( 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 .

O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_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 O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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) 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 ∆ 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 ) .

O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_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 O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_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 O O 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 ∆ 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 .

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

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

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

(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 ) 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 .

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

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

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

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

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 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 .

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

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

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

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

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 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 .

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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 : 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 .

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

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 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 .

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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 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 .

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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 , 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 ) .

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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 , 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 .

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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 and 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 , and Ubr3 - RNAi + OvoB . Samples were stained for GFP ( green ) . H ' quantification of GFP - positive cells 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 ) , and ß - catenin ( purple ) , DE - Cadherin ( white ) or Scribble ( yellow ) . Top and 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 , 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 .

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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 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 .

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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 , 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 ) .

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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 , 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 ) .

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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 ) and Prospero ( red ) . Closeups correspond to boxed regions , with DAPI shown in purple and GFP - positive cells 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 ) 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 .

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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 , and Notch - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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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 and STAT - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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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 and mCherry - RNAi ) , or expressing svb - RNAi , SvbACT , and Pri . Samples were stained for F - actin ( white ) , GFP ( green ) and 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 ) and DAPI ( Blue ) . A ' pictures display cross sections of the 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 ) 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) The interaction between TRB1 and ARID2 . Arabidopsis plants carrying TRB1 - Flag and / 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 ) The interaction between TRB1 and EPCR1 . Arabidopsis plants carrying TRB1 - Flag and / 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 ) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull - down assay .

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


( 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 .

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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 ) 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 .

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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 ) The developmental phenotype of the pwwp1 / 2 / 3 + / - mutant as compared to the wild type . One - month - old plants 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.


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 .

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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.


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 .

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 ) 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 .

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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 and TEs in the arid2 / 3 / 4 and epcr1 / 2 mutants relative to the 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 the transcript patterns of ROS1 and IBM1 in arid2 / 3 / 4 and epcr1 / 2 relative to the 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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 , and CHH methylation on chromosome 3 of Arabidopsis in wild type , epcr1 / 2 , and arid2 / 3 / 4 plants .

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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 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 .

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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 , 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 .

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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 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 .

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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 , 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 .

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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 the silencing of EPCR1 / 2 target loci in the 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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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.


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 ) .

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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 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 .

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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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O 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 ) .

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


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 .

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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.


Human T cells from two healthy donors , P1 and his mother were expanded and cultured in the presence of C . albicans and IL - 2 for 2 - 4 weeks . ( C ) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK - 7975A and analyzed for cytokine production by flow cytometry . Bar graphs represent the mean ± SEM from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (C) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK-7975A and analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.


Human T cells from two healthy donors , P1 and his mother were expanded and cultured in the presence of C . albicans and IL - 2 for 2 - 4 weeks . ( D ) Fura - 2 loaded T cells were stimulated with thapsigargin ( TG ) in Ca2 + - free buffer followed by readdition of 2 mM Ca2 + to induce SOCE . Bar graphs represent the mean ± SEM from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (D) Fura-2 loaded T cells were stimulated with thapsigargin (TG) in Ca2+-free buffer followed by readdition of 2 mM Ca2+ to induce SOCE. Bar graphs represent the mean ± SEM from two independent experiments.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( A ) Body weight ( BW ) measured for 7 days and normalized to BW at day 0 of infection . Data are mean ± SEM measured from 4 repeat experiments and 13 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (A) Body weight (BW) measured for 7 days and normalized to BW at day 0 of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( B ) Quantification of CFU of C . albicans per gram tongue tissue of the indicated mice at day 7 p . i . Data are mean ± SEM from 4 repeat experiments and 12 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( C ) Macroscopic images of the C . albicans - infected tongues of mice at day 7 p . i . Arrows and dashes lines indicate C . albicans infiltrated areas .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. Arrows and dashes lines indicate C. albicans infiltrated areas.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( D ) Histological images of H & E ( left ) and PAS ( right ) stained tongue tissue at day 7 p . i . Clusters of polymorphonuclear ( PMN ) cells ( neutrophils , white arrows ) and Candida ( black arrows ) are indicated .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (D) Histological images of H&E (left) and PAS (right) stained tongue tissue at day 7 p.i. Clusters of polymorphonuclear (PMN) cells (neutrophils, white arrows) and Candida (black arrows) are indicated.


