( C ) DCC depletion mitigates netrin - 1 depletion - induced apoptosis in Netrin - 1fl / fl mice . TH - positive cell loss was assessed by TUNEL assay in the SN . Upper panel , TUNEL ( red ) and TH ( Cy5 - white ) ( Scale bar , 50 µm ) . Apoptotic index ( bar graph , bottom panel ) expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . N = 3 each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * P < 0 . 05 ; P * * < 0 . 01 .

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(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.


( D ) Immunoblot of SN lysates ( upper panel ) and corresponding quantification ( bottom panel ) . Quantification of band intensity ( bottom ) . Mouse N = 2 . N = 3 independent immunoblot analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 .

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(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( E ) Motor behaviour tests at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * P < 0 . 05 ; N . S . , not significant .

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(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.


( B ) Amphetamine - induced rotations at two and six weeks after lesion . Individual values and Mean ± SD are shown , n = 11 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 compared to WT .

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(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.


( C ) Optical density of TH - positive fibres in the striatum . Individual values and mean ± SD , n = 10 animals , unpaired t - test , * P < 0 . 05 .

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(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.


( D ) TH - positive cell count in the SN . Individual values and mean ± SD , n = 10 animals , unpaired t - test .

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(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.


( F ) TH staining by immunohistochemistry ( Scale bar , 2000µm ) in the SN ( left ) and striatum ( right ) of WT and SNCA Tg mice , 6 days after treatment .

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(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.


( G ) TH - positive cell count bar graph . All experiments were repeated 3 times . We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * * P < 0 . 01 ; N . S . , not significant .

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(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.


Motor behavioural assays ( Rotarod rod test ( H ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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Motor behavioural assays (Rotarod rod test (H) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


Motor behavioural assays Cylinder test ( I ) , Grid test ( J ) ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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Motor behavioural assays Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


( B ) Cumulative amphetamine - induced ipsilateral rotations . Progression of amphetamine - induced ipsilateral rotations over time ( right panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * * * * P < 0 . 0001 compared to the initial rotation score at week 2 . Comparison of the treatment ' s effect between groups ( left panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group .

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(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.


( C ) Dopamine fibres in the striatum of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the striatum showing TH IHC staining ( left panel ) , intact side on the left and 6 - OHDA lesioned side on the right . ( Scale bar , 2000 µm ) . Optical density measurement of TH - positive fibres in the lesioned side of the striatum ( bar graph , right panel ) . Individual values and mean ± SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the

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(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom


( D ) Dopamine cell bodies in the SN of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the SN ( lesioned side ) showing TH - positive cell bodies stained by IHC ( left panel ) ( Scale bar , 2000 µm ) . TH - positive cell count in the SN ( right panel ) . Individual values and mean + SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the bottom .

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(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.


( A ) Netrin - 1 gene expression profiling by array of substantiae nigrae from PD and non - PD ( Control ) patients using the GEO dataset GSE7621 that has a total of 25 samples ( n = 9 control and n = 16 PD cases ) . Unpaired t - test , * * * P < 0 . 0005 , Mean ± SD are shown .

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(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.


( B ) Immunoblot of netrin - 1 , DCC , UNC5B , active caspase - 3 , alpha synuclein FL , alpha synuclein pS129 , and Tyrosine Hydroxylase ( TH ) levels in age - matched controls ( n = 3 ) vs Parkinson ' s disease patients brain samples ( n = 3 ) ( left panel ) . Band quantification bar graph ( right panel ) . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . P * * < 0 . 01 . N = 3

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(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates


( C ) Immunohistochemistry for C . burnetii in the lung on d7 after i . t . infection . Representative images and quantitation of lung tissue positive for C . burnetii ( n = 12 - 13 mice , pooled from three experiments , each normalized to the mean of WT or Acod1 + / - infected mice ) . Each dot represents one mouse .

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(C) Immunohistochemistry for C. burnetii in the lung on d7 after i.t. infection. Representative images and quantitation of lung tissue positive for C. burnetii (n=12-13 mice, pooled from three experiments, each normalized to the mean of WT or Acod1+/- infected mice). Each dot represents one mouse.


( E ) Immunohistochemistry for C . burnetii on day 7 after intraperitoneal infection . Each dot represents one mouse .

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(E) Immunohistochemistry for C. burnetii on day 7 after intraperitoneal infection. Each dot represents one mouse.


representative images of liver sections and quantification of immunohistochemistry for C . burnetii in liver sections ( G ) . n = 6 - 11 mice ( for NMII infections ) , pooled from two experiments . Each dot represents one mouse . ( Mann - Whitney test comparing treatment and control group of NMII infected Acod1 - / - mice in specified organs , *

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representative images of liver sections and quantification of immunohistochemistry for C. burnetii in liver sections (G). n=6-11 mice (for NMII infections), pooled from two experiments. Each dot represents one mouse. (Mann-Whitney test comparing treatment and control group of NMII infected Acod1-/- mice in specified organs, *


A . qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice ; n = 4 / group ; * P < 0 . 05 , Unpaired t - test , two - tailed . Note that the transgene is not detected in the brain .

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A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.


C . Ejection fraction , cardiac output and index are significantly decreased in CamkIIδc TG mice ( n = 8 ) when compared to control mice ( n = 5 ; * P < 0 . 05 , , * * P < 0 . 01 , * * * P < 0 . 001 , Unpaired t - test , two - tailed ) . Heart to body ratio , left ventricle to body ratio , and lung to body weight ratio are increased in CamkIIδc TG ( n = 8 ) compared to control ( n = 5 ; * P < 0 . 05 , * * P < 0 . 01 , Unpaired t - test ; two - tailed ) .

