Figure 3a ) Region of 1H - 15N - HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid : free 15N - Ub ( blue ) , UbcH7 - O - 15N - Ub ( black ) , and HHARI RING1 - bound UbcH7 - O - 15N - Ub ( red ) . Black arrow highlights perturbation upon conjugation to UbcH7 ( black arrow ) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7 . b ) Region of 1H - 13C - HSQC spectra of 13C - UbcH7 ( blue ) , 13C - UbcH7 - O - Ub ( black ) , and HHARI RING1 - bound 13C - UbcH7 - O - Ub ( red ) that includes the methyl ( 13CH3 ) resonance of the surface - exposed UbcH7 cross - over helix residue , Ala 110 ( black arrow ) is shown . The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C - UbcH7 - O - Ub ( black ) and reappears at its position in free UbcH7 in the presence of HHARI RING1 , consistent with disruption of closed UbcH7 ~ Ub conformations . Pairwise overlays and larger spectra are provided in Appendix Fig S3 . c ) Regions of 1H - 15N - HSQC - TROSY spectra of UbcH7 - O - 15N - Ub in the absence ( black ) and presence ( red ) of a RING1 domain from the RBR E3s HHARI ( left ) or RNF144 ( middle ) or a canonical RING domain of BRCA1 / BARD1 ( right ) . The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar , while binding of the canonical RING domain of BRCA1 / BARD1 has no observable effect on the Ub spectrum . Gray boxes mark area expanded in a )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7.b) Region of 1H-13C-HSQC spectra of 13C-UbcH7 (blue), 13C-UbcH7-O-Ub (black), and HHARI RING1-bound 13C-UbcH7-O-Ub (red) that includes the methyl (13CH3) resonance of the surface-exposed UbcH7 cross-over helix residue, Ala 110 (black arrow) is shown. The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C-UbcH7-O-Ub (black) and reappears at its position in free UbcH7 in the presence of HHARI RING1, consistent with disruption of closed UbcH7~Ub conformations. Pairwise overlays and larger spectra are provided in Appendix Fig S3.c) Regions of 1H-15N-HSQC-TROSY spectra of UbcH7-O-15N-Ub in the absence (black) and presence (red) of a RING1 domain from the RBR E3s HHARI (left) or RNF144 (middle) or a canonical RING domain of BRCA1/BARD1 (right). The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar, while binding of the canonical RING domain of BRCA1/BARD1 has no observable effect on the Ub spectrum. Gray boxes mark area expanded in a)


Figure 4b ) LEFT . Auto - ubiquitination assays were performed with wild - type HHARIRBR or an active site - dead mutant ( C357A - HHARIRBR ) . Products were visualized by western blotting against GST . The zero time point was taken immediately prior to ATP addition , all other times are post - ATP addition . RIGHT . Identical assays as shown on left were performed with the UBR - hybrid . The active site - dead mutant ( C357A ) retains substantial auto - ubiquitination activity signifying that UbcH5 ~ Ub is able to transfer Ub directly onto the GST - UBR - hybrid construct , bypassing the RING2 active site Cys .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.


Figure 5a ) LEFT . E3 auto - ubiquitination assays were performed using various Ub mutants ( I44A , R42A , Q49A , Q49E , and V70A ) and the RBR E3s HHARIRBR ( left ) and ParkinRBR ( right ) . Products were visualized by western blotting against HA - Ub . Samples were analyzed 30 minutes after ATP addition . c ) UbcH7 ~ Ub conjugates were preformed with either with WT - Ub or I44A - Ub . After the addition of apyrase to quench the charging reaction , UbcH7 ~ UbWT ( left ) or UbcH7 ~ UbI44A ( right ) was incubated with ParkinRBR . The disappearance of each UbcH7 ~ Ub species and appearance of auto - ubiquitinated E3 was visualized under non - reducing conditions by western blotting for HA - Ub . Time was recorded post addition of ParkinRBR . E2 ~ Ub conjugated with I44A - Ub does not disappear over the time course of the reaction . d ) UbcH7 conjugated with WT - Ub , I44A - Ub , or V70A Ub as indicated was incubated with either H887A - HOIPRBR - LDD ( left ) or H359A - HHARIRBR ( right ) mutants that allow trapping of the E3 ~ Ub thioester with WT - Ub ( Stieglitz et al , 2013 ; [ 29 ] ) . While a HOIP ~ Ub thioester species is observed when UbcH7 was charged with WT - Ub , no detectable transfer occurs with the I44A - Ub conjugate ( left ) . Similarly , V70A - Ub shows reduced formation of the HHARI ~ Ub thioester ( right ) . In addition to blotting for HA - Ub , the blot on the right was also blotted for GST - HHARI .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition.c) UbcH7~Ub conjugates were preformed with either with WT-Ub or I44A-Ub. After the addition of apyrase to quench the charging reaction, UbcH7~UbWT (left) or UbcH7~UbI44A (right) was incubated with ParkinRBR. The disappearance of each UbcH7~Ub species and appearance of auto-ubiquitinated E3 was visualized under non-reducing conditions by western blotting for HA-Ub. Time was recorded post addition of ParkinRBR. E2~Ub conjugated with I44A-Ub does not disappear over the time course of the reaction.d) UbcH7 conjugated with WT-Ub, I44A-Ub, or V70A Ub as indicated was incubated with either H887A-HOIPRBR-LDD (left) or H359A-HHARIRBR (right) mutants that allow trapping of the E3~Ub thioester with WT-Ub (Stieglitz et al, 2013;[29]). While a HOIP~Ub thioester species is observed when UbcH7 was charged with WT-Ub, no detectable transfer occurs with the I44A-Ub conjugate (left). Similarly, V70A-Ub shows reduced formation of the HHARI~Ub thioester (right). In addition to blotting for HA-Ub, the blot on the right was also blotted for GST-HHARI.


