( E , F ) The number proliferating progenitor neurons identified by BrdU and Ki - 67 double staining is restored in the SGZ of Ts65Dn / DSCR1 + / - mouse compared to that of Ts65Dn mouse . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ . Scale bars : 100 μm in the large image and 10 μm in magnified image . Each hippocampal section was 40 μm in thickness , and total 6 hippocampi were used for analysis . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 05 .

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(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.


( G - J ) Ts65Dn has learning and memory defects , but Ts65Dn / DSCR1 + / - mouse clearly rescues learning and memory defects to control levels . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 8 ( control ) , 5 ( Ts65Dn ) , 10 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 001 ( G , H ) . * P < 0 . 05 ( I ) , * * P < 0 . 01 ( I ) . ( J ) Open - field analysis shows no defects in movement of tested animals . Values are shown as mean ± SEM , N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals .

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(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.


C , Microscale thermophoresis ( MST ) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711 . D , dDATmfc did not show any interactions with NO711 in the MST experiments . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions with NO711 in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


E , SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM . F , MST experiments display no interactions of purified dDATmfc with SKF89976a . The MST profiles shown are one of two independent measurements . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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E, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


G , Michaelis - Menten uptake kinetics of 3H - GABA by the GAT1WT used in the study displaying a KM value of 11 . 4 μM .

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G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.


H , Inhibition of 3H - GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14 . 4 μM . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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H, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


I , Inhibition of 3H - GABA uptake through GAT1WT by tiagabine ( Ki = 725 nM ) , NO711 ( 1 . 07 μM ) and SKF89976a ( 7 . 3 μM ) . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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I, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM). Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


B , Substitution of L300 to other hydrophobic side chains and the consequences on transport activity . L300W has the maximal effect on the transport activity . The histogram represents mean of a total 6 measurements ( n = 6 ) done as two independent measurements . Individual datapoints are represented as black dots around the error bar that represents s . e . m .

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B, Substitution of L300 to other hydrophobic side chains and the consequences on transport activity. L300W has the maximal effect on the transport activity. The histogram represents mean of a total 6 measurements (n=6) done as two independent measurements. Individual datapoints are represented as black dots around the error bar that represents s.e.m.


Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . A near 10 - fold loss of nipecotic acid inhibition potency was observed at a value of 134 . 3 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows ; nipecotic acid ,

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Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. A near 10-fold loss of nipecotic acid inhibition potency was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows; nipecotic acid,


Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows panel E - 0 . 0001 ; tiagabine , panel F - 0 . 0017 ; NO711 , panel G - 0 . 018 ;

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Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a


E , F , Eadie - Hofstee plots of E , tiagabine and F , NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT . G , Eadie - Hofstee plot of SKF89976a displaying mixed ( competitive + non - competitive ) inhibition of 3H - GABA uptake in GAT1WT . Data information : ( E - G ) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors . Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates . Error bars represent s . e . m .

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E, F, Eadie-Hofstee plots of E, tiagabine and F, NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT. G, Eadie-Hofstee plot of SKF89976a displaying mixed (competitive+non-competitive) inhibition of 3H-GABA uptake in GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m.


C , Michaelis - Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value ( 28 . 54 ± 2 . 3 μM ) . The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

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C, Michaelis-Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value (28.54 ± 2.3 μM). The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


D , 3H - GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30 . 1 μM Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

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D, 3H-GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30.1 μM Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


E , The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

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E, The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


a , GFP - MAP1LC3B foci after RNAi and / or rapamycin treatment in U2OS cells . Scale bar , 20 µm .

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a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.


A LC3 punctum formation in WT and GCN5 KO HeLa cells ( Scale bars , 10 µm ) .

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A LC3 punctum formation in WT and GCN5 KO HeLa cells (Scale bars, 10 µm).


Immunoblot showing LC3 - II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.


Immunoblot showing LC3 - II formation MB - 3 - treated HeLa cells in the presence or absence of the lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation MB-3-treated HeLa cells in the presence or absence of the lysosome inhibitor Baf.


D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E Quantification of LC3 puncta in cells The cells were treated with or without Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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E Quantification of LC3 puncta in cells The cells were treated with or without Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Formation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment


Formation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf treatment ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bar , 10 µm ) .

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bar, 10 µm).


H LC3 - II formation in GFP - GCN5 - overexpressing HeLa cells .

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H LC3-II formation in GFP-GCN5-overexpressing HeLa cells.


I GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were cultured with GCN5 siRNA with or without CQ .

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I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ.


J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP - GCN5 and addition of CQ .

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J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ.