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C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).


H . qPCR quantification of selected genes reflecting ER - stress or protein methylation - related processes in CamkIIδc TG mice ( n = 4 ) and control mice ( n = 5 ) . * p < 0 . 05 , ns = non - significant , Unpaired t - test ; two - tailed . Data is normalized to Hprt1 expression .

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H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.


D . The cumulative score of hippocampus - dependent search strategies during Barnes maze training is impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . E . Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . Data information : Bars and error bar indicates mean ± SEM .

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D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.


A . Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A , Kmt2A and Kmt2b knock out mice . Fisher ' s hypergeometric test , Benjamini - Hochberg ( BH ) correction .

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A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.


E . Cumulative hippocampus - dependent strategy scores during the Barnes maze training ( One - way ANOVA ) . n = 10 per group . F . Number of visits to the escape hole during probe test ( n = 10 / group , One - Way ANOVA ) . Data information : Bars and error bars indicate mean ± SEM .

E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.


E . H3K4me3 at TSS region of genes from RNA module1 . Inset shows statistical comparisons among groups ( One - way ANOVA , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Bars and error bars indicate mean ± SEM . Number of replicates analyzed for H3K4me3 profile at TSS ( Wildtype vehicle : 3 ; CamkIIδc TG - vehicle n = 4 ; CamkIIδc TG - Vorinostat n = 4 ) .

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E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).


( A ) Raw 264 . 7 cells were pre - treated with DMSO or the EGFR inhibitor gefitinib ( 10 µM ) before cGAMP transfection ; after 5h , IFNβ mRNA was measured by qRT - PCR ( n = 3 ) .

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(A) Raw 264.7 cells were pre-treated with DMSO or the EGFR inhibitor gefitinib (10 µM) before cGAMP transfection; after 5h, IFNβ mRNA was measured by qRT-PCR (n=3).


( B ) WT and EGFR knockdown Raw 264 . 7 cells were transfected with cGAMP ; 4 . 5h post treatment , IFNβ mRNA was measured ( n = 3 ) .

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(B) WT and EGFR knockdown Raw 264.7 cells were transfected with cGAMP; 4.5h post treatment, IFNβ mRNA was measured (n=3).


( C ) WT and EGFR - / - BMDMs were transfected with cGAMP ; 5h post transfection , IFNα mRNA induction was measured ( n = 4 ) .

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(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4).


( D ) WT and EGFR - / - HeLa cells were transfected with cGAMP ; after 5h IFNβ mRNA was measured ( n = 3 ) .

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(D) WT and EGFR-/- HeLa cells were transfected with cGAMP; after 5h IFNβ mRNA was measured (n=3).


MEF cells were pre - treated with gefitinib for 1h and then transfected with cGAMP ; 4 . 5h post treatment , total RNA was harvested for analysis . ( E ) RNA - seq analysis ( n = 2 ) . For RNA - seq analysis , poly T enriched cDNA library was prepared and 30 M single - end 50 bp reads were analyzed . The heat map for some of the genes is shown .

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MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (E) RNA-seq analysis (n=2). For RNA-seq analysis, poly T enriched cDNA library was prepared and 30 M single-end 50 bp reads were analyzed. The heat map for some of the genes is shown.


MEF cells were pre - treated with gefitinib for 1h and then transfected with cGAMP ; 4 . 5h post treatment , total RNA was harvested for analysis . ( G ) The RNA - seq analysis results were confirmed by qRT - PCR analysis of a gefitinib - sensitive and an insensitive mRNA ( n = 3 ) .

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MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (G) The RNA-seq analysis results were confirmed by qRT-PCR analysis of a gefitinib-sensitive and an insensitive mRNA (n=3).


( H ) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly ( I : C ) transfection ; 4 . 5h post treatment , total RNA was harvested and IFNα mRNA induction was measured ( n = 3 ) .

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(H) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly (I:C) transfection; 4.5h post treatment, total RNA was harvested and IFNα mRNA induction was measured (n=3).


( A ) MEF cells were pre - treated with gefitinib or DMSO for 1 hour , and then infected with HSV1 at m . o . i . of 1 . 4 hours post infection ( h . p . i . ) , IFNβ mRNA induction was monitored by qRT - PCR ( n = 3 ) . Uninfected cells were used as negative control ( U ) .

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(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U).


( B ) DNA was extracted from gefitinib - treated or untreated infected MEFs 0 and 16 h . p . i . and viral DNA was measured by qPCR ( n = 3 ) .

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(B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3).


( C ) WT and EGFR KO HeLa cells were infected with HSV1 , RNA was extracted at 4 h . p . i . and IFNα mRNA induction was measured ( n = 3 ) .

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(C) WT and EGFR KO HeLa cells were infected with HSV1, RNA was extracted at 4 h.p.i. and IFNα mRNA induction was measured (n=3).


( D ) Survival of uninfected mice treated with gefitinib and HSV1 - infected mice treated or untreated with gefitinib .

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(D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib.


( E ) Survival of WT and EGFRfl + / - LysMCre + / + mice infected with HSV1 .

(E) Survival of WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1.


WT and EGFRfl + / - LysMCre + / + mice infected with HSV1 . HSV1 titers in the brains of the live mice at day 9 were determined .