Figure 6a ) Top panel . Regions of 1H - 15N - HSQC TROSY spectra of 15N - HHARI RING2 in the absence ( black ) and presence of excess Ub ( red ) . Middle panel . Identical spectral region , but in the presence of Ub - V70A ( red ) . Bottom panel . Identical spectral region , but spectra are from a truncated HHARI RING2 construct that lacks N - terminal residues 325 - 335 ( HHARI RING2 - ΔL ) . d ) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto - ubiquitination assays . Time points ( 10min ) are visualized by western blotting for the GST - tag on HHARI . Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared . e ) 1H - 15N - HSQC TROSY spectra of 15N - Ub demonstrate binding by WT HHARI RING2 , but not by mutant HHARI RING2 constructs that exhibit decreased auto - ubiquitination activity . Overlay of 15N - Ub spectra in the absence ( black ) and presence of WT - HHARI RING2 ( red ) , W336A - HHARI RING2 ( blue ) , T341N - HHARI RING2 ( green ) , or E352A - HHARI RING2 ( purple ) . HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL).d) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto-ubiquitination assays. Time points (10min) are visualized by western blotting for the GST-tag on HHARI. Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared.e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).


figf1 ( a ) UVRAG interaction with C - Vps . 293T cells were co - transfected with Flag - UVRAG and HA - Vps16 , HA - Vps11 , HA - Vps18 , HA - Vps33 or HA - Vps39 . Whole - cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with an anti - Flag antibody , followed by immunoblotting ( IB ) with an anti - HA antibody . 1 % WCL was used as the input . ( b ) Interaction between endogenous UVRAG and C - Vps subunits . WCLs of 293T cells were used for IP with control serum ( control ) or an anti - UVRAG antibody , followed by IB with the indicated antibodies . The right panel shows endogenous protein expression . ( c ) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells . Insets highlight colocalization . ( d ) Confocal microscopy analysis of the colocalization of UVRAG with C - Vps subunits in HeLa cells transfected with Flag - UVRAG , and epitope - tagged C - Vps subunits as indicated . ( e ) Colocalization of UVRAG - C - Vps in early endosomes . HeLa cells were co - transfected with EGFP - Vps16 and Flag - UVRAG , stained with antibodies to LAMP1 , LAMP2 , EEA1 and Rab5 , followed by confocal microscopy . All images are representative of at least three independent experiments . Scale bars , 5 μm . The raw data for a and b are shown in Supplementary Information , Fig .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag-UVRAG and HA-Vps16, HA-Vps11, HA-Vps18, HA-Vps33 or HA-Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input.(b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression.(c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization.(d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag-UVRAG, and epitope-tagged C-Vps subunits as indicated.(e) Colocalization of UVRAG-C-Vps in early endosomes. HeLa cells were co-transfected with EGFP-Vps16 and Flag-UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6


figf2 ( a ) The N - terminal C2 domain or the C - terminal region of UVRAG interacts individually with Vps16 . 293T cells were co - transfected with HA - Vps16 , together with vector , Flag - UVRAG or Flag - UVRAG mutants . WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody . ( b ) HeLa cells were transfected with EGFP - Vps16 together with Flag - UVRAG mutants as indicated and stained with anti - Flag ( red ) , followed by confocal microscopy . Scale bars , 5 μm . ( c ) Interactions of the UVRAG C - terminal truncated mutants with Vps16 . At 48 h post - transfection with HA - Vps16 and Flag - UVRAG C - terminal mutants , 293T WCLs were immunoprecipitated with anti - Flag , followed by immunoblotting with anti - HA . ( d ) UVRAG C2 domain or C - terminal region deletion abolishes Vps16 binding . At 48 h post - transfection with HA - Vps16 together with Flag - UVRAG or its mutants , 293T WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA-Vps16, together with vector, Flag-UVRAG or Flag-UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.(b) HeLa cells were transfected with EGFP-Vps16 together with Flag-UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm.(c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA-Vps16 and Flag-UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA.(d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA-Vps16 together with Flag-UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.


figf3 ( a ) HeLa . Vec and HeLa . UVRAG cells transfected with HA - Vps16 and GFP - LC3 were treated with 2 μM rapamycin for 2 h , and processed for confocal microscopy ( left panel ; scale bars , 5 μm ) . The percentage of GFP - LC3 punctae positive for HA - Vps16 staining was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 01 ) . ( b ) Effects of Vps16 on UVRAG - mediated autophagosome formation . Light microscopic quantification of autophagosomes in HeLa . Vec and HeLa . UVRAG cells ( upper left panel ) or in HCT116 . Vec and HCT116 . UVRAG cells ( bottom left panel ) when transfected with GFP - LC3 together with control siRNA or Vps16 siRNA ( data are mean ± s . e . m . , n = 60 for HeLa cells ; n = 450 for HCT116 cells , * P 0 . 01 ; * * P 0 . 001 ) . Immunoblotting of Vps16 and tubulin are shown in the right panel .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA-Vps16 and GFP-LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP-LC3 punctae positive for HA-Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P 0.01).(b) Effects of Vps16 on UVRAG-mediated autophagosome formation. Light microscopic quantification of autophagosomes in HeLa.Vec and HeLa.UVRAG cells (upper left panel) or in HCT116.Vec and HCT116.UVRAG cells (bottom left panel) when transfected with GFP-LC3 together with control siRNA or Vps16 siRNA (data are mean ± s.e.m., n = 60 for HeLa cells; n = 450 for HCT116 cells, *P 0.01; **P 0.001). Immunoblotting of Vps16 and tubulin are shown in the right panel.


figf4 ( a ) UVRAG enhances the colocalization efficiency of GFP - LC3 with LAMP1 . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - LC3 were either untreated or treated with rapamycin ( 2 μM ) in the absence or presence of bafilomycin A1 ( 0 . 1 μM ) and stained for LAMP1 . Insets highlight the colocalization . The percentage of LAMP1 - positive autophagosomes was calculated in each setting ( data are mean ± s . e . m . , n = 200 ; 5 independent experiments , * * P 0 . 01 ; * P 0 . 05 ) . ( b ) UVRAG enhances the acquisition of CD63 by autophagosomes . The GFP - LC3 - transfected HeLa . Vec and HeLa . UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63 - positive autophagosomes was calculated ( data are mean ± s . e . m . , n = 200 , * * P 0 . 01 ; * P 0 . 05 ) . ( c ) UVRAG promotes autophagic degradation of long - lived proteins . HeLa . Vec and HeLa . UVRAG cells were incubated for 16 h with L - 3H - Leu ( 1 μCi ml - 1 ) . The degradation of long - lived proteins was measured at the indicated time points in complete medium ( NC ) , EBSS alone ( Starv . ) , or EBSS + 0 . 1 μM bafilomycin A1 ( Starv . + BalfA1 ) . Results ( data are mean ± s . e . m . of triplicates ) are representative of 2 independent experiments . Scale bars , 5 μm .

