 ; Student t - p - value = 0 . 5 ) .

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F. Depressive-like behavior was measured in the Porsolt forced swim test. There was no significant difference in the % time spent immobile between groups (Control, n = 8; cKO, n = 8; Student t-test p-value = 0.5).


G . Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice ( Control : n = 15 , cKO : n = 15 . Repeated measures ANOVA , genotype effect : F ( 1 , 28 ) = 82 . 34 , * * * * p - value < 0 . 0001 ) .

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G. Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice (Control: n = 15, cKO: n = 15. Repeated measures ANOVA, genotype effect: F (1,28) = 82.34, **** p-value < 0.0001).


H . Plots showing specific search strategies during water maze training . Note failure of Setd1b cKO mice to adopt hippocampus - dependent search strategies . I . cognitive score calculated on basis of hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice ( Student t - : * * * p - value < 0 . 001 ) .

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H. Plots showing the specific search strategies during water maze training. Note the failure of Setd1b cKO mice to adopt hippocampus-dependent search strategies. I. The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice (Student t-test: *** p-value < 0.001).


F . Left panel shows representative of RNAscope performed for Kmt2a , Kmt2b Setd1b . Right panel shows a bar chart quantifying dots / ( n = 1500 from 2 mice ) indicative of corresponding expression level . Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b ( * * * p - value < 0 , 0001 , Student t - ) .

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F. Left panel shows representative images of RNAscope performed for Kmt2a, Kmt2b and Setd1b. Right panel shows a bar chart quantifying the dots/cell (n = 1500 cells from 2 mice) indicative of the corresponding expression level. Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b (*** p-value < 0,0001, Student t-test).


( A ) transcript level of solo LTR in wild type epcr1 mutant . Error bars are standard deviations of three technical replicates . experiment was independently performed three times similar results were obtained .

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(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained.


( B ) developmental phenotype of epcr1 epcr2 single mutants epcr1 / 2 double mutant relative to wild type . Two - week - old seedlings grown on vertical MS medium plates are shown .

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(B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown.


( C ) transcript of solo LTR , SDC , AtSN1 , AtGP1 AtCOPIA28 in wild type , epcr1 epcr2 single mutants , epcr1 / 2 double mutant , two EPCR1 transgenic lines in epcr1 / 2 double mutant background . Error bars are standard deviations of three biological replicates .

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(C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.


( A ) interaction between EPCR1 ARID2 or ARID3 . Arabidopsis carrying EPCR1 - Flag ARID2 - Myc or ARID3 - Myc transgenes were used for co - IP .

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(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.


( B ) interaction between TRB1 ARID2 . Arabidopsis carrying TRB1 - Flag / or ARID2 - Myc transgenes were used for co - IP .

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(B) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP.


( C ) interaction between TRB1 EPCR1 . Arabidopsis carrying TRB1 - Flag / or EPCR1 - Myc transgenes were used for co - IP .

(C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP.


( E ) interaction between TRB1 PWWP2 or ARID2 was detected by an GST pull - down .

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(E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay.


( F ) interaction between EPCR1 , PWWP2 , ARID2 , TRB1 was detected by an HIS pull - down . full - PWWP2 , ARID2 , TRB1 , EPCR1 - N terminal ( 1 - 500 aa ) were used for interaction .

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(F) The interaction between EPCR1, PWWP2, ARID2, and TRB1 was detected by an HIS pull-down assay. The full-length PWWP2, ARID2, and TRB1, and the EPCR1-N terminal (1-500 aa) were used for the interaction assay.


( A ) developmental phenotype of arid2 / 3 / 4 mutant as compared to wild type . Two - week - old seedlings grown on MS medium are shown .

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(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown.


( B ) developmental phenotype of pwwp1 / 2 / 3 + / - mutant as compared to wild type . One - month - old grown in soil are shown .

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(B) The developmental phenotype of the pwwp1/2/3+/- mutant as compared to the wild type. One-month-old plants grown in soil are shown.


effect of arid2 , arid3 , arid4 mutations on silencing of solo LTR , SDC , FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations .

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The effect of the arid2, arid3, and arid4 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.


effect of pwwp1 , pwwp2 , pwwp3 mutations on silencing of solo LTR , SDC , FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations .

The effect of the pwwp1, pwwp2, and pwwp3 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.


( E ) effect of trb1 mutation TRB2 knockdown ( TRB2 - KD ) on silencing of solo LTR , as determined by qPCR ( right panel ) . transcript level of TRB2 was evaluated to confirm TRB2 knockdown ( left panel ) . Error bars represent standard deviations of three biological replicates .

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(E) The effect of the trb1 mutation and the TRB2 knockdown (TRB2-KD) on the silencing of solo LTR, as determined by qPCR (right panel). The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown (left panel). Error bars represent standard deviations of three biological replicates.


( B ) Heat maps showing differentially transcribed genes TEs in arid2 / 3 / 4 epcr1 / 2 mutants relative to wild type .

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(B) Heat maps showing differentially transcribed genes and TEs in the arid2/3/4 and epcr1/2 mutants relative to the wild type.


( D ) Genome browser snapshots showing transcript patterns of ROS1 IBM1 in arid2 / 3 / 4 epcr1 / 2 relative to wild type .

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(D) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2/3/4 and epcr1/2 relative to the wild type.


( E ) Effects of arid2 / 3 / 4 epcr1 / 2 on transcript of ROS1 IBM1 - L as determined by qPCR . expression of ROS1 longer version of IBM1 ( IBM1 - L ) was evaluated by qPCR . ACT7 was amplified as an internal control . Bars represent SD from two independent experiments , each with three technical replications .

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(E) Effects of arid2/3/4 epcr1/2 on the transcript levels of ROS1 and IBM1-L as determined by qPCR. The expression of ROS1 and the longer version of IBM1 (IBM1-L) was evaluated by qPCR. ACT7 was amplified as an internal control. Bars represent SD from two independent experiments, each with three technical replications.


( A ) interaction of EPCR1 with HAM1 HAM2 . EPCR1 - Flag transgenic were crossed to HAM1 - Myc HAM2 - Myc transgenic . progeny were used to evaluate interaction between EPCR1 with HAM1 HAM2 by co - IP .

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(A) The interaction of EPCR1 with HAM1 and HAM2. EPCR1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants. The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co-IP.


( B ) interaction of ARID2 , ARID3 , ARID4 with HAM1 HAM2 . ARID2 - Myc , ARID3 - Myc , ARID4 - Myc transgenic were crossed to HAM1 - Flag HAM2 - Flag transgenic their progeny were used for co - IP .

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(B) The interaction of ARID2, ARID3, and ARID4 with HAM1 and HAM2. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HAM1-Flag and HAM2-Flag transgenic plants and their progeny were used for co-IP.


( C ) interaction of TRB1 with HAM1 HAM2 . TRB1 - Flag transgenic were crossed to HAM1 - Myc HAM2 - Myc transgenic their progeny were used for co - IP .

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(C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP.


( D ) interaction of HDA9 with ARID2 , ARID3 , ARID4 . ARID2 - Myc , ARID3 - Myc , ARID4 - Myc transgenic were crossed to HDA9 - Flag transgenic their progeny were used for co - IP .

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(D) The interaction of HDA9 with ARID2, ARID3, and ARID4. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HDA9-Flag transgenic plants and their progeny were used for co-IP.


( E ) interaction of HDA6 with EPCR1 ARID2 . EPCR1 - Flag ARID2 - Flag transgenic were crossed to HDA6 - Myc transgenic their progeny were used for co - IP .

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(E) The interaction of HDA6 with EPCR1 and ARID2. EPCR1-Flag and ARID2-Flag transgenic plants were crossed to HDA6-Myc transgenic plants and their progeny were used for co-IP.