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WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1. HSV1 titers in the brains of the live mice at day 9 were determined.


( A ) EGFR was not required for ligand - induced STING dimerization : MEFs were transfected with HA - tagged STING and treated with gefitinib or DMSO before cGAMP transfection , 4h later STING was immunoprecipitated with anti - HA antibodies and analyzed by Western blot .

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(A) EGFR was not required for ligand-induced STING dimerization: MEFs were transfected with HA-tagged STING and treated with gefitinib or DMSO before cGAMP transfection, 4h later STING was immunoprecipitated with anti-HA antibodies and analyzed by Western blot.


( B ) EGFR was required for the activation of IRF3 , but not TBK1 : MEFs were pretreated with gefitinib for 1h , then treated with cGAMP for 2h ; cell lysate were subjected to Western blot analysis using the indicated antibodies .

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(B) EGFR was required for the activation of IRF3, but not TBK1: MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; cell lysate were subjected to Western blot analysis using the indicated antibodies.


( C ) Nuclear translocation of IRF3 , but not NFκB p65 , required EGFR : MEFs were pre - treated with gefitinib ( + ) or DMSO ( - ) for 1 hour before transfecting cGAMP . Cells were harvested and nuclei were isolated at the indicated time points ; total cell extracts ( T ) and nuclear extracts ( N ) were analyzed by Western blot .

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(C) Nuclear translocation of IRF3, but not NFκB p65, required EGFR: MEFs were pre-treated with gefitinib (+) or DMSO (-) for 1 hour before transfecting cGAMP. Cells were harvested and nuclei were isolated at the indicated time points; total cell extracts (T) and nuclear extracts (N) were analyzed by Western blot.


MEFs were pretreated with gefitinib for 1h , then treated with cGAMP for 2h ; ( D ) IRF3 , but not TBK1 , binding to STING required EGFR cell lysates were immunoprecipitated with anti - STING antibody and analyzed by Western blot .

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MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; (D) IRF3, but not TBK1, binding to STING required EGFR cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.


( E ) STING - IRF3 interaction in human cells required EGFR : WT and EGFR - / - ( KO ) HeLa cells were transfected with cGAMP , 2h later cells were lysed , immunoprecipitated with anti - STING antibody and analyzed by Western blot .

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(E) STING-IRF3 interaction in human cells required EGFR: WT and EGFR-/- (KO) HeLa cells were transfected with cGAMP, 2h later cells were lysed, immunoprecipitated with anti-STING antibody and analyzed by Western blot.


( F ) STING and IRF3 co - localized in the endosomes of stimulated cells : STING - / - MEF cells were transfected with STING - HA for 24 hours then treated with cGAMP for 1h . The cells were then stained with the HA ( purple ) , CD63 ( red ) and IRF3 ( green ) antibodies . Imaging data were analyzed by the Fuji software to reveal co - localization as white dots . Scale bars represent 10 µ m .

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(F) STING and IRF3 co-localized in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours then treated with cGAMP for 1h. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.


( G ) EGFR was not required for ligand - induced phosphorylation of STING Ser366 : EGFR - / - HeLa cells or gefitinib - treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot .

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(G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot.


( H ) Kinetics of STING and IRF3 co - localization in the endosomes of stimulated cells : STING - / - MEF cells were transfected with STING - HA for 24 hours and treated with cGAMP for the indicated time . The cells were then stained with the HA ( purple ) , CD63 ( red ) and IRF3 ( green ) antibodies and DAPI . Imaging data were analyzed by the Fuji software to reveal co - localization as white dots . Scale bars represent 10 µ m .

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(H) Kinetics of STING and IRF3 co-localization in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours and treated with cGAMP for the indicated time. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies and DAPI. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.


( A ) STING was Tyr - phosphorylated in cGAMP - treated cells . Raw 264 . 7 cells were tranfected with cGAMP ; after 60 min , cell lysates were immuno - precipitated with anti - pTyr antibodies and then subjected to anti - STING Western blot .

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(A) STING was Tyr-phosphorylated in cGAMP-treated cells. Raw 264.7 cells were tranfected with cGAMP; after 60 min, cell lysates were immuno-precipitated with anti-pTyr antibodies and then subjected to anti-STING Western blot.


( B ) STING Tyr - phosphorylation was inhibited by gefitinib . MEF cells were pre - treated with gefitinib before cGAMP - stimulation for 60 min . Cell lysates were immuno - precipitated with anti - pTyr antibody and then analyzed by anti - STING Western blot . NTC : no treatment control .

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(B) STING Tyr-phosphorylation was inhibited by gefitinib. MEF cells were pre-treated with gefitinib before cGAMP-stimulation for 60 min. Cell lysates were immuno-precipitated with anti-pTyr antibody and then analyzed by anti-STING Western blot. NTC: no treatment control.


( C ) STING Tyr - phosphorylation required EGFR . Raw 264 . 7 cells expressing EGFR shRNA ( KD ) or non - target shRNA ( CO ) were transfected with cGAMP for 2 hours ; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti - pTyr antibodies .

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(C) STING Tyr-phosphorylation required EGFR. Raw 264.7 cells expressing EGFR shRNA (KD) or non-target shRNA (CO) were transfected with cGAMP for 2 hours; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti-pTyr antibodies.


( D ) cGAMP stimulation triggered EGFR - binding to STING . Raw 264 . 7 cells were transfected with cGAMP for 1h ; cell lysates were immunoprecipitated with anti - STING antibody and analyzed by Western blot .