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figf4(a) UVRAG enhances the colocalization efficiency of GFP-LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P 0.01; *P 0.05).(b) UVRAG enhances the acquisition of CD63 by autophagosomes. The GFP-LC3-transfected HeLa.Vec and HeLa.UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63-positive autophagosomes was calculated (data are mean ± s.e.m., n = 200, **P 0.01; *P 0.05).(c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with L-3H-Leu (1 μCi ml-1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.


figf5 ( a ) Colocalization of GFP - LC3 and LAMP1 . HeLa cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 and incubated under normal conditions or treated with rapamycin ( 2 μM ) . Autophagosome fusion with LAMP + structures was analysed by confocal microscopy ( left panel ; rapamycin - treated ) and quantified ( right upper panel ; data are mean ± s . e . m . , n = 100 ) . Arrows and insets show the LAMP1 + autophagosomes . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope ( right bottom panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 05 ; * * P 0 . 0001 ) . ( b ) Vps16 or Vps18 knockdown inhibits UVRAG - mediated autophagosome maturation . HeLa . Vec ( left ) and HeLa . UVRAG ( right ) cells were transfected with GFP - LC3 together with control siRNA , Vps16 siRNA , or Vps18 siRNA , followed by confocal microscopy . Arrows in the top panel denote the autophagosomes with LAMP1 staining . Immunoblotting of Vps16 , or Vps18 and tubulin are shown in the middle panel . The percentage of LAMP1 + autophagosomes was calculated ( data are mean ± s . e . m . , n = 50 , * P 0 . 05 ) . Scale bars , 5 μm . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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figf5(a) Colocalization of GFP-LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P 0.05; **P 0.0001).(b) Vps16 or Vps18 knockdown inhibits UVRAG-mediated autophagosome maturation. HeLa.Vec (left) and HeLa.UVRAG (right) cells were transfected with GFP-LC3 together with control siRNA, Vps16 siRNA, or Vps18 siRNA, followed by confocal microscopy. Arrows in the top panel denote the autophagosomes with LAMP1 staining. Immunoblotting of Vps16, or Vps18 and tubulin are shown in the middle panel. The percentage of LAMP1+ autophagosomes was calculated (data are mean ± s.e.m., n = 50, *P 0.05). Scale bars, 5 μm. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


figf6 ( a ) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - Rab7 and RFP - LC3 were maintained either under normal conditions ( NC ) or treated with rapamycin , followed by confocal microscopy . Insets highlight the Rab7 acquisition of autophagosomes . The percentage of Rab7 + - autophagosomes was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * * * P 0 . 001 ; * * P 0 . 01 ) . Scale bars , 5 μm . ( b ) Rab7 GTPase activity is increased with UVRAG expression . At 48 h post - transfection with GFP - Rab7 together with vector , UVRAG or UVRAGΔN ( 1 - 147 ) vector , 293T WCLs were used for immunoprecipitation with anti - GFP , followed by the Rab7 GTPase activity assay . Left panel : Autoradiographs of the GTP hydrolysis products analysed by TLC . Right panel : Quantification of the percentage of GTP hydrolyzed by Rab7 ( data are mean n = 2 independent experiments ) . ( c ) Increased Rab7 - RILP interaction UVRAG is expressed . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were transfected with GFP - Rab7 together with the GST - tagged Rab7 binding domain of RILP ( GST - RILPRBD , residues 243 - 318 ) . WCLs were used for GST pulldown , followed by immunoblotting with an anti - GFP antibody . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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figf6(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-Rab7 and RFP-LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P 0.001; **P 0.01). Scale bars, 5 μm.(b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP-Rab7 together with vector, UVRAG or UVRAGΔN(1-147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments).(c) Increased Rab7-RILP interaction UVRAG is expressed. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were transfected with GFP-Rab7 together with the GST-tagged Rab7 binding domain of RILP (GST-RILPRBD, residues 243-318). WCLs were used for GST pulldown, followed by immunoblotting with an anti-GFP antibody. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


figf7 ( a ) EGF - stimulated endocytosis and post - endocytic trafficking . Uptake of Alexa Fluor 488 - EGF ( green ) by HCT116 . Vec ( left panel ) and HCT116 . UVRAG ( right panel ) cells was followed by confocal microscopy . At the indicated times , cells were fixed and stained with antibodies to LAMP1 ( red ) , and Topro - 3 ( blue , to stain the nucleus ) . Magnified images of the insets ( fourth row ) highlight trafficking of EGF towards LAMP1 - positive structures . ( b ) EGFR degradation . HCT116 . Vec , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were treated with EGF ( 200 ng ml − 1 ) at 37 ° C for the indicated times . WCLs were subjected to immunoblotting with an anti - EGFR antibody . EGFR at each time point was quantified and indicated as a percentage relative to that at time zero ( right panel ; data are mean ± s . e . m . , n = 3 ) . ( c ) DQ - green BSA dequenching assay . Representative confocal microscopy images of DQ - BSA proteolysis ( left panel ) and flow cytometry analysis ( right panel ) of RAW264 . 7 cells expressing an empty vector ( RAW264 . 7 . Vec ; dotted black line ) , wild - type UVRAG ( red line ) or UVRAGΔN ( 1 - 147 ) mutant ( green line ) loaded with 10 μg ml − 1 DQ green - BSA for the indicated times . Background ( grey line ) represents samples without the BSA load . ( d ) Effect of UVRAG on endosome fusion . RAW264 . 7 . Vec and RAW264 . 7 . UVRAG cells were pulse - labelled with Oregon green 514 - avidin and dextran - 568 for 10 min and chased for 30 min . Cells were then labelled with biotin - BSA for 10 min , and processed for confocal microscopy ( left panel ) . In the absence of biotin - BSA , Oregon green514 - avidin showed very low fluorescence ( first row of confocal image ) . The percentage of dextran - 568 - labelled endosomes with visible Oregon green staining ( denoted by arrows in the left panel ) was calculated from approximately 20 different cells in 2 independent experiments ( mean , right panel ) . Scale bars , 5 μm . The raw data of the immunoblots in b are shown in the Supplementary Information , Fig . S6 .