( A ) Overlap among H4K5 hyperacetylated regions in arid2 / 3 / 4 epcr1 / 2 mutants relative to wild type . overlap is significant ( P = 4 . 410 - 78 ) as determined by hypergeometric . ( B ) Metaplot of histone H4K5 acetylation of TEs in wild type , arid2 / 3 / 4 , epcr1 / 2 . Histone H4K5 acetylation are represented by normalized reads that obtained in H4K5Ac ChIP - seq . TSS represents transcription start site TTS represents transcription termination site . ( C ) Boxplot of histone H4K5 acetylation of TEs in wild type , arid2 / 3 / 4 , epcr1 / 2 . Asterisks indicate that H4K5Ac is significantly higher in arid2 / 3 / 4 ( P = 2 . 4210 - 13 ) epcr1 / 2 ( P = 1 . 5210 - 8 ) mutants than in wild type as determined by Welch ' s Two - Sample t - . Horizontal lines represent median , bottom top of box represent 25th 75th percentile . whiskers represent data range within 1 . 5 × of interquantile range .

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(A) Overlap among H4K5 hyperacetylated regions in the arid2/3/4 and epcr1/2 mutants relative to the wild type. The overlap is significant (P=4.410-78) as determined by hypergeometric test. (B) Metaplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP-seq analysis. TSS represents transcription start site and TTS represents transcription termination site. (C) Boxplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Asterisks indicate that H4K5Ac levels is significantly higher in the arid2/3/4 (P=2.4210-13) and epcr1/2 (P=1.5210-8) mutants than in the wild type as determined by Welch's Two-Sample t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( D ) nuclei with condensed , intermediate , or decondensed heterochromatin status , as marked by DAPI H3K27me signals . ( E ) Percentages of nuclei with condensed , intermediate , or decondensed heterochromatin signals in wild type arid2 / 3 / 4 mutant . n = 113 .

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(D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals. (E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113.


( F ) Abundance of Pol IV - dependent siRNAs in epcr1 / 2 , arid2 / 3 / 4 , nrpd1 , nrpe1 , hda6 mutants relative to wild type [ ( Mut - WT ) / ( Mut + WT ) ] from chromosome 3 of Arabidopsis .

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(F) Abundance of Pol IV-dependent siRNAs in the epcr1/2, arid2/3/4, nrpd1, nrpe1, and hda6 mutants relative to the wild type [(Mut-WT)/(Mut+WT)] from chromosome 3 of Arabidopsis.


( H ) Boxplot showing of Pol IV - dependent siRNAs that are up - regulated in hda6 mutant . Asterisks indicate that siRNA in mutants are significantly ( P → 0 ) up - regulated compared to wild type as determined by paired student t - . Horizontal lines represent median , bottom top of box represent 25th 75th percentile . whiskers represent data range within 1 . 5 × of interquantile range .

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(H) Boxplot showing levels of Pol IV-dependent siRNAs that are up-regulated in the hda6 mutant. Asterisks indicate that siRNA levels in the mutants are significantly (P→0) up-regulated compared to the wild type as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( A ) Metaplot of CG , CHG , CHH methylation of genes TEs in genomes of wild type , epcr1 / 2 , arid2 / 3 / 4 . H indicates C , A , or T .

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(A) Metaplot of CG, CHG, and CHH methylation of genes and TEs in the genomes of wild type, epcr1/2, and arid2/3/4 plants. H indicates C, A, or T.


( B ) Metaplot of CG , CHG , CHH methylation on chromosome 3 of Arabidopsis in wild type , epcr1 / 2 , arid2 / 3 / 4 .

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(B) Metaplot of CG, CHG, and CHH methylation on chromosome 3 of Arabidopsis in wild type, epcr1/2, and arid2/3/4 plants.


( D ) Boxplot of methylation in wild type , nrpe1 , epcr1 / 2 , arid2 / 3 / 4 mutants . hypo - DMRs identified in nrpe1 mutant was defined as RdDM target loci . Asterisks indicate that methylation is significantly ( P < 0 . 001 ) decreased in mutants as determined by paired student t - . Horizontal lines represent median , bottom top of box represent 25th 75th percentile . whiskers represent data range within 1 . 5 × of interquantile range .

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(D) Boxplot of DNA methylation in the wild type, nrpe1, epcr1/2, and arid2/3/4 mutants. The hypo-DMRs identified in the nrpe1 mutant was defined as RdDM target loci. Asterisks indicate that DNA methylation is significantly (P<0.001) decreased in the mutants as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( E ) Heat maps of CG , CHG , CHH methylation at RdDM target loci . hypo - DMRs identified in nrpe1 mutant were defined as RdDM target loci analyzed in wild type , nrpe1 , eprc1 / 2 , arid2 / 3 / 4 mutants . Black yellow indicate low methylation high methylation , respectively .