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(D) cGAMP stimulation triggered EGFR-binding to STING. Raw 264.7 cells were transfected with cGAMP for 1h; cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.


( E ) STING co - localized with EGFR after cGAMP stimulation . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24hours and stimulated with cGAMP for 1h , cells were fixed and labeled with EGFR ( Red ) and Calnexin ( Purple ) antibodies . Co - localization was analyzed by Fuji and co - localized dots are shown in white . Scale bars represent 10 µ m .

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(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.


( F ) STING - EGFR interaction was transient . Raw 264 . 7 cell lysates were prepared at indicated time points after cGAMP transfection , immunoprecipitated with anti - EGFR antibodies and subjected to Western blot .

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(F) STING-EGFR interaction was transient. Raw 264.7 cell lysates were prepared at indicated time points after cGAMP transfection, immunoprecipitated with anti-EGFR antibodies and subjected to Western blot.


( G ) Gefitinib did not block , but prolonged STING - EGFR interaction . Raw 264 . 7 cells were pre - treated with gefitinib or DMSO for 1 hour before cGAMP stimulation . Cell lysates were prepared at indicated time points , immuno - precipitated with anti - STING antibody and subjected to Western blot .

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(G) Gefitinib did not block, but prolonged STING-EGFR interaction. Raw 264.7 cells were pre-treated with gefitinib or DMSO for 1 hour before cGAMP stimulation. Cell lysates were prepared at indicated time points, immuno-precipitated with anti-STING antibody and subjected to Western blot.


( H ) Poly I : C did not activate EGFR . STING - / - MEF cells were transfected with cGAMP or Poly ( I : C ) ; after 3h , p - EGFR were detected by Western blot .

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(H) Poly I:C did not activate EGFR. STING-/- MEF cells were transfected with cGAMP or Poly (I:C); after 3h, p-EGFR were detected by Western blot.


( I ) Kinetics of EGFR activation . Raw 264 . 7 cells were treated with cGAMP for the indicated time . Cell lysates were analyzed for p - EGFR via Western blot .

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(I) Kinetics of EGFR activation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were analyzed for p-EGFR via Western blot.


( J ) STING - bound EGFR was phosphorylated . Raw 264 . 7 cells were stimulated for 1h , cell lysates were immunoprecipitated with anti - STING antibody and subjected to Western blot .

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(J) STING-bound EGFR was phosphorylated. Raw 264.7 cells were stimulated for 1h, cell lysates were immunoprecipitated with anti-STING antibody and subjected to Western blot.


( K ) Kinetics of phosphorylation . Raw 264 . 7 cells were treated with cGAMP for the indicated time . Cell lysates were subjected to Western blot using the indicated antibodies .

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(K) Kinetics of phosphorylation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were subjected to Western blot using the indicated antibodies.


( L ) Poly I : C did not trigger Tyr or Ser phosphorylation of STING . HeLa cells were transfected with cGAMP or Poly ( I : C ) ; after 3h , cell lysates were analyzed by Western blot using the indicated antibodies .

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(L) Poly I:C did not trigger Tyr or Ser phosphorylation of STING. HeLa cells were transfected with cGAMP or Poly (I:C); after 3h, cell lysates were analyzed by Western blot using the indicated antibodies.


( A ) In the absence of EGFR , after cGAMP stimulation , STING translocated out of the ER . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h ; cells were fixed and labeled with Calreticulin ( ER marker , Red ) antibody . Co - localization ( Col ) is indicated by white dots .

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(A) In the absence of EGFR, after cGAMP stimulation, STING translocated out of the ER. STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h; cells were fixed and labeled with Calreticulin (ER marker, Red) antibody. Co-localization (Col) is indicated by white dots.


STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by white dots . ( B ) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib . , the cells were labeled with P58 ( ERGIC marker , Red ) antibody .

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STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (B) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib. , the cells were labeled with P58 (ERGIC marker, Red) antibody.


MEF cells were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h and transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by white dots . ( C ) STING translocated to the autophagosomes in the absence of EGFR . the cells were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( E ) In gefitinib - treated cells , STING was detected in the autophagosomes as early as 0 . 5h . The procedures were as in C .

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MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (C) STING translocated to the autophagosomes in the absence of EGFR. the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (E) In gefitinib-treated cells, STING was detected in the autophagosomes as early as 0.5h. The procedures were as in C.


Co - localization ( Col ) is indicated by white dots . STING translocated to the autophagosomes in the absence of EGFR . , the cells were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( D ) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited .

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Co-localization (Col) is indicated by white dots. STING translocated to the autophagosomes in the absence of EGFR. , the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (D) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited.


( F ) STING translocation to late endosomes required EGFR activity . STING - / - MEF cells were transfected with STING - GFP ( Green ) for 24hours , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods ; cells were fixed and labeled with CD63 ( late endosome marker , Red ) antibody .

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(F) STING translocation to late endosomes required EGFR activity. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods; cells were fixed and labeled with CD63 (late endosome marker, Red) antibody.


( A ) EGFR phosphorylates STING in vitro . Purified STING - GFP was incubated with increasing amounts of EGFR for in vitro kinase assays ; the products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . The substrate was analyzed , before the assay incubations , by the same method ( lower panel ) .

