C PHIP - 1 dephosphorylates GPB - 1 in vitro . COS - 7 cells were co - transfected with HA - GPB - 1 and T7 - GPC - 2 , and cell lysates were immunoprecipitated ( IP ) with anti - HA antibodies . The immunopurified HA - GPB - 1 was first subjected to the in vitro kinase assay with recombinant GST - NDK - 1 . Phosphorylated GPB - 1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST - PHIP - 1 or its variants . Phosphorylated GPB - 1 was detected by immunoblotting ( IB ) with anti - 3 - pHis antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST-PHIP-1 or its variants. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.


A PHIP - 1 interaction with UNC - 51 by yeast two - hybrid assay . The reporter strain PJ69 - 4A was co - transformed with expression vectors encoding GAL4 DBD - PHIP - 1 ( H45A ) and GAL4 AD - UNC - 51 ( 274 - 856 ) , as indicated . Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5 - aminotriazole for 4 days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.


C UNC - 51 phosphorylates PHIP - 1 at Ser - 112 . COS - 7 cells were co - transfected with Flag - PHIP - 1 ( WT or mutants ) and GFP - UNC - 51 [ WT or ∆ AIKAI ( KD ) ] , and cell lysates were analyzed using Phos - tag SDS - PAGE . Total lysates were immunoblotted ( IB ) with antibodies , as indicated . Filled and open arrowheads indicate unmodified and phosphorylated PHIP - 1 , respectively . Asterisk indicates non - specific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were co-transfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or ∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled and open arrowheads indicate unmodified and phosphorylated PHIP-1, respectively. Asterisk indicates non-specific band.


( A - B ) THP1 over - expressing IFITM3 , IFITM3 - Y20F , IFITM3 - ∆ 21 or control were pre - treated or not with Amphotericin B for 1 hour and then transduced with a VSV - gp ( A ) or Measles - gp ( B ) pseudotyped LV . Transduction efficiencies were evaluated at FACS five days later . Fold rescue of transduction over the DMSO control was calculated . Data are shown as mean ± SEM ( n = 5 - 8 biological replicates run in technical duplicate ) . P values are for one sample t test versus Oe - Luc DMSO = 1 , * * for p < 0 . 01 , * * * for p < 0 . 001 .

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

(A-B) THP1 over-expressing IFITM3, IFITM3-Y20F, IFITM3-∆21 or control were pre-treated or not with Amphotericin B for 1 hour and then transduced with a VSV-gp (A) or Measles-gp (B) pseudotyped LV. Transduction efficiencies were evaluated at FACS five days later. Fold rescue of transduction over the DMSO control was calculated. Data are shown as mean ± SEM (n=5-8 biological replicates run in technical duplicate). P values are for one sample t test versus Oe-Luc DMSO =1, ** for p<0.01, *** for p<0.001.


( E - F ) THP1 over - expressing IFITM3 , IFITM3 lysin - less mutant ( 4KR ) ( E ) , IFITM3 CIL - lysin mutant ( 3KR ) ( F ) or control were transduced with VSV - gp pseudotyped LV . Transduction efficiencies were evaluated by flow cytometry five days post - TD . Data represent the mean ± SEM ( n = 9 - 6 biological replicates run in technical duplicate ) . p values are for one sample t test verso Oe - Luc DMSO = 1 . * for p < 0 . 05 * * for p < 0 . 01 . * * * * for p < 0 . 0001 and ns for not significant .

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

(E-F) THP1 over-expressing IFITM3, IFITM3 lysin-less mutant (4KR) (E), IFITM3 CIL-lysin mutant (3KR) (F) or control were transduced with VSV-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=9-6 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01. **** for p<0.0001 and ns for not significant.


( G - H ) THP1 over - expressing IFITM1 , IFITM1 - 3KR ( G ) , IFITM3 , IFITM3 - Y20F , IFITM3 - Y20F - 3KR ( H ) or control were transduced with Measles - gp pseudotyped LV . Transduction efficiencies were evaluated by flow cytometry five days post - TD . Data represent the mean ± SEM ( n = 4 - 9 biological replicates run in technical duplicate ) . p values are for one sample t test verso Oe - Luc DMSO = 1 . * for p < 0 . 05 * * for p < 0 . 01 . * * * * for p < 0 . 0001 .