K Representative images of mCherry - Atg8a ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) using the pan - fat body driver ( cg - GAL4 ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ; Scale bars , 10 µm ) .

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K Representative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, ***p < 0.001, Student's t-test; Scale bars, 10 µm).


A LAMP1 puncta ( green ) and DAPI ( blue ) in WT and GCN5 KO HEK293 cells ( Scale bars , 10 µm ) .

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A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm).


B Fluorescence - activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker . Fluorescence intensity of 10 , 000 cells per sample was measured .

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B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured.


C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells . CTSD HC , cathepsin D heavy chain .

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C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells. CTSD HC, cathepsin D heavy chain.


D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E Quantification of LAMP1 puncta in ( A ) and ( D ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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E Quantification of LAMP1 puncta in (A) and (D) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


F Hexosaminidase activity in GCN5 KO HEK293 cells ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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F Hexosaminidase activity in GCN5 KO HEK293 cells (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ .

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G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ.


H Representative images of LysoTracker staining ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bars , 10 µm ) .

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H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).


A Quantification of LC3 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


B Immunoblot showing LC3 - II formation in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf .

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B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf.


C Quantification of LAMP1 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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C Quantification of LAMP1 puncta in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


D Immunoblot showing LAMP1 levels in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells .

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D Immunoblot showing LAMP1 levels in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells.


E Acetylation of TFEB - Flag in stable TFEB - Flag - expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated . TFEB - Flag was immunoprecipitated with anti - Flag beads and analyzed by immunoblot using anti - acetyl - lysine ( Ace - lys ) .

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E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys).


F TFEB - Flag acetylation in the stable TFEB - Flag - expressing HEK293 cells transfected with Myc - tagged GCN5 or GCN5 - E575Q after incubation with GCN5 siRNA for 48 h .

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F TFEB-Flag acetylation in the stable TFEB-Flag-expressing HEK293 cells transfected with Myc-tagged GCN5 or GCN5-E575Q after incubation with GCN5 siRNA for 48 h.


G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB - 3 or the p300 / CBP inhibitor C646 . H3 , histone H3 . Ace - H3 ( K9 ) and Ace - H3 ( K27 ) were used to show the activity of GCN5 and p300 , respectively .

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G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively.


H Co - precipitation of GCN5 with TFEB . TFEB was immunoprecipitated from MEF cells using anti - TFEB , and the precipitates were analyzed using anti - GCN5 .

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H Co-precipitation of GCN5 with TFEB. TFEB was immunoprecipitated from MEF cells using anti-TFEB, and the precipitates were analyzed using anti-GCN5.


I In vitro acetylation assays of TFEB . Purified recombinant TFEB was incubated with Myc - GCN5 or Myc - GCN5 - E575Q immunoprecipitated from HEK293 cells .

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I In vitro acetylation assays of TFEB. Purified recombinant TFEB was incubated with Myc-GCN5 or Myc-GCN5-E575Q immunoprecipitated from HEK293 cells.


J Acetylation of Flag - tagged TFEB or TFEB mutants expressed in HEK293 cells . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg .

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J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.


A RT - qPCR analysis of the expression of TFEB target genes in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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A RT-qPCR analysis of the expression of TFEB target genes in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


B RT - qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5 . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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B RT-qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB - WT or TFEB - 3KR . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB-WT or TFEB-3KR. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


D , E Luciferase activity measured in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( D ) , and in HEK293 cells with TFEB - WT or TFEB - 3KR transfection ( E ) . The cells were transfected or co - transfected with a TFEB - luciferase reporter . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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D, E Luciferase activity measured in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (D), and in HEK293 cells with TFEB-WT or TFEB-3KR transfection (E). The cells were transfected or co-transfected with a TFEB-luciferase reporter. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 ( Scale bars , 10 µm ) .

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A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm).


B Subcellular localization of Flag - tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1 . Cells were stained with anti - Flag antibody ( Scale bars , 10 µm ) .

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B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm).


C ChIP - qPCR analysis of TFEB binding to the promoter of its target genes CLCN7 , GLA and CTSD . Normal mouse IgG and primers against the upstream region lacking CLEAR sites ( up ) were used as negative controls ( mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test ) .

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C ChIP-qPCR analysis of TFEB binding to the promoter of its target genes CLCN7, GLA and CTSD. Normal mouse IgG and primers against the upstream region lacking CLEAR sites (up) were used as negative controls (mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test).


D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA . A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants , and was subjected to electrophoresis . Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non - binding DNA control .

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D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control.


F Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - TFEB in HEK293T cells . Immunoprecipitation was carried out with anti - Myc beads , and the precipitates were analyzed using anti - Flag .

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F Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-TFEB in HEK293T cells. Immunoprecipitation was carried out with anti-Myc beads, and the precipitates were analyzed using anti-Flag.


Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged MITF ( G ) in HEK293T cells .

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Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged MITF (G) in HEK293T cells.


Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged TFE3 ( H ) in HEK293T cells .

Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged TFE3 (H) in HEK293T cells.


I TFEB homodimer formation detected by glutaraldehyde ( GA ) crosslinking . Purified recombinant GST - tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde , then the products were analyzed by immunoblotting using anti - TFEB .

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I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.


A Acetylation of TFEB - Flag , Myc - GCN5 and histone H3 in HEK293 cells . The cells were either starved of glucose or amino acids , or treated with Torin1 or MB - 3 .

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A Acetylation of TFEB-Flag, Myc-GCN5 and histone H3 in HEK293 cells. The cells were either starved of glucose or amino acids, or treated with Torin1 or MB-3.


B Acetylation of endogenous TFEB in HeLa cells .

B Acetylation of endogenous TFEB in HeLa cells.


C GCN5 activity measured by in vitro acetylation assay using Myc - GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate . ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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C GCN5 activity measured by in vitro acetylation assay using Myc-GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate. (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


D Co - precipitation of TFEB - Flag with Myc - TFEB in HEK293

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D Co-precipitation of TFEB-Flag with Myc-TFEB in HEK293 cells


E LC3 - II formation in HeLa cells transfected with Flag - tagged TFEB or TFEB mutants after TFEB RNAi . The cells were treated with or without amino acid starvation .

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E LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.


F GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were treated with or without addition of CQ .

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F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.


Representative images of mCherry - Atg8a ( red ) ( G ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

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Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Representative images of LysoTracker ( red ) staining ( H ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

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Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Quantification of mCherry - ATG8a puncta ( I ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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Quantification of mCherry-ATG8a puncta (I) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Quantification of LysoTracker puncta ( J ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding . The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as the control .

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The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.


C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR - Gal4 driver . The Drosophila were fed with or without CQ . Drosophila ( GMR - Gal4 / + ) was used as the control .

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C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.


D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ ( 30 flies per condition were randomly selected for analyses ; * p < 0 . 05 , * * * p < 0 . 001 , ꭓ2 test ) .

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D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).


D . Quantity of Ki67 + cells in tumour and representative histology images analysed by Immunohistochemistry ( IHC ) that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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D. Quantity of Ki67+ cells in tumour and representative histology images analysed by Immunohistochemistry (IHC) that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


F . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 , Muc1 of colon tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10, Muc1 of colon tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


G . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein expression ( right ) in the tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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G. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein expression (right) in the tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


representative histological images ( B ) of wild - type control mice or BTNL2 - KO mice treated with 2 % DSS for 8 days were shown ( n = 15 ) .

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representative histological images (B) of wild-type control mice or BTNL2-KO mice treated with 2% DSS for 8 days were shown (n=15).


E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β , IL - 6 and IL - 10 of colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β, IL-6 and IL-10 of colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


representative H & E images ( H ) of wild - type control mice or BTNL2 - KO mice treated with Fc or mIL - 22 - Fc ( ip . 5 μg / mouse ) at day 0 , 2 , 4 , 6 during 2 % DSS treatment were shown ( n = 12 ) . Data information Data are representative of three biological replicates H ) .

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 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

representative H&E images (H) of wild-type control mice or BTNL2-KO mice treated with Fc or mIL-22-Fc (ip. 5 μg/mouse) at day 0, 2, 4, 6 during 2% DSS treatment were shown (n=12). Data information Data are representative of three biological replicates H).


B . IL - 22 and IL - 17a expression in lamina propria lymphocytes ( LPLs ) after cultured in plate - coated Fc , mBTNL2 - Fc ( 30 μg / mL ) with or without IL - 23 ( 10 ng / mL ) for 20 h was analyzed by ELISA . C . IL - 22 production from cells of LPLs cultured in the presence of plate - coated Fc , mBTNL2 - Fc with anti - control Ab or anti - BTNL2 mAb for 20 h was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were

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

B. IL-22 and IL-17a expression in lamina propria lymphocytes (LPLs) after cultured in plate-coated Fc, mBTNL2-Fc (30 μg/mL) with or without IL-23 (10 ng/mL) for 20 h was analyzed by ELISA. C. IL-22 production from cells of LPLs cultured in the presence of plate-coated Fc, mBTNL2-Fc with anti-control Ab or anti-BTNL2 mAb for 20 h was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shown