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(A) EGFR phosphorylates STING in vitro. Purified STING-GFP was incubated with increasing amounts of EGFR for in vitro kinase assays; the products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel).


products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . The substrate was analyzed , before the assay incubations , by the same method ( lower panel ) . ( B ) Y245F STING mutant is not a substrate of EGFR . WT and the indicated mutants of STING - GFP were purified and used as substrates for

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products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel). (B) Y245F STING mutant is not a substrate of EGFR. WT and the indicated mutants of STING-GFP were purified and used as substrates for EGFR


( C ) LC - MS / MS analysis of STING identifies phosphorylated Y245 . STING - GFP was purified from cGAMP - stimulated cells , subjected to protease digestion and LC - MS / MS analysis . Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified . The MS / MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown . This doubly charged peptide has an observed m / z of 932 . 9266 Da and is within - 0 . 96 ppm of the expected mass . The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245 .

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(C) LC-MS/MS analysis of STING identifies phosphorylated Y245. STING-GFP was purified from cGAMP-stimulated cells, subjected to protease digestion and LC-MS/MS analysis. Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified. The MS/MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown. This doubly charged peptide has an observed m/z of 932.9266 Da and is within -0.96 ppm of the expected mass. The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245.


( E ) Gefitinib pretreatment inhibits phosphorylation of Y245 . STING - GFP - 293XL cells were pretreated with gefitinib and then treated with cGAMP . LC - MS / MS analysis was done , as above , for detecting pY245 ( n = 3 ) .

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(E) Gefitinib pretreatment inhibits phosphorylation of Y245. STING-GFP-293XL cells were pretreated with gefitinib and then treated with cGAMP. LC-MS/MS analysis was done, as above, for detecting pY245 (n=3).


( F ) EGFR is required for STING Tyr phosphorylation . STING - GFP was expressed in 293XL cells in which EGFR expression had been knocked down . STING phosphorylation was analyzed by LC - MS /

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(F) EGFR is required for STING Tyr phosphorylation. STING-GFP was expressed in 293XL cells in which EGFR expression had been knocked down. STING phosphorylation was analyzed by LC-MS/MS


( G ) Y245F STING mutant cannot trigger IFNβ mRNA induction . RNA was isolated from cells , expressing WT or Y245F STING , 3h after stimulation and subjected to qRT - PCR analysis ( n = 3 ) .

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(G) Y245F STING mutant cannot trigger IFNβ mRNA induction. RNA was isolated from cells, expressing WT or Y245F STING, 3h after stimulation and subjected to qRT-PCR analysis (n=3).


( H ) Y245F STING is phosphorylated on S366 after stimulation of cells . Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time .

(H) Y245F STING is phosphorylated on S366 after stimulation of cells. Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time.


( I ) TBK1 , but not IRF3 , can bind to Y245F STING and be phosphorylated in cGAMP - stimulated cells . Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after ( upper panel ) or before ( lower panel ) GFP pull down .

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(I) TBK1, but not IRF3, can bind to Y245F STING and be phosphorylated in cGAMP-stimulated cells. Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after (upper panel) or before (lower panel) GFP pull down.


( J ) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING .

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(J) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING.


( K ) Inhibition of EGFR by gefitinib induced more LC3II after 1 . 5h cGAMP treatment of L929 cells .

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(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n


( A ) STING - / - MEF cells were transfected with mSTING WT - GFP and Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time ; cells were fixed and labeled with LC3 ( autophagosome marker , Red ) antibody . Co - localization ( Col ) is indicated by white dots .

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(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, Red) antibody. Co-localization (Col) is indicated by white dots.


( A ) STING - / - MEF cells were transfected with mSTING WT - GFP and Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time ; cells were fixed and labeled with CD63 ( late endosome marker ) .

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(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with CD63 (late endosome marker).


A - C . Immunodetection of BES1 - CFP from heat - treated plants . Plants pre - grown at 21 ° C were ( A ) exposed for 60 minutes to the indicated temperatures , ( B ) to 45 ° C for the indicated period of time , or ( C ) to 45 ° C and , following sampling after 30 minutes ( lane 2 ) and 60 minutes ( lane 3 ) , returned to 21 ° C for 3 hours ( lane 4 ) . CBB , Coomassie Brilliant Blue stained gel .

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A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel.


D . Subcellular localization of BES1 - CFP in roots of 7 - day - old plants after heat shock . 35S : BES1 - CFP seedlings were grown at 21 ° C and either left untreated or exposed for 60 minutes to 45 ° C . Left : representative photos . Right : quantification of the nuclear / cytoplasmic signal . Data show the mean ± SD . n = 25 . The asterisks indicate significant differences to the untreated plants by Student ' s t - test ( * * * P ≤ 0 . 001 ) .

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D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001).


F . Heat stress resistance of bes1 - 1 , bes1 - 2 and bes1 - D as compared to wild - type . For basal resistance assays ( top charts ) plants were directly exposed to 45 ° C for the indicated time . For acquired resistance ( bottom charts ) , plants were primed with the respective temperatures for 120 minutes before transfer to 45 ° C for 150 minutes . Data show the mean ± SE . n = 7 . The asterisks indicate significant differences compared to wild - type by Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).


B . Immunodetection of BES1 - CFP from heat - stressed plants , treated with epiBL or BRZ . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 1 μM 24 - epiBL or 5 μM BRZ and then heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes ; lane 4 : 45 ° C for 60 minutes and recovery at 21 ° C for 180 minutes .

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B. Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 1 μM 24-epiBL or 5 μM BRZ and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.