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

(G-H) THP1 over-expressing IFITM1, IFITM1-3KR (G), IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR (H) or control were transduced with Measles-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=4-9 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01.**** for p<0.0001.


( A - B ) Immunofluorescence images were performed using TCS SP5 Leica confocal microscope , 60x with oil on THP1 over - expressing IFITM3 - WT , IFITM3 - 4KR , IFITM3 - 3KR or IFITM3 - Y20F ( A ) ( n = 22 images acquired from 3 biological replicates ) and primary monocytes derived macrophages ( MDM ) over - expressing IFITM3 - WT , IFITM3 - 4KR , IFITM3 - 3KR ( B ) ( n = 12 images acquired from 3 independent donors ) . Co - localization ( purple areas marked by white arrows ) of IFITM3 ( in red ) with the lysosome associated membrane protein 1 ( LAMP1 ) marker ( in blue ) was evaluated . Representative zoomed images are shown ,

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

(A-B) Immunofluorescence images were performed using TCS SP5 Leica confocal microscope, 60x with oil on THP1 over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR or IFITM3-Y20F (A) (n=22 images acquired from 3 biological replicates) and primary monocytes derived macrophages (MDM) over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR (B) (n=12 images acquired from 3 independent donors). Co-localization (purple areas marked by white arrows) of IFITM3 (in red) with the lysosome associated membrane protein 1 (LAMP1) marker (in blue) was evaluated. Representative zoomed images are shown,


( D ) IFITM3 - IFITM3 interactions ( red dots ) were evaluated by proximity ligation assay ( PLA ) in THP1 knock out for endogenous IFITM3 and expressing either HA - IFITM3 and His - IFITM3 , HA - 3KR and His - 3KR or His - dimer mutant IFITM3 ( D . M ) and HA - dimer mutant . Images were acquired on TCS SP5 Leica confocal microscope 60x with oil . Representative images are shown ( n = 8 images of 4 biological replicates ) , scale bar 40μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IFITM3-IFITM3 interactions (red dots) were evaluated by proximity ligation assay (PLA) in THP1 knock out for endogenous IFITM3 and expressing either HA-IFITM3 and His-IFITM3, HA-3KR and His-3KR or His-dimer mutant IFITM3 (D.M) and HA-dimer mutant. Images were acquired on TCS SP5 Leica confocal microscope 60x with oil. Representative images are shown (n=8 images of 4 biological replicates), scale bar 40μm.


( F ) IFITM3 dimerization was assessed by native PAGE followed by WB analysis in THP1 . IFITM3 higher order protein complex bands are highlighted by the asterisk . IFITM3 dimers and higher molecular weight complexes were quantified over the normalizer using ImageJ ( mean ± SEM , n = 7 biological replicates ) . p values are for Mann Whitney test . * for p < 0 . 05 , * * for p < 0 . 01 .

(F) IFITM3 dimerization was assessed by native PAGE followed by WB analysis in THP1. IFITM3 higher order protein complex bands are highlighted by the asterisk. IFITM3 dimers and higher molecular weight complexes were quantified over the normalizer using ImageJ (mean ± SEM, n=7 biological replicates). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.


( B ) K562 were prestimulated with IFNα and then transduced with VSV - gp pseudotyped LV . Transduction was evaluated five days after TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . Data are represented as Folds over the Oe - Luc control . p values are for one sample t test versus - IFNα = 1 . ns for not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) K562 were prestimulated with IFNα and then transduced with VSV-gp pseudotyped LV. Transduction was evaluated five days after TD (mean ± SEM, n=4 biological replicates run in technical duplicate). Data are represented as Folds over the Oe-Luc control. p values are for one sample t test versus -IFNα = 1. ns for not significant.


( C ) Interferon stimulated genes ( ISGs ) mRNA expression were measured 24 hours post - IFNα stimulation by RT - qPCR in K562 ( mean ± SEM , n = 3 biological replicates run in technical duplicate ) .