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figf7(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488-EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures.(b) EGFR degradation. HCT116.Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3).(c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1-147) mutant (green line) loaded with 10 μg ml−1 DQ green-BSA for the indicated times. Background (grey line) represents samples without the BSA load.(d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514-avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin-BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin-BSA, Oregon green514-avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.


Figure 1 ( B - C ) Survival ( B ) and weight loss ( C ) . N = 10 mice for each group . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . ( E ) Cytokine expression levels of Tnfa , Ifng , Il1a , and Il1b were measured by RT - qPCR normalized by Ppia . Data are presented as mean ± SEM , N = 4 mice per group . * , p < 0 . 05 by unpaired Student ' s t - test with two sided .

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Figure 1(B-C) Survival (B) and weight loss (C). N = 10 mice for each group. The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown.(E) Cytokine expression levels of Tnfa, Ifng, Il1a, and Il1b were measured by RT-qPCR normalized by Ppia. Data are presented as mean ± SEM, N = 4 mice per group. *, p < 0.05 by unpaired Student's t-test with two sided.


Figure 2 ( B - C ) Catalytically active sACE22 . v2 . 4 - IgG1 ( B ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( C ) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the sensorgrams . ( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 were aerosolized ( 7 . 5 ml protein at 8 . 3 mg / ml in 25 minutes ) and delivered by inhalation to K18 - hACE2 transgenic mice at 12 h , 48 h , and 84 h post - inoculation with SARS - CoV - 2 gamma variant . 10 mice in each group were observed for survival ( D ) and weight loss ( E ) . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . Error bars for mouse weight are centered on the mean and show SEM . Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B-C) Catalytically active sACE22.v2.4-IgG1 (B) and catalytically dead sACE22.v2.4(NN)-IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the sensorgrams.(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.


Figure 3 ( B - C ) Survival ( B ) and weight loss ( C ) following treatment with an IgG1 isotype control ( red ) versus catalytically active ( blue ) and dead ( green ) sACE22 . v2 . 4 - IgG1 . N = 8 mice for IgG1 group and catalytic active group , N = 9 for catalytic dead group . Error bars for mouse weight are centered on the mean and show SEM . Gehan - Breslow - Wilcoxon test was applied to analyze the survival curves . P = 0 . 0099 for catalytic active group compared to catalytic dead group . P = 0 . 017 for catalytic dead group compared to IgG1 control . ( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 ( D ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( E ) were immobilized on BLI biosensors and transferred to solutions of RBD ( original variant ) as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the BLI sensorgrams .

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Figure 3(B-C) Survival (B) and weight loss (C) following treatment with an IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE22.v2.4-IgG1. N = 8 mice for IgG1 group and catalytic active group, N = 9 for catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. Gehan-Breslow-Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for catalytic active group compared to catalytic dead group. P = 0.017 for catalytic dead group compared to IgG1 control.(D-E) Catalytically active sACE22.v2.4-IgG1 (D) and catalytically dead sACE22.v2.4(NN)-IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.


Figure 4 ( A ) Binding of monomeric sACE2 - 8his proteins to Expi293F cells expressing S of BA . 1 omicron was measured by flow cytometry . ( B ) Binding of dimeric sACE22 - IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID - 19 patients . Binding to Expi293F cells expressing BA . 1 omicron S was measured by flow cytometry . ( C ) Neutralization of BA . 1 omicron pseudovirus . sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) were incubated with pseudovirus for 1 h before adding to HeLa - hACE2 - 11 cells . Infection 48 h later was measured by luciferase reporter gene expression . Data are mean ± SD , N = 3 independent biological replicates . ( D - E ) Authentic BA . 1 omicron virus ( isolate USA / MD - HP20874 / 2021 ) was incubated with sACE22 - IgG1 ( D ) or sACE22 . v2 . 4 - IgG1 ( E ) for 1 h and added to Calu - 3 cells . Infection 48 h later was measured by RT - qPCR for the viral N gene . 3 µM remdesivir ( black columns ) is a positive neutralization control .

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Figure 4(A) Binding of monomeric sACE2-8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry.(B) Binding of dimeric sACE22-IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID-19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry.(C) Neutralization of BA.1 omicron pseudovirus. sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa-hACE2-11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates.(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control.


Figure 5 ( B - C ) Survival ( B ) and weight loss ( C ) . N = 8 ( IgG1 control ) or 9 ( sACE22 . v2 . 4 - IgG1 treated ) mice . Error bars for mouse weight are centered on the mean and show SEM . ( E ) Viral Spike , Nsp , and Rdrp mRNA levels in lung tissue were measured by RT - qPCR at Days 8 and 14 , normalized to Ppia transcript levels . Data are presented as mean ± SEM , N = 4 mice per group . ns , not significant ; * * , p < 0 . 01 by two - ways ANOVA , corrected by Tukey .

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Figure 5(B-C) Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM.(E) Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT-qPCR at Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; **, p < 0.01 by two-ways ANOVA, corrected by Tukey.


Figure 6 ( A ) Using flow cytometry , binding to Expi293F cells expressing S of BA . 2 omicron was measured for monomeric sACE2 - 8his proteins . ( B ) Binding of antibodies and dimeric sACE22 - IgG1 proteins to Expi293F cells expressing BA . 2 omicron S measured by flow cytometry . ( C ) BA . 2 omicron pseudovirus was incubated with sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) for 1 h and added to HeLa - hACE2 - 11 cells . Infection was measured after 48 h . ( D - E ) Binding of monomeric sACE2 - 8his proteins ( D ) and dimeric IgG1 proteins ( E ) to Expi293F cells expressing BA . 4 / BA . 5 omicron S as measured by flow cytometry .

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Figure 6(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. (B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry.(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h.(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry.


Figure 1 . ( A and B ) ARPE - 19 cells were transfected with siRNA duplexes to raptor , RagA + B , p18 , or nontarget . 60 h after transfection , cells were infected with adenovirus expressing either TFEB - FLAG - WT ( A ) or TFEB - FLAG - S211A ( B ) . 12 h later , cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against FLAG ( used to detect TFEB ) and mTOR . Bars , 10 µm . ( C ) Quantification of A . ( D ) Quantification of B . Values are means ± SD of three independent experiments . * * * , P < 0 . 001 .

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Figure 1.(A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.