C , D . Immunodetection of BES1 - CFP from heat - treated bri1 - 1 or det2 - 1 plants . Left : phenotypes of 10 - day - old plants of all lines as compared to wild - type Col - 0 ( wt ) . Right : Immunoblots from the lines grown and treated as in B .

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C, D. Immunodetection of BES1-CFP from heat-treated bri1-1 or det2-1 plants. Left: phenotypes of 10-day-old plants of all lines as compared to wild-type Col-0 (wt). Right: Immunoblots from the lines grown and treated as in B.


F . Immunodetection of BES1 - CFP from heat - treated bin2 - 1 plants .

F. Immunodetection of BES1-CFP from heat-treated bin2-1 plants.


A . Immunodetection of BES1 - CFP from heat - stressed plants , treated with ABA or Fluridone ( Flu ) . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 5 μM ABA or 10 μM Fluridone and heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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A. Immunodetection of BES1-CFP from heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM Fluridone and heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


C . Immunodetection of native BES1 from heat - stressed and epiBL - treated abitM plants . abitM and wild - type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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C. Immunodetection of native BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


C . ChIP to determine BES1 - CFP enrichment on the promoters of HSP70 . 3 , HSP70 . 4 and HSP90 . 1 following heat stress . 35S : BES1 - CFP and wild - type plants grown at 21 ° C were either left untreated or treated with 45 ° C for 60 minutes . BES1 - CFP was immuno - precipitated with α - GFP beads . Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated . TA3 ( 70 . 3p and 90 . 1p ) or 5s rRNA ( 70 . 4p ) were used for normalization . UBQ5 served as an internal control . Data show the mean ± SE . n = 3 biological replicates measured in 3 technical repeats . Significant differences as compared to wild - type at 21 ° C , calculated by Student ' s t - test , are shown ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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C. ChIP to determine BES1-CFP enrichment on the promoters of HSP70.3, HSP70.4 and HSP90.1 following heat stress. 35S:BES1-CFP and wild-type plants grown at 21°C were either left untreated or treated with 45°C for 60 minutes. BES1-CFP was immuno-precipitated with α-GFP beads. Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated. TA3 (70.3p and 90.1p) or 5s rRNA (70.4p) were used for normalization. UBQ5 served as an internal control. Data show the mean ±SE. n=3 biological replicates measured in 3 technical repeats. Significant differences as compared to wild-type at 21°C, calculated by Student's t-test, are shown (*P≤0.05; **P≤0.01; ***P≤0.001).


A . EMSAs showing in vitro DNA interaction of recombinant BES1 and / or HSFA1 with HSE - containing fragments of the HSP70 . 3 and HSP90 . 1 promoters . Unlabeled competitor fragments with functional HSEs ( C1 + HSE ) or mutated versions ( C2 - HSE ) were used for competition studies in 100x molar access ( lanes 6 and 7 ) .

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A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7).


B . BiFC assays showing cells co - transformed with nYFP - BES1 and cYFP - HSFA1a . The fusion proteins were co - expressed in N . benthamiana leaves and fluorescence was visualized . Two nuclei are highlighted and shown in a 6 - fold magnification . Scale bar = 20 μm .

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B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm.


C . LUC assays in protoplasts of wild - type , bes1 - D or the hsfA1qM plants . LUC reporter constructs , driven by 2 kb of the HSP70 . 3 , HSP70 . 4 and HSP90 . 1 promoters , were tested either alone ( 0 , with empty effector plasmid ) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed . Data show the mean ± SD . n = 5 . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s t - test .

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C. LUC assays in protoplasts of wild-type, bes1-D or the hsfA1qM plants. LUC reporter constructs, driven by 2 kb of the HSP70.3, HSP70.4 and HSP90.1 promoters, were tested either alone (0, with empty effector plasmid) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed. Data show the mean ±SD. n=5. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


A , B . Constitutive and BR - responsive growth of bes1 - DxhsfA1qM seedlings . Hypocotyl elongation of 7 - day - old dark - grown ( A ) and light - grown ( B ) seedlings on medium containing either 1 μM epi - BL , 1 μM BRZ or DMSO as a control ( c ) . Left : Mean and SD of 30 plants . Significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined with a Student ' s t - test . Right : photos of representative plants .

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A, B. Constitutive and BR-responsive growth of bes1-DxhsfA1qM seedlings. Hypocotyl elongation of 7-day-old dark-grown (A) and light-grown (B) seedlings on medium containing either 1 μM epi-BL, 1 μM BRZ or DMSO as a control (c). Left: Mean and SD of 30 plants. Significant difference at P≤0.05 of results is indicated with different letters and was determined with a Student's t-test. Right: photos of representative plants.


C . Expression of BR - regulated genes in bes1 - DxhsfA1qM , as compared to its parents and respective wild - types analyzed by qPCRs . Data show the mean ± SD . n = 3 biological repeats , each measured in 3 technical replicates , normalized to UBC . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s t - test .

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C. Expression of BR-regulated genes in bes1-DxhsfA1qM, as compared to its parents and respective wild-types analyzed by qPCRs. Data show the mean ±SD. n=3 biological repeats, each measured in 3 technical replicates, normalized to UBC. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


( B ) The degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using the 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of the phenyl and indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency and the discovery of the 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) and n = 25 ( GSK716 ) .

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(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).