O O O O O O O O O O O O O O O O O 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) Interferon stimulated genes (ISGs) mRNA expression were measured 24 hours post-IFNα stimulation by RT-qPCR in K562 (mean ± SEM, n=3 biological replicates run in technical duplicate).


( E ) K562 were pre - stimulated or not with IFNα for 24 hours and then inoculated with the infectious ZIKV ( mean ± SEM , n = 3 biological replicates run in technical triplicates ) . Viral supernatant was collected at different time points and titered on Vero E6 cells . p values are for Mann Whitney test . * * for p < 0 . 01 , * * * for p < 0 . 001 .

(E) K562 were pre-stimulated or not with IFNα for 24 hours and then inoculated with the infectious ZIKV (mean ± SEM, n=3 biological replicates run in technical triplicates). Viral supernatant was collected at different time points and titered on Vero E6 cells. p values are for Mann Whitney test. ** for p<0.01, *** for p<0.001.


( F ) K562 over - expressing IFITM1 , IFITM1 - 3KR or control were transduced with Measles - gp pseudotyped LV . Transduction efficiency was evaluated five days post - TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc = 1 . * * * * for p < 0 . 0001 .

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

(F) K562 over-expressing IFITM1, IFITM1-3KR or control were transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc = 1. **** for p<0.0001.


K562 over - expressing IFITM3 , IFITM3 - Y20F , IFITM3 - Y20F - 3KR or control were pre - exposed or not to Ampho B and then transduced with Measles - gp ( G ) , BaEV - gp ( H ) pseudotyped LV . Transduction efficiencies were measured by flow cytometry five days after TD ( mean ± SEM , n = 5 - 4 - 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc DMSO = 1 or unpaired t test for Ampho B versus DMSO . * for p < 0 . 05 , * * for p < 0 . 01 , * * * for p < 0 . 001 , * * * * for p < 0 . 0001 .

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

K562 over-expressing IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR or control were pre-exposed or not to Ampho B and then transduced with Measles-gp (G), BaEV-gp (H) pseudotyped LV. Transduction efficiencies were measured by flow cytometry five days after TD (mean ± SEM, n=5-4-4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1 or unpaired t test for Ampho B versus DMSO. * for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001.


( A ) Interaction between IFITM3 wild - type or 3KR and Phosphatydinositol - 3 phosphate ( PIP3 ) ( red dots ) was evaluated by proximity ligation assay . Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild - type or IFITM3 - 3KR . Number of foci were counted on imageJ and normalized over the number of nuclei ( mean ± SEM , n = 5 biological replicates ) . White arrows indicate foci , scale bar 40μm .

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

(A) Interaction between IFITM3 wild-type or 3KR and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay. Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild-type or IFITM3-3KR. Number of foci were counted on imageJ and normalized over the number of nuclei (mean ± SEM, n=5 biological replicates). White arrows indicate foci, scale bar 40μm.


( B ) Phosphatydinositol - 3 phosphate ( PIP3 ) levels were measured by immunofluorescence in THP1 over - expressing the His - IFITM3 / HA - IFITM3 and His - 3KR / HA - 3KR . PIP3 was quantified via Imagej ( mean ± SEM , n = 5 biological replicates ) .

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

(B) Phosphatydinositol-3 phosphate (PIP3) levels were measured by immunofluorescence in THP1 over-expressing the His-IFITM3/HA-IFITM3 and His-3KR/HA-3KR. PIP3 was quantified via Imagej (mean ± SEM, n=5 biological replicates).


( C ) THP1 over - expressing IFITM3 or control were transduced with VSV - gp pseudotyped LV in presence or absence of Rapamycin ( Rapa ) . Transduction efficiency was measured at FACS five days post - TD ( mean ± SEM , n = 6 biological replicates run in technical duplicate . p values are for one sample t test versus Oe - Luc DMSO = 1 and Mann Whitney test for Rapa vs DMSO . * * for p < 0 . 01 , * * * * for p < 0 . 0001 .

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

(C) THP1 over-expressing IFITM3 or control were transduced with VSV-gp pseudotyped LV in presence or absence of Rapamycin (Rapa). Transduction efficiency was measured at FACS five days post-TD (mean ± SEM, n=6 biological replicates run in technical duplicate. p values are for one sample t test versus Oe-Luc DMSO=1 and Mann Whitney test for Rapa vs DMSO. ** for p<0.01, **** for p<0.0001.


( D ) THP1 over - expressing IFITM3 - Y20F - 3KR or control were exposed or not to Rapamycin and transduced with Measles - gp pseudotyped LV . Transduction efficiency was evaluated by flow cytometry five days post - TD ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for one sample t test versus Oe - Luc DMSO = 1 . * * for p < 0 . 0 .

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

(D) THP1 over-expressing IFITM3-Y20F-3KR or control were exposed or not to Rapamycin and transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated by flow cytometry five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1. ** for p<0.0.


( E ) Hematopoietic stem and progenitor cells ( HSPC ) were transduced with VSV - gp or Measles - gp pseudotyped LV in presence or absence of Rapamycin . Transduction was evaluated at FACS five days later measuring the % of BFP expression ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Mann Whitney test . * for p < 0 . 05 .

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

(E) Hematopoietic stem and progenitor cells (HSPC) were transduced with VSV-gp or Measles-gp pseudotyped LV in presence or absence of Rapamycin. Transduction was evaluated at FACS five days later measuring the % of BFP expression (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05.


Levels of phosphatidylinositol ( PI ) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562 . The results are presented as log plots of the PI ratio in K562 over HSPC ( D ) .

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-MEASURED_VAR O O O O O O O O O

Levels of phosphatidylinositol (PI) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562. The results are presented as log plots of the PI ratio in K562 over HSPC (D).


( E ) PIP3 levels were measured by immunofluorescence ( scale bar 40μm ) and quantified as integrated density with ImageJ software in THP1 and K562 over - expressing IFITM3 ( mean ± SEM , n = 3 biological replicates ) . p values are for Mann Whitney test , * for p < 0 . 05 .

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

(E) PIP3 levels were measured by immunofluorescence (scale bar 40μm) and quantified as integrated density with ImageJ software in THP1 and K562 over-expressing IFITM3 (mean ± SEM, n=3 biological replicates). p values are for Mann Whitney test, * for p<0.05.


( F ) Interaction between IFITM3 and Phosphatydinositol - 3 phosphate ( PIP3 ) ( red dots ) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3 . Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) , scale bar 40μm . p values are for Mann Whitney test , * * for p < 0 . 01 , * * * for p < 0 . 001 . ) .

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

(F) Interaction between IFITM3 and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3. Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei (mean ± SEM, n=4 biological replicates run in technical duplicate), scale bar 40μm. p values are for Mann Whitney test, ** for p<0.01, *** for p<0.001.).


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 ( G ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

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

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 (G) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC ( H ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

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

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC (H) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM ( I ) pre - stimulated or not with IFN - α ( mean ± SEM , n = 3 biological replicates . p values are for Mann Whitney test , * * * for p < 0 . 001 .

PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM (I) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.


( A ) Calu3 over - expressing IFITM1 , IFITM2 , IFITM3 , PM - mutant IFITM3 Y20A or control were transduced with SARS - CoV2 Spike - pseudotyped LV ( mean ± SEM , n > 6 biological replicates run in technical duplicate ) . Transduction efficiency was measured by FACS five days after transduction , p values are for one sample paired T test versus Oe - Luc Ctrl = 1 , * * for p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Calu3 over-expressing IFITM1, IFITM2, IFITM3, PM-mutant IFITM3 Y20A or control were transduced with SARS-CoV2 Spike-pseudotyped LV (mean ± SEM, n>6 biological replicates run in technical duplicate). Transduction efficiency was measured by FACS five days after transduction, p values are for one sample paired T test versus Oe-Luc Ctrl=1, **for p<0.01.


( B - C ) Calu3 cells over - expressing IFITM1 , IFITM2 , IFITM3 , IFITM3 Y20A or control were infected with SARS - CoV2 . Viral titer was calculated by plaque assay in Vero E6 cells ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Mann Whitney test , * for p < 0 . 05 , * * * for p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3, IFITM3 Y20A or control were infected with SARS-CoV2. Viral titer was calculated by plaque assay in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test, * for p<0.05, *** for p<0.001.


( G ) Calu3 over - expressing IFITM1 , IFITM3 or control were challenged to SARS - CoV2 Omicron pseudotyped LV . Transduction efficiency was evaluated 5 days post - transduction ( mean ± SEM , n = 7 biological replicates run in technical duplicate ) . p values are for one sample T test versus Oe - Luc Ctrl = 1 , * * for p < 0 . 01 , * * * * for p < 0 . 0001 .

(G) Calu3 over-expressing IFITM1, IFITM3 or control were challenged to SARS-CoV2 Omicron pseudotyped LV. Transduction efficiency was evaluated 5 days post-transduction (mean ± SEM, n=7 biological replicates run in technical duplicate). p values are for one sample T test versus Oe-Luc Ctrl=1, **for p<0.01, ****for p<0.0001.


( H - I ) Calu3 cells over - expressing IFITM1 , IFITM2 , IFITM3 or control ( H ) or IFITM3 , Y20A or control ( I ) were infected with Omicron at MOI 1 . Viral supernatants were collected at different time points and tittered on Vero E6 cells . Data are presented as mean ± SEM ( n = 4 - 4 biological replicates run in technical triplicate ) . p values are for Mann Whitney test , * * for p < 0 . 01 , * * * for p < 0 . 001 , * * * * for p < 0 . 0001 .

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

(H-I) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3 or control (H) or IFITM3, Y20A or control (I) were infected with Omicron at MOI 1. Viral supernatants were collected at different time points and tittered on Vero E6 cells. Data are presented as mean ± SEM (n=4-4 biological replicates run in technical triplicate). p values are for Mann Whitney test , **for p<0.01, ***for p<0.001, ****for p<0.0001.


( A - B ) VSV ( A ) or ZIKV ( B ) viral supernatants were collected one day post - infection in THP1 ( A ) or Calu3 ( B ) over - expressing IFITM3 , IFITM3 lysin - less mutant ( 4KR ) , IFITM3 CIL - lysin mutant ( 3KR ) or control and titered on VERO E6 cells . Data represent the mean ± SEM ( n = 5 - 3 biological replicates run in technical duplicate ) . p values are for Mann Whitney test . * for p < 0 . 05 , * * for p < 0 . 01 .

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(A-B) VSV (A) or ZIKV (B) viral supernatants were collected one day post-infection in THP1 (A) or Calu3 (B) over-expressing IFITM3, IFITM3 lysin-less mutant (4KR), IFITM3 CIL-lysin mutant (3KR) or control and titered on VERO E6 cells. Data represent the mean ± SEM (n=5-3 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.


( C - D ) K562 were inoculated with the infectious VSV ( C ) or ZIKV ( D ) ( mean ± SEM , n = 3 biological replicates run in technical duplicate ) . Viral supernatant was collected at different time points and titered on Vero E6 cells .

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(C-D) K562 were inoculated with the infectious VSV (C) or ZIKV (D) (mean ± SEM, n=3 biological replicates run in technical duplicate). Viral supernatant was collected at different time points and titered on Vero E6 cells.


( I ) IFITM3 and PIP3 level were measured by immunofluorescence ( scale bar 100µm ) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron ( n = 3 biological replicates ) . p values are for Mann Whitney test , * * for p < 0 . 01 . ( J ) PIP3 coupled to a specific PIP carrier ( PIP3 + Carr Ctrl ) or Carr Ctrl were provided to Calu3 over - expressing IFITM3 , control or KO for IFITM3 prior to infection with SARS - CoV2 Omicron . Infectious supernatants were collected after three days and tittered in Vero E6 cells ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Wilcoxon signed ranked test , * for p < 0 . 05 . The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl ( mean ± SEM , n = 4 biological replicates run in technical duplicate ) . p values are for Mann Whitney test , * for p < 0 . 05 .