Figure 2 . ( A ) ARPE - 19 cells were nucleofected with the indicated Rag - expressing plasmids . 6 h after nucleofection , cells were infected with adenovirus expressing TFEB - FLAG - WT . 12 h later , cells were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG ( used to detect TFEB - WT ) . ( B ) ARPE - 19 cells were nucleofected with the indicated Rag - and TFEB - GFP - expressing plasmids . 18 h later , cells were lysed and subjected to immunoprecipitation with the anti - HA antibody ( used to immunoprecipitate Rag proteins ) . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP ( used to detect Rag proteins and TFEB - WT , respectively ) . ( C ) ARPE - 19 coexpressing TFEB and the indicated Rag - expressing plasmids were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG ( used to detect Rag proteins and TFEB - WT , respectively ) . ( D ) Immunoblotting analysis of coimmunoprecipitated TFEB - FLAG - S211A with Rag heterodimers . IP , immunoprecipitation .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 2.(A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT).(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP-expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively).(C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively).(D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.


Figure 3 . ( A ) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin - 1 for 1 h . Cells were then lysed and subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) and actin . ( B ) HeLa cells were treated as indicated in A , and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation . Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) , Lamp1 , or Histone H3 . ( C ) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin - 1 as indicated in A . ( D ) HeLa cells were starved in serum - and amino acid - free medium ( starvation ) for 3 h and analyzed by immunoblotting with antibodies against TFEB . ( E ) HeLa cells were incubated in normal medium ( control ) and serum - and amino acid - free medium ( Starvation ) for 4 h or starved for 4 h followed by restimulation with amino acids ( starvation + amino acids ) for 30 min and analyzed by immunofluorescence with antibodies against TFEB ( endogenous TFEB is shown in green ) and Lamp1 ( red ) . The region within the dotted box is magnified in the insets . ( F ) HeLa cells were nucleofected with the indicated Rag - expressing plasmids . 18 h later , cells were lysed , and RagB / D heterodimers were pulled down using glutathione - Sepharose beads . Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST ( used to detect endogenous TFEB and Rag proteins , respectively ) . Bars , 10 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin.(B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3.(C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A.(D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB.(E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets.(F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.


Figure 4 . ( A ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - WT . 16 h later , cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 3 h . Cells were then fixed , permeabilized with 0 . 2 % saponin , and double stained with antibodies against TFEB ( used to detect recombinant TFEB ) and Lamp1 . ( B ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - S211A . 12 h later , cells were starved in serum - and amino acid - free medium for 4 h ( starvation ) or kept in complete medium ( control ) . Cells were then fixed , permeabilized with 0 . 2 % Triton X - 100 , and stained with antibodies against FLAG ( used to detect TFEB - S211A ) . ( C ) HeLa cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 4 h with the addition of Torin - 1 during the last hour of starvation . Cells were then washed , fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against TFEB ( used to detect endogenous TFEB ) and Lamp1 . Regions within the dotted boxes are magnified in the insets .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1.(B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid-free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A).(C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets.


Figure 5 . ( A and B ) ARPE - 19 cells were transfected with either active or inactive RagB / D heterodimers and infected with adenovirus expressing TFEB - FLAG - WT ( A ) or TFEB - FLAG - S211A ( B ) . After 12 h , cells were double stained with antibodies against FLAG and GST ( used to detect TFEB or Rag proteins , respectively ) . ( C ) Quantification of TFEB - WT nuclear localization from A . ( D ) Quantification of TFEB - S211A lysosomal localization from B . ( E and F ) ARPE - 19 cells were cotransfected with plasmids expressing TFEB - GFP together with inactive ( E ) or active Rag heterodimers ( F ) . Cells were then incubated with 250 nM Torin - 1 for 1 h ( E ) or starved in serum - and amino acid - free medium for 3 h ( F ) . Anti - GST antibodies were used to detect Rag proteins . Arrows point to cells that do not express Rag heterodimers .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.(A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B.(E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid-free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers.


Figure 6 . ( A ) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE - 19 cells treated with either DMSO or Torin - 1 . ( B ) ARPE - 19 cells were nucleofected with the indicated Rag - and TFEB - expressing plasmids . After 12 h , cell lysates were immunoprecipitated with the anti - FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST ( used to detect TFEB and Rag proteins , respectively ) . ( C ) FLAG - tagged TFEB - WT or TFEB deletion mutants were immunoprecipitated with the anti - FLAG antibody and analyzed by immunoblotting with antibodies against FLAG , 14 - 3 - 3 binding motif , or 14 - 3 - 3 . ( D ) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB - WT and TFEB - S3A / R4A mutant upon incubation with DMSO ( vehicle ) or 250 nM Torin - 1 for 1 h . Cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and stained with antibodies against FLAG ( used to detect TFEB ) . ( E ) ARPE - 19 cells expressing either TFEB - S211A or TFEB - S211A - Δ30 were double stained with antibodies against TFEB and Lamp1 . Regions within the dotted boxes are magnified in the insets .

O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1.(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively).(C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3.(D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB).(E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets.


Figure 7 . ( A ) ARPE - 19 cells coexpressing active RagB / D heterodimer and the indicated TFEB plasmids were fixed , permeabilized with 0 . 2 % saponin , and stained with antibodies against GST ( used to detect Rag proteins ) . Regions within the dotted boxes are magnified in the insets . Bars , 10 µm . ( B ) ARPE - 19 cells were cotransfected with active RagB / D heterodimers and the indicated TFEB constructs . After 18 h , cell lysates were immunoprecipitated with the anti - HA antibody ( used to immunoprecipitate Rag proteins ) and immunoblotted with antibodies against GFP and GST ( used to detect TFEB - GFP and Rag proteins , respectively ) . The white line indicates that intervening lanes have been spliced out . ( C ) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB - ( 1 - 30 ) - GFP in ARPE - 19 cells coexpressing active RagB / D heterodimers ( antibodies against GST were used to detect Rags ) . Bar , 4 µm . ( D ) Relative RT - PCR analysis of the mRNA expression of autophagy ( ATG9B and UVRAG ) and lysosomal ( MCOLN1 ) genes in ARPE - 19 cells infected with the indicated adenovirus for 48 h . The values are expressed as a ratio to RNA from cells infected with control adenovirus ( Ad . - Null ) . Values are means ± SD of two independent experiments . * * * , P < 0 . 001 ; * * , P < 0 . 01 . ( E ) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies . IP , immunoprecipitation .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm.(B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out.(C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1-30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm.(D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01.(E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.