Sublingual infection of WT , Stim1fl / flVav - iCre and Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( E ) Frequencies of Cd11b + Gr1 + neutrophils in the tongues from the indicated mice at day 7 p . i . Bar graphs represent the mean ± SEM from 3 experiments and 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (E) Frequencies of Cd11b+Gr1+ neutrophils in the tongues from the indicated mice at day 7 p.i. Bar graphs represent the mean ± SEM from 3 experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


( F , G ) Cytokine production by CD4 + T cells isolated from submandibular lymph nodes of WT , Stim1fl / flVav - iCre , Stim1fl / flStim2fl / flVav - iCre and Stim1fl / flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C . albicans . Representative contour plots of cytokine production ( F ) and percentages of CD4 + T cells producing TNF - α , IFN - γ , IL - 17A , IL - 17F and GM - CSF ( G ) . Data are the mean ± SEM from two independent experiments and 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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

(F,G) Cytokine production by CD4+ T cells isolated from submandibular lymph nodes of WT, Stim1fl/flVav-iCre, Stim1fl/flStim2fl/flVav-iCre and Stim1fl/flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C. albicans. Representative contour plots of cytokine production (F) and percentages of CD4+ T cells producing TNF-α, IFN-γ, IL-17A, IL-17F and GM-CSF (G). Data are the mean ± SEM from two independent experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Candicidal function of neutrophils depend on SOCE . ( H ) Coculture of Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre and Stim1fl / flStim2fl / flMx1Cre mice with C . albicans in vitro for 0 , 30 , 60 and 90 min ( MOI 0 . 5 ) . Percentages of live C . albicans isolated from neutrophils that were lysed at the indicated time points . Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments .

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Candicidal function of neutrophils depend on SOCE. (H) Coculture of Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice with C.albicans in vitro for 0, 30, 60 and 90 min (MOI 0.5). Percentages of live C.albicans isolated from neutrophils that were lysed at the indicated time points. Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments.


ROS production of neutrophils depend on SOCE . ( I ) ROS production by Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre and Stim1fl / flStim2fl / flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes . Representative histogram plots and bar graphs indicating the percentages of ROS + neutrophils normalized to WT neutrophils stimulated with PMA .

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

ROS production of neutrophils depend on SOCE. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( A ) Body weight ( BW ) change measured for 7 days p . i . relative to BW at the day of infection . Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl / flCd4Cre mice .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (A) Body weight (BW) change measured for 7 days p.i. relative to BW at the day of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl/flCd4Cre mice.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( B ) Quantification of CFU of C . albicans isolated from the tongues of mice at day 7 post - infection ( p . i . ) . Data are mean ± SEM from 4 repeat experiments and 7 - 13 mice per genotype .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (B) Quantification of CFU of C. albicans isolated from the tongues of mice at day 7 post-infection (p.i.). Data are mean ± SEM from 4 repeat experiments and 7-13 mice per genotype.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( C ) Macroscopic images of the C . albicans - infected tongues of mice at day 7 p . i .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i.


Sublingual C . albicans infection of WT and Stim1fl / flCd4Cre mice as described in Figure 5 . ( D ) Histological images of H & E ( left ) and PAS - stained ( right ) tongue tissue at day 7 p . i . Scale bars 100 μm . Arrows point to clusters of polymorphonuclear ( PMN ) cell ( neutrophil ) infiltrates .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (D) Histological images of H&E (left) and PAS-stained (right) tongue tissue at day 7 p.i. Scale bars 100 μm. Arrows point to clusters of polymorphonuclear (PMN) cell (neutrophil) infiltrates.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( E ) Kaplan - Meier survival plot showing survival of mice . Data from 5 Stim1fl / fl Cd4Cre mice and 12 WT mice is shown .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (E) Kaplan-Meier survival plot showing survival of mice. Data from 5 Stim1fl/fl Cd4Cre mice and 12 WT mice is shown.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( F ) Body weight ( BW ) of mice normalized to BW at the day of infection . Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (F) Body weight (BW) of mice normalized to BW at the day of infection. Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl/flCd4Cre mice.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( G ) Representative macroscopic images of kidneys at day 6 p . i .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (G) Representative macroscopic images of kidneys at day 6 p.i.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( H ) CFUs of C . albicans per gram tissue isolated from the liver , the right kidney and the lung of mice at day 6 p . i . Each dot represents one mouse .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (H) CFUs of C. albicans per gram tissue isolated from the liver, the right kidney and the lung of mice at day 6 p.i. Each dot represents one mouse.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( I ) Representative histological images of H & E and PAS stained kidneys of mice at day 6 p . i . Magnification 6 . 4x ( left ) and 40x ( right ) . White arrows define areas infiltrated with leukocytes ( H & E ) and C . albicans ( PAS ) .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (I) Representative histological images of H&E and PAS stained kidneys of mice at day 6 p.i. Magnification 6.4x (left) and 40x (right). White arrows define areas infiltrated with leukocytes (H&E) and C. albicans (PAS).


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( J ) Frequencies of CD4 + T cells isolated from the spleens of mice at day 6 p . i . producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (J) Frequencies of CD4+ T cells isolated from the spleens of mice at day 6 p.i. producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry.


Systemic C . albicans infection . WT and Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( K ) Frequencies CD45 + Cd11b + Gr - 1 + neutrophils in the blood of mice at day 6 post - infection analyzed by flow cytometry . Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl / flCd4Cre mice ; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (K) Frequencies CD45+Cd11b+Gr-1+ neutrophils in the blood of mice at day 6 post-infection analyzed by flow cytometry. Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl/flCd4Cre mice; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl/flCd4Cre mice.


RNA - seq analysis of CD4 + T cells isolated from 3 WT and 3 Stim1fl / flCd4Cre mice that were differentiated into pathogenic Th17 cells ( pTh17 : IL - 1β , IL - 6 , IL - 23 ) and non - pathogenic Th17 cells ( npTh17 : IL - 6 , TGFβ1 ) . ( B ) Volcano plots of DEGs in npTh17 cells ( gray dots ) . Highlighted in red ( upregulated in Stim1fl / flCd4Cre ) and blue ( downregulated in Stim1fl / flCd4Cre ) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously ( Gaublomme et al . , 2015 , Lee et al . , 2012 ) .

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

RNA-seq analysis of CD4+ T cells isolated from 3 WT and 3 Stim1fl/flCd4Cre mice that were differentiated into pathogenic Th17 cells (pTh17: IL-1β, IL-6, IL-23) and non-pathogenic Th17 cells (npTh17: IL-6, TGFβ1). (B) Volcano plots of DEGs in npTh17 cells (gray dots). Highlighted in red (upregulated in Stim1fl/flCd4Cre) and blue (downregulated in Stim1fl/flCd4Cre) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously (Gaublomme et al., 2015, Lee et al., 2012).


( C ) Normalized expression of Th17 cell - associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl / flCd4Cre mice . Bar graphs represent the mean ± SEM from 3 mice per group . Statistical analysis by unpaired Student ' s t - test with the following significance levels : * p < 0 . 05 , * * * p < 0 . 001 .

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(C) Normalized expression of Th17 cell-associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl/flCd4Cre mice. Bar graphs represent the mean ± SEM from 3 mice per group. Statistical analysis by unpaired Student's t-test with the following significance levels: *p<0.05, ***p<0.001.


( F ) Heat map of DEGs ( p ( adj . ) < 0 . 1 ) within the KEGG pathways " Foxo signaling " and " HIF - 1 signaling " that are specifically deregulated in npTh17 cells . Numerical values are relative gene expression .

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(F) Heat map of DEGs (p (adj.) < 0.1) within the KEGG pathways "Foxo signaling" and "HIF-1 signaling" that are specifically deregulated in npTh17 cells. Numerical values are relative gene expression.


( G ) Gene set enrichment analysis ( GSEA ) of DEGs in WT and STIM1 - deficient npTh17 cells . Normalized enrichment score ( NES ) and p - values are as indicated for the following genesets : Myc targets : " Menssen _ myc _ targets " ; Glycolysis : " Humancyc _ mm _ glycolysis _ i " ; Mitochondrial function : " Wong _ mitochondria _ gene _ module " ; TCA cycle : " Kegg _ mm _ citrate _ cycle " .

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(G) Gene set enrichment analysis (GSEA) of DEGs in WT and STIM1-deficient npTh17 cells. Normalized enrichment score (NES) and p-values are as indicated for the following genesets: Myc targets: "Menssen_myc_targets"; Glycolysis: "Humancyc_mm_glycolysis_i"; Mitochondrial function: "Wong_mitochondria_gene_module"; TCA cycle: "Kegg_mm_citrate_cycle".