D . CD4 + T cells , γδ T cells , ILC3s , CD8 + T cells sorted by FACS were cultured in the plate - coated Fc , mBTNL2 - Fc with or without IL - 23 ( 10 ng / mL ) for 20 h , and IL - 22 production was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were

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

D. CD4+ T cells, γδ T cells, ILC3s, CD8+ T cells sorted by FACS were cultured in the plate-coated Fc, mBTNL2-Fc with or without IL-23 (10 ng/mL) for 20 h, and IL-22 production was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shown


F . IL - 22 production was analyzed by Flow cytometry in LPLs of wild - type mice treated with Fc or mBTNL2 - Fc ( ip . 50 μg / mouse ) on day 0 , 2 , 4 , 6 during 2 % DSS treatment . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 12 for the Fc group and n = 14 for the mBTL2 - Fc group ( F ) . Pooled data from three biological replicates were shown F .

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

F. IL-22 production was analyzed by Flow cytometry in LPLs of wild-type mice treated with Fc or mBTNL2-Fc (ip. 50 μg/mouse) on day 0, 2, 4, 6 during 2%DSS treatment. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=12 for the Fc group and n=14 for the mBTL2-Fc group (F). Pooled data from three biological replicates were shown F.


G . IL - 22 , IL - 17a , RORC and HIF - 1α expression in splenocytes after cultured in plate - coated Fc or mBTNL2 - Fc ( 30 μg / mL ) for 20 h was analyzed by real - time PCR . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Data are representative of three biological

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 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 O O O O O O O O O O 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. IL-22, IL-17a, RORC and HIF-1α expression in splenocytes after cultured in plate-coated Fc or mBTNL2-Fc (30 μg/mL) for 20 h was analyzed by real-time PCR. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Data are representative of three biological replicates


E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 from colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 15 Pooled data from three biological

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

E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 from colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=15 Pooled data from three biological replicates


J . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 of colonic tissue described in ( F ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 12 Pooled data from three biological

J. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 of colonic tissue described in (F) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=12 Pooled data from three biological replicates


Representative colonic tumour burden ( B ) that described in ( A ) were shown ( n = 12 ) . Data information : Data are representative of three biological replicates (

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

Representative colonic tumour burden (B) that described in (A) were shown (n=12). Data information: Data are representative of three biological replicates (B


F . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 and Muc1 of colonic tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for F , Each dot represents one mouse , n = 12 . Pooled data from three biological

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

F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10 and Muc1 of colonic tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for F, Each dot represents one mouse, n=12. Pooled data from three biological replicates


G . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein ( right ) expression of tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for G , Each dot represents one mouse , n = 12 . Data are representative of three biological replicates left panel of G ) . Pooled data from three biological replicates are shown in right panel of

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein (right) expression of tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for G, Each dot represents one mouse, n=12. Data are representative of three biological replicates left panel of G). Pooled data from three biological replicates are shown in right panel of G


H , Quantity of Ki67 + cells in tumours and representative histology images that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for H ) . Each dot represents one mouse , n = 12 . Pooled data from three biological replicates are shown in H .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantity of Ki67+ cells in tumours and representative histology images that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for H). Each dot represents one mouse, n=12. Pooled data from three biological replicates are shown in H.


A . Survival of patients with colorectal cancer from Kaplan - Meier database was analyzed Left panel of A were representative of 150 technical replicates , and right panel of A were representative of 499 technical replicates .

O O O O O O O O O O O O O O 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. Survival of patients with colorectal cancer from Kaplan-Meier database was analyzed Left panel of A were representative of 150 technical replicates, and right panel of A were representative of 499 technical replicates.


representative histology images in human colorectal cancer specimens ( 1 # - 5 # ) and human colon polyp specimens ( 6 # - 7 # ) were shown . Number of cells expressed BTNL2 or IL - 22 was analysed in high power field ( HPF ) , arrows indicate cells expressed BTNL2 or IL - 22 ( B ) .

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

representative histology images in human colorectal cancer specimens (1#-5#) and human colon polyp specimens (6#-7#) were shown. Number of cells expressed BTNL2 or IL-22 was analysed in high power field (HPF), arrows indicate cells expressed BTNL2 or IL-22 (B).


D - E . Expression of BTNL2 in normal colon tissue or DSS + AOM induced tumour tissue were measured by immunoblot ( D ) and real - time PCR ( E ) ( n = 5 ) . Data information : All data are mean ± s . e . m . NS , not significant . , * * P < 0 . 01 based on two - tailed Student ' s t test for ( E ) . Each dot represents one mouse , n = 5 .