Figure 8 . ( B - D ) ARPE - 19 cells were infected with adenovirus expressing MITF1 - FLAG . 16 h later , cells were incubated with DMSO or 250 nM Torin - 1 for 1 h and analyzed by immunoblotting ( C ) or immunofluorescence ( B and D ) with anti - FLAG antibodies . The region within the dotted box is magnified in the insets . Bars : ( B and D , main images ) 10 µm ; ( D , insets ) 5 µm . ( E ) ARPE - 19 cells were incubated with 250 nM Torin - 1 for 1 h . Cells were then lysed and analyzed by immunoblotting with antibodies against MITF . ( F ) HEK - 293T cells were incubated in medium containing DMSO or Torin - 1 for 1 h . Cells were lysed , and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation . Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF , Lamp1 , and Histone H3 . ( G ) ARPE - 19 cells expressing active ( RagBGTP / RagDGDP ) or inactive ( RagBGDP / RagDGTP ) Rag heterodimers were immunoprecipitated with the anti - FLAG antibody and immunoblotted with antibodies against FLAG and GST ( used to detect MITF and Rag proteins , respectively ) . ( H ) HEK - 293T cells expressing active ( RagBGTP / RagDGDP ) or inactive ( RagBGDP / RagDGTP ) Rag heterodimers were pulled down using glutathione - Sepharose beads and immunoblotted with antibodies against GST and MITF ( used to detect Rag proteins and endogenous MITF , respectively ) . IP , immunoprecipitation .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8.(B-D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm.(E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF.(F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3.(G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively).(H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione-Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.


Figure 9 . ( A and B ) ARPE - 19 cells were nucleofected with the indicated Rag - and MITF - expressing plasmids . 12 h later , cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against FLAG and GST ( used to detect MITF and Rag proteins , respectively ) . Bars , 10 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9.(A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm.


Figure 1 ( C ) Electrophoretic mobility - shift assay ( EMSA ) with biotin - labeled RORA consensus , rs2978980T or rs2978980G probes and HGC - 27 or MGC80 - 3 nuclear extracts . HGC - 27 or MGC80 - 3 left panel : EMSA with RORA consensus or rs2978980T probes . Lane 1 - 8 : from the first lane at the left side to the right side . Lanes 4 and 8 , probe only ; lanes 2 and 6 , probe and nuclear extracts ; lanes 1 , 3 , 5 and 7 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 1 and 5 ) or RORA consensus probes ( lanes 3 and 7 ) . HGC - 27 or MGC80 - 3 right panel : EMSA with rs2978980T or rs2978980G probes . Lanes 1 and 6 , probe only ; lanes 2 and 7 , probe and nuclear extracts ; lanes 3 - 5 and 8 - 10 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 5 and 8 ) , rs2978980G ( lanes 3 and 10 ) or RORA consensus probes ( lanes 4 and 9 ) . ( F ) Suppression of RORA using siRNAs ( siRORA - 1 and siRORA - 2 ) could markedly down - regulate lncPSCA expression in MGC80 - 3 cells but not in HGC - 27 cells . In detail , MGC80 - 3 and HGC - 27 cells were transfected with siRORA - 1 and siRORA - 2 , lncPSCA and RORA expression levels were measured by RT - qPCR and Western Blot .

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

Figure 1  (C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).   (F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.


Figure 2 ( E ) lncPSCA - knockout significantly promoted growth of MGC80 - 3 ( n = 8 ) or HGC - 27 ( n = 7 ) xenografts compared with control xenografts after 27 or 18 days . ( F ) HE and Ki67 staining of MGC80 - 3 or HGC - 27 xenografts . Scale bar = 100 μm .

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Figure 2 (E) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.  (F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.


Figure 3 ( B ) lncPSCA inhibits invasion abilities of MGC80 - 3 and HGC - 27 cells . Cells on the lower surface of the chamber were stained by crystal violet . Scale bar = 100 μm . ( C ) In MGC80 - 3 and HGC - 27 cells , expression changes of different markers ( β - catenin , ZO - 1 , SNAIL and VIMENTIN ) of epithelial - to - mesenchymal transition were examined after overexpression or knockout of lncPSCA .

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

Figure 3 (B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.  (C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.


Figure 4 ( F ) Gastric cancer cells after lncPSCA - knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide ( CHX ) or vehicle for the indicated periods of time . DDX5 levels were analyzed by Western blot . ( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . ( J ) Silencing of DDX5 with shRNA ( shD ) significantly reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . (

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

Figure 4 (F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot.  (I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (J) Silencing of DDX5 with shRNA (shD) significantly reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (


Figure 5 ( E ) RNA Pol II ChIPseq and RNAseq signals of PMAIP1 , CDKN1A , SESN2 and SESN3 in HGC - 27 cells . Red bars represent the RNA Pol II ChIPseq signal . Orange or blue bars represent the RNAseq signals of HGC - 27 cells overexpressing lncPSCA ( lncP ) or with silenced DDX5 ( shD ) , respectively . ( F ) Validation of candidate downstream genes of the lncPSCA - DDX5 axis identified through the integrated analyses . qRT - PCR was performed for PMAIP1 , CDKN1A and SESN2 genes upon overexpression of lncPSCA ( lncP ) , silencing of DDX5 ( shD ) or lncPSCA konckout . Error bars indicate the SEM . Data show one representative example of three biological replicates .

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

Figure 5 (E) RNA Pol II ChIPseq and RNAseq signals of PMAIP1, CDKN1A, SESN2 and SESN3 in HGC-27 cells. Red bars represent the RNA Pol II ChIPseq signal. Orange or blue bars represent the RNAseq signals of HGC-27 cells overexpressing lncPSCA (lncP) or with silenced DDX5 (shD), respectively.  (F) Validation of candidate downstream genes of the lncPSCA-DDX5 axis identified through the integrated analyses. qRT-PCR was performed for PMAIP1, CDKN1A and SESN2 genes upon overexpression of lncPSCA (lncP), silencing of DDX5 (shD) or lncPSCA konckout. Error bars indicate the SEM. Data show one representative example of three biological replicates.


Figure 1A . ( Top ) USP44 transcript levels across CD4 + T cell populations . Suspension of the lymph node and spleen cells of 6 - 8 week old female C57BL / 6 mice ( n = 5 experiments ) were obtained , and naïve ( CD62Lhigh / CD25 - ) CD4 + T cells and nTregs ( CD4 + / CD25high ) were obtained by FACS . The indicated effector T cell populations were generated by activating 1x10 ^ 6 naïve CD4 + T precursors with anti - CD3 and anti - CD28 antibodies ( 1 and 4 ug / ml , respectively ) for 4 days under the indicated in vitro skewing conditions ( described in the Methods section ) . After harvesting RNA by Trizol reagent and generating cDNA , the Usp44 mRNA levels expressed by the resulting Teff and iTreg , as well as freshly isolated nTreg cells were determined by qRT - PCR . ( Bottom ) Suspension of lymph node and spleen cells of 6 - 8 week old female C57BL / 6 mice were obtained , and naïve ( CD62LhighCD25 - ) CD4 + T cells and nTregs ( CD4 + / CD25high ) were sorted by FACS . iTregs were generated by activating 1x10 ^ 6 naïve CD4 + T cells with anti - CD3 and anti - CD28 antibodies ( 1 and 4 ug / ml , respectively ) for 4 days in the presence of IL - 2 ( 100u / ml ) and TGF - β ( 5ng / ml ) before the cells were harvested for SDS - PAGE . Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH . B . Naïve murine CD4 + T cells were obtained and differentiated into the iTreg lineage as in A . Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT - PCR . C . Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured . After 1 - 4 days of iTreg skewing , cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies . D . Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα , IL - 6 , or IL - 1β ( each at a 10 ng / ml concentration ) , or LPS ( 1µg / ml ) for 24 hours . Treated Tregs were then harvested and USP44 protein levels were determined as above .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. (Top) USP44 transcript levels across CD4+ T cell populations. Suspension of the lymph node and spleen cells of 6-8 week old female C57BL/6 mice (n= 5 experiments) were obtained, and naïve (CD62Lhigh/CD25-) CD4+ T cells and nTregs (CD4+/CD25high) were obtained by FACS. The indicated effector T cell populations were generated by activating 1x10^6 naïve CD4+ T precursors with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days under the indicated in vitro skewing conditions (described in the Methods section). After harvesting RNA by Trizol reagent and generating cDNA, the Usp44 mRNA levels expressed by the resulting Teff and iTreg, as well as freshly isolated nTreg cells were determined by qRT-PCR. (Bottom) Suspension of lymph node and spleen cells of 6-8 week old female C57BL/6 mice were obtained, and naïve (CD62LhighCD25-) CD4+ T cells and nTregs (CD4+/CD25high) were sorted by FACS. iTregs were generated by activating 1x10^6 naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days in the presence of IL-2 (100u/ml) and TGF-β (5ng/ml) before the cells were harvested for SDS-PAGE. Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH.B. Naïve murine CD4+ T cells were obtained and differentiated into the iTreg lineage as in A. Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT-PCR.C. Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured. After 1-4 days of iTreg skewing, cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies.D. Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα, IL-6, or IL-1β (each at a 10 ng/ml concentration), or LPS (1µg/ml) for 24 hours. Treated Tregs were then harvested and USP44 protein levels were determined as above.


Figure 2A . HEK293T cells were transfected with MYC - FOXP3 and FLAG - USP44 encoding expression constructs using Polyethylenimine . 48hrs post - transfection , cells were harvested , lysed , and anti - FLAG or anti - MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner . Co - IP ' ed proteins were subjected to SDS PAGE followed by immunoblot analysis . Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3 , respectively . B . Endogenous co - IP of USP44 and FOXP3 in murine iTregs . iTregs were generated as in Fig . 1 from naïve CD4 + T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL / 6 mice ( n = 2 - 3 / experiment ) . iTregs were lysed and key proteins were immunoprecipitated using either anti - USP44 ( right panel ) or anti - FOXP3 ( left panel ) antibody . Proteins pulled - down in this experiment were then resolved and analyzed by immunoblot using anti - FOXP3 or anti - USP44 antibodies . C . Endogenous co - IP of USP44 and FOXP3 in murine nTregs . nTregs ( CD4 + CD25high ) isolated by FACS were activated by anti - CD3 and anti - CD28 ( 1 and 4 ug / ml , respectively ) overnight in the presence of IL - 2 ( 100 U / ml ) . The cells were lysed and proteins were immunoprecipitated using either anti - Foxp3 ( left panel ) or anti - Usp44 ( right panel ) . Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies . D . Naïve murine CD4 + T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl / fl CD4Cre + mice and that of their wild type littermates ( USP44fl / fl CD4Cre - mice ; n = 2 - 3 / group / experiment ) . iTreg cells were generated from these mice as described for Fig . 1 before incubation on a microscope slide pre - coated with poly - L lysine for 1h . Adhered cells were then fixed by PFA for 0 . 5 followed by blocking with 1 % BSA for 1h , then incubation with the specified antibodies . Representative confocal microscopy images ( 40X ) were visualized for endogenous USP44 ( red ) and FOXP3 Baxter et al ( ) . DAPI was used to visualize cell nuclei ( blue ) ; scale bar 50μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. HEK293T cells were transfected with MYC-FOXP3 and FLAG-USP44 encoding expression constructs using Polyethylenimine. 48hrs post-transfection, cells were harvested, lysed, and anti-FLAG or anti-MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner. Co-IP'ed proteins were subjected to SDS PAGE followed by immunoblot analysis. Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3, respectively.B. Endogenous co-IP of USP44 and FOXP3 in murine iTregs. iTregs were generated as in Fig. 1 from naïve CD4+ T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL/6 mice (n=2-3/experiment). iTregs were lysed and key proteins were immunoprecipitated using either anti-USP44 (right panel) or anti-FOXP3 (left panel) antibody. Proteins pulled-down in this experiment were then resolved and analyzed by immunoblot using anti-FOXP3 or anti-USP44 antibodies.C. Endogenous co-IP of USP44 and FOXP3 in murine nTregs. nTregs (CD4+CD25high) isolated by FACS were activated by anti-CD3 and anti-CD28 (1 and 4 ug/ml, respectively) overnight in the presence of IL-2 (100 U/ml). The cells were lysed and proteins were immunoprecipitated using either anti-Foxp3 (left panel) or anti-Usp44 (right panel). Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies.D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.


Figure 3A . HEK293T cells were transfected with expression constructs encoding MYC - FOXP3 , His - Ubiquitin , and either a wild type or catalytically inactive ( C282S , " cs " ) version of USP44 tagged with FLAG . Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis . Pull - down of His - labeled proteins using Ni - NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC . B . Effect of USP44 knock down on FOXP3 ubiquitination . Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells . After selected for 7d , cells were transfected with MYC - FOXP3 and His - Ubiquitin , then treated with 20 μM MG132 for 4 hrs before harvest and lysis . Ubiquitinated FOXP3 proteins were visualized as in A . C . Levels of polyubiquitinated FOXP3 in the presence or absence of USP44 . As in Fig 2b , murine iTregs were generated from naïve CD4 + precursors isolated from wild type and mice globally deficient in USP44 - / - mice ( n = 3 / group / experiment ) . Ubiquitinated proteins were extracted from iTreg lysates with anti - ubiquitin antibodies ( anti - Ubi ) prior to resolution by SDS PAGE and immunoblot analysis , probing for FOXP3 . D , E . HEK293T cells were transfected with plasmids encoding MYC - FOXP3 , FLAG - USP44 ( either wild type USP44 , " wt " or the C282S mutant ) . These cells were subsequently treated with cycloheximide ( CHX ; 5μg / ml ) for the indicated time points before harvested and cell lysis . FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti - MYC antibodies . F , USP44 knock down in human Tregs . Naïve CD4 + T cells ( CD4 + CD25 - CD45RA + ) were isolated from the peripheral blood of healthy donors by FACS , and iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot ( F ) G . USP44 knock down in human Tregs . Naïve CD4 + T cells ( CD4 + CD25 - CD45RA + ) were isolated from the peripheral blood of healthy donors by FACS , and iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . FOXP3 expression was assessed by intracellular staining followed by flow cytometry ( G ) . H . Murine naïve CD4 T cells ( CD62L + / CD25 - / CD4 + ) from WT and USP44 - / - mice ( n = 2 - 3 / group / experiment ) were FACS purified and activated in vitro for four days by CD3 / CD28 cross - linking antibodies ( 1ug and 4ug / ml , respectively ) in the presence of 100 U / ml IL - 2 and the indicated dose of TGF - β . Down - regulation of FOXP3 was observed in USP44 - / - mice by intracellular immunostaining and flow cytometry .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC.B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A.C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3.D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies.F, USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot (F)G. USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. FOXP3 expression was assessed by intracellular staining followed by flow cytometry (G).H. Murine naïve CD4 T cells (CD62L+/CD25-/CD4+) from WT and USP44-/- mice (n=2-3/group/experiment) were FACS purified and activated in vitro for four days by CD3/CD28 cross-linking antibodies (1ug and 4ug/ml, respectively) in the presence of 100 U/ml IL-2 and the indicated dose of TGF-β. Down-regulation of FOXP3 was observed in USP44-/- mice by intracellular immunostaining and flow cytometry.


Figure 4A . Reciprocal Co - IP of USP44 and USP7 . HEK293T cells were transfected with constructs encoding MYC - USP44 and FLAG - USP7 . These cells were lysed and pertinent factors were immunoprecipitated using either anti - FLAG or anti - MYC . Pulled - down proteins were observed by immunoblot analysis using either anti - MYC or anti - FLAG antibodies . B . Effect of USP44 on USP7 levels . HEK293T cells were transfected with MYC - FOXP3 . Cells also received combinations of FLAG - USP7 and varying doses of pLVX - USP44 encoding plasmids . FOXP3 was immunoprecipitated using anti - MYC , and immunoblots were analyzed using anti - FLAG , anti - USP44 or anti - MYC antibodies . C . Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination . HEK293T cells were transfected with plasmids encoding MYC - FOXP3 , either FLAG - USP44 or FLAG - USP7 or both , His - ubiquitin . Cells were treated with 20 μM MG132 for 4 hrs before being harvested , lysed , and incubated on Ni - NTA beads to pull down ubiquitinated FOXP3 , which was visualized by immunoblotting using antibodies specific for MYC . D . ( left panel ) iTreg were polarized from human naïve CD4 + T cells over 7 days ( n = 3 donors / experiment ) receiving control ( shCK ) , or shRNA knockdown constructs targeting USP44 ( shUSP44 - 1 , - 2 ) , or USP7 ( shUSP7 - 1 , - 2 ) , or both shRNA constructs . Freshly isolated nTregs were treated with either control or shRNA constructs . After transduction with lentivirus for 48 hours , cells were harvested and run SDS - PAGE gels . Protein levels were visualized by immunoblotting using antibodies specific for FOXP3 , USP44 , USP7 and GAPDH . Shown are representative findings from 3 independent experiments ( biological replicates ) .

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Figure 4A. Reciprocal Co-IP of USP44 and USP7. HEK293T cells were transfected with constructs encoding MYC-USP44 and FLAG-USP7. These cells were lysed and pertinent factors were immunoprecipitated using either anti-FLAG or anti-MYC. Pulled-down proteins were observed by immunoblot analysis using either anti-MYC or anti-FLAG antibodies.B. Effect of USP44 on USP7 levels. HEK293T cells were transfected with MYC-FOXP3. Cells also received combinations of FLAG-USP7 and varying doses of pLVX-USP44 encoding plasmids. FOXP3 was immunoprecipitated using anti-MYC, and immunoblots were analyzed using anti-FLAG, anti-USP44 or anti-MYC antibodies.C. Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, either FLAG-USP44 or FLAG-USP7 or both, His-ubiquitin. Cells were treated with 20 μM MG132 for 4 hrs before being harvested, lysed, and incubated on Ni-NTA beads to pull down ubiquitinated FOXP3, which was visualized by immunoblotting using antibodies specific for MYC.D. (left panel) iTreg were polarized from human naïve CD4+ T cells over 7 days (n=3 donors /experiment) receiving control (shCK), or shRNA knockdown constructs targeting USP44 (shUSP44-1, -2), or USP7 (shUSP7-1, -2), or both shRNA constructs. Freshly isolated nTregs were treated with either control or shRNA constructs. After transduction with lentivirus for 48 hours, cells were harvested and run SDS-PAGE gels. Protein levels were visualized by immunoblotting using antibodies specific for FOXP3, USP44, USP7 and GAPDH. Shown are representative findings from 3 independent experiments (biological replicates).