( C ) GSK716 binds to Z α1 - antitrypsin with a high affinity mean pKD of 8 . 5 ± 0 . 12 ( n = 18 ) as determined by a competition binding assay with a fluorescently labelled derivative . There was a 50 - fold lower affinity for plasma - purified wild - type M α1 - antitrypsin , with a mean pKD of 6 . 8 ± 0 . 18 ( n = 10 ) . Data presented as mean ± SD .

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(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.


( D ) The compound bound to monomeric but not polymeric Z α1 - antitrypsin ( Z α1 - AT ) as reported by fluorescence polarisation of an Alexa - 488 - labelled variant of GSK716 .

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(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.


( E ) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1 - antitrypsin based on changes in intrinsic tryptophan fluorescence . Based on the concentration dependence ( inset ) , the second - order rate constant of association was found to be 4 . 1 x 104 M - 1 s - 1 . ( F ) The association of GSK716 with M α1 - antitrypsin , giving a second - order rate constant of 2 . 1 x 102 M - 1 s - 1 .

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(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.


( G ) CHO - K1 Tet - On cells expressing Z α1 - antitrypsin were induced with doxycycline ( 0 . 5μg / ml ) for 48 h . Cells were incubated with 10μM GSK716 ( or 0 . 1 % v / v DMSO for the control ) during the induction . Culture media containing either the experimental compound or DMSO were changed every 24 h . After the induction , cells were labelled for 10 minutes with 35S Met / Cys and chased at the indicated times . Culture media were collected and cells lysed in 1 % v / v NP - 40 buffer . Intracellular fractions and culture media from cells expressing Z α1 - antitrypsin were immunoprecipitated either with a mAb against total α1 - antitrypsin ( 3C11 ) or with a polymer - specific mAb ( 2C1 ) . Samples were resolved by 4 - 12 % w / v acrylamide SDS - PAGE and detected by autoradiography . ( H ) The graphs show the effect of GSK716 on intracellular and extracellular Z α1 - antitrypsin ( mean ± SEM , n = 2 )

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(G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)


( B ) M ( above ) and Z ( below ) α1 - antitrypsin , at a concentration of 0 . 2mg / ml in PBS + 5 % v / v glycerol , were heated at a range of temperatures between 48 ° C - 65 ° C for 4 hours in the presence and absence of GSK716 , as indicated . The oligomerisation state was determined by non - denaturing PAGE electrophoresis ; the lane denoted ' M ' contains the unheated monomeric control .

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(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.


( C ) M and Z α1 - antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride ( gdnhcl ) in the presence and absence of GSK716 , with bis - ANS dye added to report the presence of the unfolded intermediate . The normalised fluorescence intensity data were fitted with an equation describing a three - state unfolding curve . Values shown are the mean of three independent experiments and the error bars represent ± SEM .

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(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.


( D ) Top panel , the association of GSK716 induced a marked quenching and blue - shift of the α1 - antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein ( inset graph ) . The association of 10µM GSK716 with four α1 - antitrypsin variants that vary in their propensity to polymerise , ranging from inert ( M ) , to mild ( S ) , moderate ( Baghdad denoted ' B ' ) and severe ( Z ) . Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0 . 2mg / ml protein are shown . Bottom panel , the half - time of association calculated from three such independent experiments ( error bars are ± SEM ) show a correspondence with the polymerisation propensity of the four variants . The change in Z α1 - antitrypsin fluorescence was faster than the dead - time of the apparatus ( ~ 10s ) and scaling was with reference to unbound intensity .

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(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.


( B ) M α1 - antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS - PAGE . The position of covalent α1 - antitrypsin - chymotrypsin complex ( cpx ) , native ( nat ) and cleaved ( clv ) is shown .

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(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.


( C ) The inhibitory activity of α1 - antichymotrypsin against chymotrypsin ( ach + chym ) , neuroserpin against trypsin ( neus + tryp ) , and antithrombin against thrombin ( atiii + thrombin ) was determined in the presence and absence of GSK716 . Error bars reflect ± SD of two independent experiments .

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(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.


Z α1 - antitrypsin transgenic animals were dosed with 10 , 30 or 100 mg / kg GSK716 three times a day . Total concentrations of GSK716 were determined by LC - MS / MS ( Table 2 ) . ( A ) 100mg / kg GSK716 resulted in a mean 7 - fold increase in circulating monomeric Z α1 - antitrypsin levels demonstrating robust target engagement in the liver . The observed steady state total and free compound levels of GSK716 in the transgenic Z α1 - antitrypsin mouse were well - predicted by the in silico PK model built on : ( 1 ) in vitro metabolic clearance data , ( 2 ) plasma protein binding data , ( 3 ) in vivo PK data from wild type mice and ( 4 ) a term comprising a 5μM circulating sink for drug with an affinity of 1 . 5 nM , representing the Z α1 - antitrypsin within blood . The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay . Interestingly , 30mg / kg and 10mg / kg groups also gave significant , dose - dependent increases in circulating Z α1 - antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period . Significance at p < 0 . 05 by a student ' s t test is denoted by * , shown as pairwise comparisons ( each animal is compared with itself , treated vs pre - treatment ) and between groups ( compound treated vs vehicle ) , with n = 7 , 8 and 8 per group for day 6 dose level 10 , 30 and 100mg / Kg respectively ; day 15 , n = 10 ; day 21 , n = 5 . Data presented as mean ± SD .

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

Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.