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(E) Heat maps of CG, CHG, and CHH methylation at RdDM target loci. The hypo-DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type, nrpe1, eprc1/2, and arid2/3/4 mutants. Black and light yellow indicate low methylation and high methylation, respectively.


( A ) Scatter plots showing effect of epcr1 / 2 arid2 / 3 / 4 mutations on CG , CHG , CHH methylation at co - upregulated TEs genes in epcr1 / 2 arid2 / 3 / 4 mutants . TE methylation gene methylation refer , respectively , to methylation of TE methylation of 1 - kb - gene - promoter region .

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(A) Scatter plots showing the effect of the epcr1/2 and arid2/3/4 mutations on CG, CHG, and CHH methylation at the co-upregulated TEs and genes in the epcr1/2 and arid2/3/4 mutants. TE methylation and gene DNA methylation refer, respectively, to methylation of the TE body and methylation of the 1-kb-gene-promoter region.


( B ) Box plots of CG , CHG , CHH methylation in wild type , arid2 / 3 / 4 , epcr1 / 2 mutants at co - upregulated TEs genes in arid2 / 3 / 4 epcr1 / 2 mutants . Asterisks indicate statistical significance as determined by paired student t . CHG methylation of co - upregulated TEs is significantly increased in arid2 / 3 / 4 ( P = 1 . 2610 - 4 ) epcr1 / 2 ( P = 6 . 310 - 9 ) ; CHH methylation of co - upregulated genes is significantly decreased in arid2 / 3 / 4 ( P = 3 . 510 - 6 ) epcr1 / 2 ( P = 6 . 410 - 7 ) . Horizontal lines represent median , bottom top of box represent 25th 75th percentile . whiskers represent data range within 1 . 5 × of interquantile range .

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(B) Box plots of CG, CHG, and CHH methylation in the wild type, arid2/3/4, and epcr1/2 mutants at the co-upregulated TEs and genes in the arid2/3/4 and epcr1/2 mutants. Asterisks indicate statistical significance as determined by paired student t test. CHG methylation of co-upregulated TEs is significantly increased in arid2/3/4 (P=1.2610-4) and epcr1/2 (P=6.310-9); CHH methylation of co-upregulated genes is significantly decreased in arid2/3/4 (P=3.510-6) and epcr1/2 (P=6.410-7). Horizontal lines represent the median, the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( C ) Overexpression of DRD1 fails to restore silencing of EPCR1 / 2 target loci in epcr1 / 2 mutant background .

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(C) Overexpression of DRD1 fails to restore the silencing of EPCR1/2 target loci in the epcr1/2 mutant background.


( C ) STIM1 STIM2 mRNA expression in PBMC of P1 , P2 , mother a healthy donor ( HD ) that were left unstimulated or stimulated with anti - CD3 / CD28 for 24 hours . mRNA of STIM1 / 2 normalized to 18S . Graph shows mean ± of duplicates from one experiment .

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(C) STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother and a healthy donor (HD) that were left unstimulated or stimulated with anti-CD3/CD28 for 24 hours. mRNA levels of STIM1/2 normalized to 18S. Graph shows the mean ± SEM of duplicates from one experiment.


( D ) STIM1 expression in CD4 + CD8 + T from P1 , P2 , mother a HD analyzed by flow cytometry . Shaded histograms : polyclonal rabbit IgG control antibody ; open histograms : polyclonal anti - STIM1 antibody . Bar graphs show delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T . Bar graphs are mean ± of 2 independent repeat experiments .

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(D) STIM1 protein expression in CD4+ and CD8+ T cells from P1, P2, the mother and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti-STIM1 antibody. Bar graphs show the delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of 2 independent repeat experiments.


( E ) STIM1 expression in expanded human T analyzed by immunoblotting . One representative Western blot of 3 is shown . Bar graphs are mean ± of STIM1 expression normalized to actin from 3 independent experiments .

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(E) STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from 3 independent experiments.


( F ) Ca2 + influx in Fura - 2 loaded PBMC of P1 , P2 , mother a HD . T were stimulated with 1 μM thapsigargin ( TG ) in absence of extracellular Ca2 + followed by addition of 1 mM extracellular Ca2 + . Bar graphs show integrated Ca2 + influx response ( AUC , under curve ) from 400 - 800 sec peak Ca2 + influx normalized to baseline Ca2 + ( F340 / 380 ) at 400 sec . Data represent mean ± from 2 experiments . Statistical by unpaired Student ' s t - . * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400-800 sec and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 sec. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **p<0.01, ***p<0.001.


GFP - ORAI1 expressing HEK293 were transfected with mCherry - STIM1 ( WT or L374P ) left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( D , E ) Representative TIRF microscopy from one of two repeat experiments . Scale bars , 10 μm .

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

GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm.


GFP - ORAI1 expressing HEK293 were transfected with mCherry - STIM1 ( WT or L374P ) left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( F ) Percentages of WT or mutant mCherry - STIM1 present at plasma ( i . e . visible in TIRFM evanescent field ) before TG stimulation . Data are mean ± calculated from 550 ( WT ) 830 ( L374P ) from two independent experiments . ( G ) Change of mCherry - STIM1 ( WT or L374P mutant ) [ F ( t ) / F ( 0 ) ] in TIRFM evanescence field . normalized to values at time of TG addition in Ca2 + - free Ringer ' s buffer at 0 sec . Traces show mean ± of 40 - 50 from two independent experiments . ( H ) Colocalization of mCherry - STIM1 ( WT or L374P mutant ) GFP - ORAI1 from time of TG addition at 0 sec . Colocalization measured using Pearson ' s coefficient for GFP mCherry .

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

GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry.


( A ) of CD4 + T from P1 ( red ) , P2 ( blue ) , mother ( grey ) HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) anti - CD28 ( 10 μg / ml ) in presence or absence of 1 μM FK506 for 24 hours . ( A ) Representative histograms of FSC ( left panel ) percentages of T blasts ( defined as to right of dotted vertical ) analyzed by flow cytometry ( right panel ) .

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

Cell size (A) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel).


proliferation ( B ) of CD4 + T from P1 ( red ) , P2 ( blue ) , mother ( grey ) HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) anti - CD28 ( 10 μg / ml ) in presence or absence of 1 μM FK506 for 24 hours . ( B ) Representative histograms of CFSE dilution ( left panel ) percentages of proliferating ( defined as to left of dotted vertical ) ( right panel ) . Bar graphs in A B are mean ± from two independent experiments .

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

proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments.


( C , D ) Cytokine production by PBMC from P1 , P2 , mother an unrelated HD after stimulation with PMA ( 40 ng / ml ) ionomycin ( 500 ng / ml ) for 4 hours . Cytokines were analyzed by flow cytometry following surface with antibodies against CD3 , CD4 CD45RO , permeabilization intracellular cytokine for GM - CSF , IL - 22 IL - 17A . Representative flow cytometry plots ( C ) of Th17 ( GM - CSF , IL - 22 , IL - 17A ) , Th1 ( TNF - α , IFN - γ ) Th2 ( IL - 4 ) cytokines ( D ) . Data represent mean ± from two independent experiments .

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

(C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.


A ) Severe onychomycosis in P2 ( STIM1 p . L374P ) .

O O O B-DISEASE O O O B-GENEPROD O O O O O

A) Severe onychomycosis in P2 (STIM1 p.L374P).


B ) PBMC of a healthy donor ( HD1 ) cultured for 6 days in presence of 6 . 5 mg / ml C . albicans . At day 6 , PBMC were restimulated with PMA ionomycin in presence of 10 μM GSK - 7975A or DMSO for 6 h CD4 + CD8 + T were analyzed for cytokine production by flow cytometry . Bar graphs represent mean ± from two independent experiments .

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

B) PBMC of a healthy donor (HD1) cultured for 6 days in the presence of 6.5 mg/ml C. albicans protein. At day 6, PBMC were restimulated with PMA and ionomycin in the presence of 10 μM GSK-7975A or DMSO for 6 h and CD4+ and CD8+ T cells were analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.