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 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 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

(I) IFITM3 and PIP3 level were measured by immunofluorescence (scale bar 100µm) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron (n=3 biological replicates). p values are for Mann Whitney test, ** for p<0.01. (J) PIP3 coupled to a specific PIP carrier (PIP3+Carr Ctrl) or Carr Ctrl were provided to Calu3 over-expressing IFITM3, control or KO for IFITM3 prior to infection with SARS-CoV2 Omicron. Infectious supernatants were collected after three days and tittered in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Wilcoxon signed ranked test, * for p<0.05. The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Mann Whitney test, * for p<0.05.


Western blot analysis was performed using SETD1A fragment - expressing 293T cells . These constructs were used for the following rescue experiments . Representative images from 2 independent experiments are shown .

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Western blot analysis was performed using SETD1A fragment-expressing 293T cells. These constructs were used for the following rescue experiments. Representative images from 2 independent experiments are shown.


Setd1afl / fl ; CreER cells were transfected with SETD1A fragment and endogenous Setd1a was deleted by tamoxifen , then assayed for colony - forming potential . Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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Setd1afl/fl;CreER cells were transfected with SETD1A fragment and endogenous Setd1a was deleted by tamoxifen, then assayed for colony-forming potential. Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


qRT - PCR was performed at 3 days post - tamoxifen to analyze Fancd2 expression in human SETD1A - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells . qRT - PCR with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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qRT-PCR was performed at 3 days post-tamoxifen to analyze Fancd2 expression in human SETD1A-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


293T cells were transfected with SETD1A - GFP fusion constructs . Cell nucleus was visualized with DAPI . The percentage of GFP nuclear localization is indicated in each image . Asterisks in images indicate the cells with nuclear specific SETD1A - GFP signals . Scale bars : 10 µm . G − H . Mouse Setd1afl / fl ; CreER MLL - AF9 leukemia cells were transfected with FO3 - GFP fusion constructs . ( G ) Subcellular localization of GFP fusion constructs was visualized by immunofluorescence and nucleus was stained with DAPI . The percentage of GFP nuclear localization is indicated in each image . Scale bars : 10 µm . ( H ) FO3 - GFP fusion expressing cells were treated with tamoxifen and colony assay with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test ) .

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

293T cells were transfected with SETD1A-GFP fusion constructs. Cell nucleus was visualized with DAPI. The percentage of GFP nuclear localization is indicated in each image. Asterisks in images indicate the cells with nuclear specific SETD1A-GFP signals. Scale bars: 10 µm. G−H. Mouse Setd1afl/fl;CreER MLL-AF9 leukemia cells were transfected with FO3-GFP fusion constructs. (G) Subcellular localization of GFP fusion constructs was visualized by immunofluorescence and nucleus was stained with DAPI. The percentage of GFP nuclear localization is indicated in each image. Scale bars: 10 µm. (H) FO3-GFP fusion expressing cells were treated with tamoxifen and colony assay with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).


Relative expression of Mlh1 in SETD1A rescued MLL - AF9 leukemia cells . qRT - PCR with 3 biological replicates was performed . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 , * P ≤ 0 . 05 ( Student ' s t - test for ( D )

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Relative expression of Mlh1 in SETD1A rescued MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test for (D)


Percentage of GFP + SETD1A mutant - expressing leukemia cells in PB was analyzed at 3 weeks post - transplant ( n = 5 / group ) . Data information : * * P ≤ 0 . 01 , * P ≤ 0 . 05 One - way

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Percentage of GFP+ SETD1A mutant-expressing leukemia cells in PB was analyzed at 3 weeks post-transplant (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 One-way ANOVA


Survival of recipient mice harboring SETD1A mutant - expressing leukemia cells after transplantation ( TX ) was plotted ( n = 5 / group ) . Data information : * * P ≤ 0 . 01 , * P ≤ 0 . 05 Kaplan - Meier for ( F ) ) .

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Survival of recipient mice harboring SETD1A mutant-expressing leukemia cells after transplantation (TX) was plotted (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 Kaplan-Meier for (F)).


SETD1A FLOS domain - binding proteins were identified by Co - IP MS . Full - length SETD1A and FO3 fragment were used as baits and overlapped proteins ( red dots ) were identified as SETD1A FLOS domain - binding proteins .

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SETD1A FLOS domain-binding proteins were identified by Co-IP MS. Full-length SETD1A and FO3 fragment were used as baits and overlapped proteins (red dots) were identified as SETD1A FLOS domain-binding proteins.


293T cells were transfected with full - length SETD1A alanine mutants and protein extracts were used for the Co - IP experiments .

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293T cells were transfected with full-length SETD1A alanine mutants and protein extracts were used for the Co-IP experiments.


F . MOLM - 13 leukemia cells were examined by ChIP - seq analyses against HA - tagged SETD1A , BuGZ , BUB3 , H3K4me3 and H3K4me1 . Heatmaps of SETD1A ( + ) / BuGZ ( + ) regions ( upper ) and SETD1A ( - ) / BuGZ ( + ) regions ( lower ) are shown .

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F. MOLM-13 leukemia cells were examined by ChIP-seq analyses against HA-tagged SETD1A, BuGZ, BUB3, H3K4me3 and H3K4me1. Heatmaps of SETD1A(+)/BuGZ(+) regions (upper) and SETD1A(-)/BuGZ(+) regions (lower) are shown.


Relative expression levels of Bugz were analyzed in Bugz shRNA - expressing mouse MLL - AF9 leukemia cells at day 3 post - treatment , respectively . Data from 3 biological replicates are shown . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 ( Student ' s t -

Relative expression levels of Bugz were analyzed in Bugz shRNA-expressing mouse MLL-AF9 leukemia cells at day 3 post-treatment, respectively. Data from 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01 (Student's t-test


Survival of recipient mice harboring dox - inducible shRNA - expressing leukemia cells was plotted ( n = 8 - 10 / group ) . Data information : * * P ≤ 0 . 01 Kaplan - Meier for ( H ) ) .

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Survival of recipient mice harboring dox-inducible shRNA-expressing leukemia cells was plotted (n = 8-10 / group). Data information: **P ≤ 0.01 Kaplan-Meier for (H)).


Images are shown of representative karyotypes in control , Bugz and Bub3 shRNA - expressing leukemia cells . Asterisks in images indicate the trisomy 15 in all cells analyzed . Red arrows indicate chromosomal aneuploidies . Representative images of sister - chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels .

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Images are shown of representative karyotypes in control, Bugz and Bub3 shRNA-expressing leukemia cells. Asterisks in images indicate the trisomy 15 in all cells analyzed. Red arrows indicate chromosomal aneuploidies. Representative images of sister-chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels.


293T cells were transfected with SETD1A F2 alanine mutant constructs . Myc - DDK - tagged SETD1A was used for Co - IP .

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293T cells were transfected with SETD1A F2 alanine mutant constructs. Myc-DDK-tagged SETD1A was used for Co-IP.


293T cells were transfected with BuGZ mutants . Myc - DDK - tagged BuGZ mutants illustrated on Figure S5B were used for Co - IP .

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293T cells were transfected with BuGZ mutants. Myc-DDK-tagged BuGZ mutants illustrated on Figure S5B were used for Co-IP.


293T cells were transfected with BUB3 mutants and protein extracts were used for the Co - IP experiments . Myc - DDK - tagged BUB3 mutants illustrated on Figure S5C were used .

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293T cells were transfected with BUB3 mutants and protein extracts were used for the Co-IP experiments. Myc-DDK-tagged BUB3 mutants illustrated on Figure S5C were used.


293T cells were transfected with Myc - DDK - tagged human BuGZ constructs and the protein extracts were used for a Co - IP experiment .

293T cells were transfected with Myc-DDK-tagged human BuGZ constructs and the protein extracts were used for a Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged BuGZ variant constructs and the protein extracts were used for the Co - IP experiment .

293T cells were transfected with Myc-DDK-tagged BuGZ variant constructs and the protein extracts were used for the Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged BuGZ constructs and the protein extracts were used for a Co - IP experiment .

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

293T cells were transfected with Myc-DDK-tagged BuGZ constructs and the protein extracts were used for a Co-IP experiment.


293T cells were transfected with Myc - DDK - tagged wild - type BuGZ and the cells were treated with 1 , 6 - hexanediol for 3 h . The protein extracts were used for the Co - IP experiments .

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

293T cells were transfected with Myc-DDK-tagged wild-type BuGZ and the cells were treated with 1,6-hexanediol for 3 h. The protein extracts were used for the Co-IP experiments.


SETD1A - GFP fusion - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , then assayed for colony - forming potential . The FUCCI reporter ( mCherry - Cdt1 ( 30 - 120 ) / Citrine - Geminin ( 1 - 110 ) ) - expressing cells were used as control . Data from 3 biological replicates are shown . Data are presented as mean ± SD .

SETD1A-GFP fusion-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, then assayed for colony-forming potential. The FUCCI reporter (mCherry-Cdt1(30-120)/Citrine-Geminin(1-110))-expressing cells were used as control. Data from 3 biological replicates are shown. Data are presented as mean ± SD.


SGC - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , and RNA - seq analysis was performed with sorted cells . Data are shown as log2 fold change over tamoxifen non - treated GFP - expressing control .

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SGC-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, and RNA-seq analysis was performed with sorted cells. Data are shown as log2 fold change over tamoxifen non-treated GFP-expressing control.


( A , B ) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice . Brain sections were prepared 1 day after BrdU injection . 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 .

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(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. 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.


( C , D ) Differentiating cells are identified by staining with BrdU and DCX . Brain sections were prepared 10 days after BrdU injection .

(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.


( E , F ) Maturation of progenitor cells are assessed by staining with BrdU and NeuN . Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness , and a total of 24 sections were obtained from one hippocampus . Scale bars : 100 μm in the large panel , and 10 μm in the magnified images . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.


( A ) miR - 124 expression is altered in DSCR1 mutants . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 01 .

(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.


( B ) Promoter activity of miR - 124 in Neuro2 A cells with DSCR1 reduction or overexpression . Firefly luciferase reporter under the control of miR - 124 promoter is measured . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.


( C ) Bisulfite sequencing analyses of the miR - 124 promoter . Each CpG site is indicated , and the methylation status of two different regions are shown . Open and filled circles represent unmethylated and methylated CpG sites , respectively . The percentage of methylated CpGs among total number of CpGs is also shown .

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(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.


( D ) Neuro2A cells containing DSCR1shRNA show increased activity of miR - 124 promoter measured . The miR - 124 promoter was inserted in front of the luciferase reporter . Site - directed mutation of two methylation sites ( 31 and 58 ) in the promoter shows the luciferase activity similar to that of DSCR1 reduction . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( E ) Overexpression of DSCR1 reduced the luciferase activity , while removing all methylation sites in the miR - 124 promoter increases it . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( A ) DSCR1 binds to TET1 introns . The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA .

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(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.


( B ) DSCR1 missing RNA recognition motif ( △ RRM ) does not bind to the TET1 intron 8 .

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(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.


( C , D ) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4 . DSCR1 reduction increases the activity of this luciferase construct , while DSCR1 overexpression decreases its activity . In contrast , altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . * * * P < 0 . 001 .

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(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.


( E ) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants . TET1 is increased in DSCR1 knockout , while it is decreased in DSCR1 transgenic mice . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animal for each condition , * P < 0 . 05 , * * P < 0 . 01 .

(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.


( B ) 3 weeks after lentivirus injection , neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region . The white arrow indicates Sox2 and GFP double positive cells . Scale bar , 10 μm . ( C ) The number of GFP and Sox2 double positive cells is restored to that of wild type . Scale bar : 10μm . Values are shown as mean ± SEM and tested for statistical significance by paired t - test . N = 3 animal for each condition , * P < 0 . 05 .

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(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.


( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn and DSCR1

(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO


( B ) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2 . 5 folds compared to that of wild type . However , Ts65Dn / DSCR1 + / - mouse showed normal level of DSCR1 mRNA transcript . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.


As expected , the levels of TET1 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the TET1 mRNA expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


As expected , the levels of miR - 124 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the miR - 124 expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


( 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 ,

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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O

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 ) .

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 .

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 .

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 .

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 .

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

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.