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

D-E. Expression of BTNL2 in normal colon tissue or DSS+AOM induced tumour tissue were measured by immunoblot (D) and real-time PCR (E) (n=5). Data information: All data are mean ± s.e.m. NS, not significant., **P < 0.01 based on two-tailed Student's t test for (E). Each dot represents one mouse, n=5.


( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin .

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

(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.


( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) .

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

(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).


( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) .

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

(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).


( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

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

(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag .

O O O B-MEASURED_VAR 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

(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.


( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control .

O O O B-MEASURED_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 O O O O 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) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.


( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis .

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(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.


D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins .

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

D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.


( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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 B-MEASURED_VAR 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 O O O O O O 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

(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

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

B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.


( A and B ) RNF26 targeted MITA for K11 - linked polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , cells were subjected for IP under denatured conditions with limited amount of anti - Flag ( 0 . 5 µg ) so that equal amount of Flag - MITA is pulled down . The immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , all lysine residues of ubiquitin were mutated to arginine .

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

(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.


( C ) Effects of RNF26 - RNAi plasmids on the expression of RNF26 . In the upper panels , the 293 cells ( 1 × 106 ) were transfected with the expression plasmids for RNF26 - Flag ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with the indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole cell lysates were analyzed by immunoblots with anti - Flag or anti - HA . In the lower panels , 293 cells were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . Cells ( 1 × 106 ) were lysed and whole cell lysates were analyzed by immunoblots with anti - RNF26 or anti - β - actin .

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 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 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 B-MEASURED_VAR O O O O O O O

(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.


( D ) Immunoblot analysis of Flag - tagged ubiquitin expression in THP - 1 cells stably transfected with Flag - Ub - K11O plasmid . Whole cell lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi and control cells ( 1 × 106 ) were analyzed by immunoblots with antibodies against the indicated proteins . RNF26 - RNAi # 1 was used here and in the following experiments if not noted .

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

(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.


( E and F ) Effects of RNF26 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . The immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins . In ( F ) , 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with a control or RNF26 - RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , puromycin ( 1 µg / mL ) was added into the culture medium . The cells were selected for twenty - four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in ( E ) . All experiments were repeated for at least three times with similar results .

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

(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.


A ) Effects of RNF26 knockdown on virus - induced polyubiquitination of endogenous MITA . The THP - 1 - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Ub ( K48 ) ( upper panel ) , anti - Ub ( K63 ) ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( B ) Effects of RNF5 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . The 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with the indicated RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , the cells were selected with puromycin ( 1 µg / mL ) for twenty - four hours and infected with SeV for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies .

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

(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.


( C ) RNF26 and RNF5 competed with each other on MITA polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O and HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 and RNF5 . Twenty - four hours after transfection , cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( D ) Effects of RNF26 knockdown on virus - triggered MITA degradation . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins ( upper immunoblots ) . The relative protein levels of MITA in reference to β - actin were analyzed by Quantity One program and data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

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

(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


( A ) Effects of RNF26 knockdown on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and the indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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

(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B )

O O O O O O O B-CONTROLLED_VAR 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 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

(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA analysis (

O O O O O O O B-CONTROLLED_VAR 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 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

(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C


( D ) Effects of RNF26 knockdown on virus - triggered induction of IFNB1 gene in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with VSV , EMCV or ECTV for the indicated time points or left uninfected before quantitative real - time PCR analysis was performed as in ( B ) .

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 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

(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).


( E and F ) Effects of RNF26 knockdown on virus - triggered induction of TNFα in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( E ) and ELISA analysis ( F ) .

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 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

(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).


( G ) Effects of RNF26 knockdown on virus - triggered phosphorylation of TBK1 , IRF3 and IκBα . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected , whole cell lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

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

(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


A ) Effects of RNF26 knockdown on IRF3 level . In the left panel , whole cell lysates of THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were analyzed by immunoblots with the indicated antibodies . In the right panel , cells ( 1 × 106 ) were subjected to quantitative real - time PCR analysis .

O O O O B-CONTROLLED_VAR O 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

A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.


B ) Effects of RNF26 on IRF3 stability . The 293 cells ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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-MEASURED_VAR 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 B-MEASURED_VAR O

B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( C ) Effects of RNF26 and its mutant on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( D ) Effects of RNF26 on IRF3 ubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment . The cell lysates were subjected to IP under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.