A . Confocal images of live control ( top ) and U18 - treated ( bottom ) tsA201 cells expressing P4M - YFP . Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo / lysosomal compartments , respectively . B . Quantification of perinuclear P4M - YFP mean fluorescence intensity normalized to the cytoplasm . Control : n = 19 ; U18 : n = 16 .

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

A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.


K . Heat map of PtdIns , PtdInsP and PtdInsP2 levels from 4 different murine brain regions . The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild - type brains ; n = 3 for both WT and NPC1I1061T animals .

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

K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.


C . Confocal images of control and U18 - treated tsA201 cells fixed and stained for OSBP ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of OSBP and TGN46 regions . D . Quantification of OSBP signal in TGN46 regions ( OSBPTGN46 ) . Control : n = 40 ; U18 : n = 39 .

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 B-CONTROLLED_VAR O O O O O

C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39.


J . Confocal images of control and U18 - treated GFP11 - SAC1 HEK293 cells ( top ) and control and NPC1I1061T patient fibroblasts immunolabeled with anti - SAC1 antibody ( bottom ) . Insets show an enlarged view of SAC1 centered on a perinuclear region . K . Quantification analyses of GFP11 - SAC1 ( top ; Control : n = 10 ; U18 : n = 10 ) and anti - SAC1 ( bottom ; Control : n = 7 ; NPC1I1061T : n = 8 ) mean fluorescence intensities in Golgi normalized to the cytoplasm .

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 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

J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.


C . Left : Confocal images of control ( left ) and U18 - treated ( right ) tsA201 cells expressing PI4KIIα - GFP . Bottom panels show an enlarged view of the probe centered on a perinuclear region . Right : Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm . Control : n = 12 ; U18 : n = 14 . D . Same as ( C ) only PI4KIIIβ . Control : n = 12 ; U18 : n = 15 .

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

C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15.


E . Confocal images of control and U18 - treated tsA201 cells fixed and stained for PI4KIIα ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of PI4KIIα and TGN46 regions . F . Quantification of PI4KIIα signal in TGN46 regions ( PI4KIIα TGN46 ) . Control : n = 36 ; U18 : n = 36 .

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 B-CONTROLLED_VAR O O O O O

E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.


B , Confocal images of wild - type and PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups .

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

B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.


H . Control or GFP - DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . Quantification of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) intensity relative to cytoplasm .

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

H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.


M . Confocal images of control ( top row ) , NPC1I1061T ( middle row ) , and NPC1I1061T treated with sgDHHC3 ( bottom row ) patient cells expressing P4M , LAMP1 and CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . Quantification of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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

M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


C . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3 . Black and orange squares show expanded views of anti - ACBD3 in Golgi and endomembrane compartments , respectively . D . Analyses of ACBD3 intensity distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 cells ) , normalized to the cytoplasm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.


I . Confocal images of control and sgACBD3 HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments . J . Quantitative analyses of P4M - YFP mean fluorescence intensity at Golgi ( left graph ) or endomembranes ( right graph ) normalized to the cytoplasm . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups . For left graph ; Control : n = 19 ; U18 : n = 13 ; sgPI4K3B : n = 12 ; sgPI4K3B + U18 : n = 11 ; sgPI4K2A + sgPI4K3B : n = 12 ; sgPI4K2A + sgPI4K3B + U18 : n = 12 ; sgACBD3 : n = 15 ; sgACBD3 + U18 : n = 16 . For right graph ; control : n = 149 ; U18 : n = 251 ; sgACBD3 : n = 138 ; sgACBD3 + U18 : n = 190 . Please note that ' control ' and ' U18 ' datasets in Fig 5J are identical to those in Fig . 4C . Data for all CRISPR experiments were collected on the same days .

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

I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.


A . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts expressing P4M - YFP . Black and orange squares show expanded views of PtdIns4P in Golgi and endo / lysosomal compartments respectively .

O O O O 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

A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.


C . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody . Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images . White dashed lines represent the outline of each cell . D . Quantification of mTORC1 mean integrated density normalized bycell area in control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts . Control : n = 15 ; NPC1I1061T : n = 14 ; NPC1I1061T + sgACBD3 : n = 11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell. D. Quantification of mTORC1 mean integrated density normalized bycell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.


B . Confocal images of tsA201 cells expressing PI4KIIα - GFP . Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . C . Quantification of PI4KIIα - GFP mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . D , E . Same as ( B , C ) but in tsA - 201 cells transfected with PI4KIIIβ - GFP . Control : n = 18 , U18 : n = 22 , U18 + PF : n = 23 .

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

B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.


Confocal images of tsA201 cells Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . F , G . Same as ( B , C ) but in tsA - 201 cells transfected with P4M - YFP . Control : n = 39 , U18 : n = 42 , U18 + PF : n = 32 . H , I . Same as ( F , G ) but in wild - type and SCAP - / - CHO cells . Control : n = 12 , U18 : n = 11 , SCAP - / - + U18 : n = 10 .

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

Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.


A . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI - M6PR . Insets show expanded views of each immunolabeling in the Golgi region ( upper insets ) or centered on endomembranes ( bottom inset , merged signals ) . Dashed grey and white lines represent the outline of each cell . B . Analysis of the mean area of M6P and Giantin colocalizing puncta ( top graph ; Control : n = 23 ; NPC1I1061T : n = 18 ) and of their mean fluorescence intensity ( bottom graph ; Control : n = 18 ; NPC1I1061T : n = 18 ) in control and NPC1I1061T patient fibroblasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell. B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts.