The symptoms of infection after BSMV inoculation of a different N . benthamiana plant at 8 dpi . Magnified images of leaves ( in white boxes ) are shown in the lower panels . Scale bars , 2 cm . Statistical analysis of symptom appearance in different N . benthamiana plants after inoculation with BSMV . Western blot analysis of the virus accumulation in Figure 4D with anti - TGB1 antibody . RbcL served as loading control . Data information : , representative data are shown and three eplicates similar results . In ( E ) , error bars indicate mean ± SD from three independent experiments .

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The symptoms of infection after BSMV inoculation of a different N. benthamiana plant at 8 dpi. Magnified images of leaves (in white boxes) are shown in the lower panels. Scale bars, 2 cm.   Statistical analysis of symptom appearance in different N. benthamiana plants after inoculation with BSMV.   Western blot analysis of the virus accumulation in Figure 4D with anti-TGB1 antibody. RbcL served as loading control.   Data information: , representative data are shown and three eplicates similar results. In (E), error bars indicate mean ± SD from three independent experiments.


Left panel : systemic symptoms of N . benthamiana plants inoculated with BSMV and BSMV γb mutants . Photographs were taken at 7 dpi . Right panel : schematic representation of inoculated ( ino . ) , older systemic leaves ( o - SL ) , and younger systemic leaves ( y - SL ) in N . benthamiana plants . Scale bars , 2 cm .

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Left panel: systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV γb mutants. Photographs were taken at 7 dpi. Right panel: schematic representation of inoculated (ino.), older systemic leaves (o-SL), and younger systemic leaves (y-SL) in N. benthamiana plants. Scale bars, 2 cm.


Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N . benthamiana leaves . Anti - TGB1 and anti - CP were used for protein detection . Data information : , representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N. benthamiana leaves. Anti-TGB1 and anti-CP were used for protein detection. Data information: , representative data are shown, and three replicates had similar results. RbcL served as loading control.


Western blot detection of BSMV with anti - CP antibodies in upper N . benthamiana leaves at 7 days ( o - SL ) or 28 days ( y - SL ) after infiltration . o - SL , older systemic leaves ; y - SL , younger systemic leaves . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Western blot detection of BSMV with anti-CP antibodies in upper N. benthamiana leaves at 7 days (o-SL) or 28 days (y-SL) after infiltration. o-SL, older systemic leaves; y-SL, younger systemic leaves. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants . A . tumefaciens harboring the indicated plasmids were infiltrated into N . benthamiana leaves . Note : Agrobacterium containing NbNTRC constructs were diluted 5 - fold compared to normal use ( OD600 = 0 . 3 ) . BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1 . 5 dpi . Three days later , Western blots were conducted with anti - TGB1 and anti - Flag antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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

Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants. A. tumefaciens harboring the indicated plasmids were infiltrated into N. benthamiana leaves. Note: Agrobacterium containing NbNTRC constructs were diluted 5-fold compared to normal use (OD600 = 0.3). BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blots were conducted with anti-TGB1 and anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Left panel : Western blot detection of BSMV CP accumulation in N . benthamiana leaves inoculated with BSMV and γb movement deficient mutants . Leaf samples were collected from different regions of the same leaf ( right panel ) Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Left panel: Western blot detection of BSMV CP accumulation in N. benthamiana leaves inoculated with BSMV and γb movement deficient mutants. Leaf samples were collected from different regions of the same leaf (right panel) Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of γb and γb mutants on 2 - Cys Prx in N . benthamiana . Different combinantions of the pGD empty vector ( EV ) or γb and γb mutants are indicated above the panel . NbNTRC and γb proteins were detected by Western blotting with anti - Flag and anti - γb antibodies under reducing conditions , Western blot analyses of 2 - Cys Prx abundance with anti - 2 - Cys Prx antibody under non - reducing conditions . The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Effects of γb and γb mutants on 2-Cys Prx in N. benthamiana. Different combinantions of the pGD empty vector (EV) or γb and γb mutants are indicated above the panel. NbNTRC and γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies under reducing conditions, Western blot analyses of 2-Cys Prx abundance with anti-2-Cys Prx antibody under non-reducing conditions. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


BiFC assays to identify interactions between NbNTRC and 2 - Cys Prx in N . benthamiana during BSMV infection . NbNTRC - YFPc and 2 - Cys Prx - YFPn were co - expressed with BSMV and γb mutant derivatives as indicated on the left . The EV was used as negative control . Scale bars , 30 μm . Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software . The NbNTRC - 2 - Cys Prx interaction was normalized to that of the EV control , which was set to 1 . 0 . Data information : Representative data were shown in and three replicates had similar results . In B , error bars indicate the mean ± SEM , and a and b indicate statistically significant differences among different groups ( n = 5 , ANOVA , p - value < 0 . 05 ) .

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BiFC assays to identify interactions between NbNTRC and 2-Cys Prx in N. benthamiana during BSMV infection. NbNTRC-YFPc and 2-Cys Prx-YFPn were co-expressed with BSMV and γb mutant derivatives as indicated on the left. The EV was used as negative control. Scale bars, 30 μm.   Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software. The NbNTRC-2-Cys Prx interaction was normalized to that of the EV control, which was set to 1.0.   Data information: Representative data were shown in and three replicates had similar results. In B, error bars indicate the mean ± SEM, and a and b indicate statistically significant differences among different groups (n = 5, ANOVA, p-value < 0.05).


Split - LUC assays to determine NbNTRC - 2 - Cys Prx interactions in N . benthamiana during BSMV infection . NbNTRC - cLUC and 2 - Cys Prx - nLUC were co - expressed in N . benthamiana leaves infected with the indicated BSMV derivatives , Bioluminescence was observed at 3 dpi . The white dashed circles indicate the infiltrated regions . Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software . Data information : Representative data were shown , and three replicates had similar results . In ( D ) , error bars indicate mean ± SEM ( n = 6 ) . c indicate statistically significant differences among different groups ( ANOVA , p - value < 0 . 05 ) .

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Split-LUC assays to determine NbNTRC-2-Cys Prx interactions in N. benthamiana during BSMV infection. NbNTRC-cLUC and 2-Cys Prx-nLUC were co-expressed in N. benthamiana leaves infected with the indicated BSMV derivatives, Bioluminescence was observed at 3 dpi. The white dashed circles indicate the infiltrated regions.   Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software.   Data information: Representative data were shown , and three replicates had similar results. In (D), error bars indicate mean ± SEM (n = 6). c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05).


Competitive GST pull - down assays showing the effects of γb on interactions between NbNTRC and 2 - Cys Prx . GST - NbNTRC and 2 - Cys Prx - His were incubated with increasing amounts ( 5 , 10 , 20 , and 40 μg ) of γb and its mutant derivatives . Glutathione - Sepharose beads were washed and Western blot analysis was performed using anti - GST and anti - His antibodies . Data information : representative data are shown , and three independent experiments had similar results .

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Competitive GST pull-down assays showing the effects of γb on interactions between NbNTRC and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot analysis was performed using anti-GST and anti-His antibodies. Data information: representative data are shown, and three independent experiments had similar results.


Analysis of NbNTRC - 2 - Cys Prx interactions in the presence of the γb protein . NbNTRC - 3xFlag and 2 - Cys Prx - GFP were co - expressed in N . benthamiana leaves . Total proteins were extracted at 4 dpi and immunoprecipitated with anti - Flag beads , co - incubated with increasing amounts ( 20 , 40 , and 60 μg ) of γb - His and γb - His mutant derivatives . Input and IP proteins were analyzed by Western blotting with anti - His , anti - GFP or anti - Flag antibodies . The uncleaved 2 - Cys Prx with cTP and cleaved 2 - Cys Prx without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three independent experiments had similar results .

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Analysis of NbNTRC-2-Cys Prx interactions in the presence of the γb protein. NbNTRC-3xFlag and 2-Cys Prx-GFP were co-expressed in N. benthamiana leaves. Total proteins were extracted at 4 dpi and immunoprecipitated with anti-Flag beads, co-incubated with increasing amounts (20, 40, and 60 μg) of γb-His and γb-His mutant derivatives. Input and IP proteins were analyzed by Western blotting with anti-His, anti-GFP or anti-Flag antibodies. The uncleaved 2-Cys Prx with cTP and cleaved 2-Cys Prx without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three independent experiments had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N . benthamiana epidermal cells . The pCB301 empty vector ( EV ) was used as a negative control . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of LRSV - dependent changes in Chl - HyPer2 fluorescence at 3 dpi . Data information : , data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.   Data information: , data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot analyses of 2 - Cys Prx abundance in EV and LRSV inoculated leaves under non - reducing conditions at 3 dpi . RbcL served as a loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analyses of 2-Cys Prx abundance in EV and LRSV inoculated leaves under non-reducing conditions at 3 dpi. RbcL served as a loading control. Data information: representative data are shown, and three replicates had similar results.


Western blot analyses to detect LRSV γb in NbNTRC - OE and NbNTRC - KO N . benthamiana plants challenged with LRSV at 8 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analyses to detect LRSV γb in NbNTRC-OE and NbNTRC-KO N. benthamiana plants challenged with LRSV at 8 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N . benthamiana epidermal cells . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of the TYMV - dependent changes in Chl - HyPer2 fluorescence at 5 dpi . Data information : data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of the TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.   Data information: data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot analysis of 2 - Cys Prx abundance in EV or TYMV inoculated leaves under non - reducing conditions at 5 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analysis of 2-Cys Prx abundance in EV or TYMV inoculated leaves under non-reducing conditions at 5 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


RT - qPCR analysis of TYMV RNA accumulation at 8 dpi . NbEF1α served as a internal control . Data information : representative data are shown , and three replicates had similar results .

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RT-qPCR analysis of TYMV RNA accumulation at 8 dpi. NbEF1α served as a internal control. Data information: representative data are shown, and three replicates had similar results.


( a , b ) Dual positive cells are more prevalent in epithelial organs and cells . ( a ) Proportion of ACE2 + TMPRSS2 + cells ( y axis ) per dataset ( dots ) from 21 tissues and organs ( rows ) .

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(a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows).


( e - i ) In situ validation of dual positive cells in the lung , airways , and submucosal gland . ( e - g ) PLISH and immunostaining in human adult lung alveoli for ( e ) ACE2 ( white ) , TMPRSS2 ( green ) and CTSL ( red ) , ( f ) ACE2 ( white ) , TMPRSS2 ( green ) and HTII - 280 ( red ) , ( g ) ACE2 ( red ) and Pro - SFTPC ( green ) . ( h ) PLISH and immunostaining in human large airway submucosal glands . ACE2 ( red ) , ACTA2 ( green ) and DAPI ( blue ) . ( i ) PLISH in human adult large airway . ACE2 ( green ) and DAPI ( blue ) .

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(e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).


( a , c ) Single - cell ATAC - seq of lung samples from primary carina ( 1C ) and subpleural parenchyme ( RPL ) ( n = 1 patient , k = 3 samples , 3 , 366 cells from 1C , 8 , 340 cells from RPL ) . Uniform Manifold Approximation and Projection ( UMAP ) embedding of scATAC - seq profiles ( dots ) colored by ( a , left ) cell types , ( a , right ) cells with at least 1 fragment ( indicating accessibility , open chromatin ) mapping to the ACE2 gene locus ( defined as − 2kb upstream the Transcription Start Site to Transcription End Site ) , or by sample

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(a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location


( g ) Low expression in pediatric samples . Mean expression level ( log CPM , y axis ) of ACE2 ( blue ) , TMPRSS2 ( orange ) , and CTSL ( green ) across age bins ( x axis ) in AT2 ( left ) and ciliated ( right ) cells . Pediatric samples : 0 - 10 years . Samples from past or current smokers were removed from this plot to avoid smoking confounders . Error bars are omitted due to y - axis limitations . They are typically 10 - fold the mean value ( Supplementary Table 5 ) . Multiciliated and AT2 cells are shown as these cell types are present in fetal data , and show significant age associations with ACE2 expression .

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(g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression.


( a ) Gradual increase in Ace2 expression by airway epithelial cell type with age . Mean expression ( y axis ) of Ace2 in different airway epithelial cells ( x axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( standard error of the mean ( SEM ) ) .

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(a) Gradual increase in Ace2 expression by airway epithelial cell type with age. Mean expression (y axis) of Ace2 in different airway epithelial cells (x axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (standard error of the mean (SEM)).


( b ) Increase in proportion of Ace2 + Ctsl + goblet and club cells with age . Percent of Ace2 + Ctsl + cells ( x axis ) in different airway epithelial cell types ( y axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( SEM ) .

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(b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM).


( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( c , d ) UMAP of scRNA - seq profiles ( dots ) colored by experimental group ( c ) or by Ace2 + cells and indicated double positive cells ( d ) . Alveolar epithelial cells ( AT1 and AT2 ) and airway epithelial secretory and ciliated cells are marked .

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

(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (c,d) UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2+ cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked.


( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( e ) The relative frequency of Ace2 + cells is increased by smoking in airway secretory cells but not AT2 cells . Relative proportion ( y axis ) of Ace2 + ( red ) and Ace2 - ( grey ) cells in smoking and control mice of different cell types ( x axis ) .

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

(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (e) The relative frequency of Ace2+ cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2+ (red) and Ace2- (grey) cells in smoking and control mice of different cell types (x axis).


( f , g ) Expression of Ace2 is increased in airway secretory cells , but not in AT2 cells . Distribution of Ace2 expression ( y axis ) in secretory ( f ) and AT2 ( g ) cells from control and smoking mice ( x axis ) .

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

(f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis).


( h - j ) Re - analysis of published bulk mRNA - Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of ( h ) Ace2 , ( i ) Tmprss2 , and ( j ) Ctsl after five months of chronic exposure .

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

(h-j) Re-analysis of published bulk mRNA-Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure.


( k ) Expression in placenta . UMAP plots showing expression of Ace2 , Tmprss2 and Ctsl in single and double positive cells from embryonic days 9 . 5 to 18 ( left ) and embryonic day 14 . 5 ( right ) of mouse placenta

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

(k) Expression in placenta. UMAP plots showing expression of Ace2, Tmprss2 and Ctsl in single and double positive cells from embryonic days 9.5 to 18 (left) and embryonic day 14.5 (right) of mouse placenta development


( a ) Multiple proteases are co - expressed with ACE2 in human lung scRNA - seq . Scatter plot of significance ( y axis , - log10 ( adjusted P value ) ) and effect size ( x axis ) of co - expression of each protease gene ( dot ) with ACE within each indicated epithelial cell type ( color ) . Dashed line : significance threshold . TMPRSS2 and PCSKs that significantly co - expressed with ACE2 are marked .

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

(a) Multiple proteases are co-expressed with ACE2 in human lung scRNA-seq. Scatter plot of significance (y axis, -log10(adjusted P value)) and effect size (x axis) of co-expression of each protease gene (dot) with ACE within each indicated epithelial cell type (color). Dashed line: significance threshold. TMPRSS2 and PCSKs that significantly co-expressed with ACE2 are marked.


A CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( - ) or stimulated for 2 min with anti - CD3 and anti - CD4 antibodies ( + ) . Equal amounts of cell lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads , followed by elution of proteins with D - biotin . Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL . Equal amounts of proteins from whole cell lysates ( WCL ) were also probed for CBLB and CBL . Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N - terminus ( http : / / www . uniprot . org / uniprot / Q3TTA7 ) . Also shown are loading control corresponding to the same immunoblot probed with anti - CD5 , and molecular masses ( kDa ) . Data are representative of at least three experiments .

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

A CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 2 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin. Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL. Equal amounts of proteins from whole cell lysates (WCL) were also probed for CBLB and CBL. Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N-terminus (http://www.uniprot.org/uniprot/Q3TTA7). Also shown are loading control corresponding to the same immunoblot probed with anti-CD5, and molecular masses (kDa). Data are representative of at least three experiments.


B CD4 + CD8 + thymocytes and mature CD4 + T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep - Tactin - APC and analyzed by flow cytometry . Background staining was deduced using cells from WT mouse . Data are representative of 2 experiments .

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

B CD4+CD8+thymocytes and mature CD4+T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep-Tactin-APC and analyzed by flow cytometry. Background staining was deduced using cells from WT mouse. Data are representative of 2 experiments.


CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . A Histogram comparing the normalized CBL protein intensities resulting from AP - MS analysis of CD4 + T cells from WT and CBLOSTmice prior to and after the specified stimulation times . For each time point , the distributions of the normalized intensities of the 9 data points ( 3 biological replicates x 3 technical replicates ) corresponding to CD4 + T from wild - type ( WT ) and CBLOSTmice were compared using the adjusted p - value from a one - way ANOVA test ( denoted as p ( t ) ) and the value r ( t ) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples . Different colors represent distinct biological replicates .

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

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.A Histogram comparing the normalized CBL protein intensities resulting from AP-MS analysis of CD4+T cells from WT and CBLOSTmice prior to and after the specified stimulation times. For each time point, the distributions of the normalized intensities of the 9 data points (3 biological replicates x 3 technical replicates) corresponding to CD4+ T from wild-type (WT) and CBLOSTmice were compared using the adjusted p-value from a one-way ANOVA test (denoted as p(t)) and the value r(t) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples. Different colors represent distinct biological replicates.


CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p - valueis plotted against the corresponding mean enrichment for CBL ( t ) > ( t ) > OST versus WT samples . The time point shown corresponds to the unstimulated condition ( t = 0s ) . Mean was calculated using bootstrap resampling ( see Results and Material and Methods ) . Proteins that displayed an enrichment r ( t ) greater than 2 - fold and a corresponding p - value p ( t ) lower than a set threshold ( see Material and Methods ) in more than 90 % of the bootstrap iterations were selected as specific partners ( indicated in red ) . Names of four of the most significantly enriched proteins are indicated . Dashed lines represent thresholds on the p - value p * = 10 - 3 and the enrichment r * = 2 used to identify specific interactors of CBL .

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

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p-valueis plotted against the corresponding mean enrichment for CBL(t)>(t)>OST versus WT samples. The time point shown corresponds to the unstimulated condition (t=0s). Mean was calculated using bootstrap resampling (see Results and Material and Methods). Proteins that displayed an enrichment r(t) greater than 2-fold and a corresponding p-value p(t) lower than a set threshold (see Material and Methods) in more than 90% of the bootstrap iterations were selected as specific partners (indicated in red). Names of four of the most significantly enriched proteins are indicated. Dashed lines represent thresholds on the p-value p*=10-3 and the enrichment r*=2 used to identify specific interactors of CBL.


CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . C , D List of specific interacting partners of CBLB ( C ) and CBL ( D ) ranked according to their maximum mean enrichment across all time points . Heat maps show , for each time point , the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate the proteins that have been previously reported as CBLB ( C ) interactors in the BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow .

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

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBLB (C) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.


CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . C , D List of specific interacting partners of CBLB ( C ) and CBL ( D ) ranked according to their maximum mean enrichment across all time points . Heat maps show , for each time point , the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate the proteins that have been previously reported as CBL ( D ) interactors in the BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow .

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

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBL (D) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.


CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 and anti - CD4 antibodies . Equal amounts of cell lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads and subjected to MS analysis as described in Material and Methods . E Histograms showing the numbers of specific interactors binding to the CBL - OST or CBLB - OST proteins prior to and after TCR stimulation for different times and for which P ( Detection ) > 0 . 9 .

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

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.E Histograms showing the numbers of specific interactors binding to the CBL-OST or CBLB-OST proteins prior to and after TCR stimulation for different times and for which P(Detection) >0.9.


D , E , F , G , H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C . CD4 + T cells were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 and anti - CD4 antibodies and subsequently lysed . Equal amounts of cell lysates were incubated with isotype control ( Iso Ctrl ) or the specified antibodies and the resulting immunoprecipitates ( IP ) analyzed by immunoblot with the antibodies specified in the left margin . The validated PPIs predicted in C correspond to CSK - CBLB and CSK - CD5 ( D ) , CRKL - PI3Kp85β and CSK - PI3Kp85β ( E ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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

D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85β and CSK- PI3Kp85β (E). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


D , E , F , G , H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C . CD4 + T cells were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 and anti - CD4 antibodies and subsequently lysed . Equal amounts of cell lysates were incubated with isotype control ( Iso Ctrl ) or the specified antibodies and the resulting immunoprecipitates ( IP ) analyzed by immunoblot with the antibodies specified in the left margin . The validated PPIs predicted in C correspond to PI3Kp85β - PI3Kp110α , CSK - PI3Kp110α ( F ) CRKL - PI3Kp110α ( G ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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

D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to PI3Kp85β-PI3Kp110α, CSK-PI3Kp110α (F) CRKL-PI3Kp110α (G). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


A CD4 + T cells from wild - type ( WT ) , CBLOST and CBLBOSTmice were left unstimulated ( - ) or stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . Equal amounts of cell lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti - ubiquitin ( UB ) , anti - CBLB or anti - CBL antibodies . Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates ( WCL ) probed with anti - CD5 , and molecular masses ( kDa ) . TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form , likely due to its ubiquitylation ( marked with an asterisk ) . In contrast , no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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

A CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti-ubiquitin (UB), anti-CBLB or anti-CBL antibodies. Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates (WCL) probed with anti-CD5, and molecular masses (kDa). TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form, likely due to its ubiquitylation (marked with an asterisk). In contrast, no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


C Immunoprecipitation of CBL from cell lysates of CD4 + T cells isolated from WT or CBLB - deficient mouse ( Cblb - / - ) and kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates and whole cell lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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

C Immunoprecipitation of CBL from cell lysates of CD4+T cells isolated from WT or CBLB-deficient mouse (Cblb-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


D Immunoprecipitation of CBLB from cell lysates of CD4 + T cells isolated from WT or CBL - deficient mouse ( Cbl - / - ) and kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates and whole cell lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 3 experiments .

D Immunoprecipitation of CBLB from cell lysates of CD4+T cells isolated from WT or CBL-deficient mouse (Cbl-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 3 experiments.


A , B CD4 + T cells from wild - type , Cblb - / - and Cd5 - / - mice were stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . CBL ( A ) were immunoprecipitated from equal amounts of protein lysates . Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin . Whole cell lystates ( WCLs ) were also analyzed with the specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). CBL (A) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


A , B CD4 + T cells from wild - type , Cblb - / - and Cd5 - / - mice were stimulated for 1 min with anti - CD3 and anti - CD4 antibodies ( + ) . PI3KR1 ( PI3Kp85α ) ( B ) were immunoprecipitated from equal amounts of protein lysates . Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin . Whole cell lystates ( WCLs ) were also analyzed with the specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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

A, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). PI3KR1 (PI3Kp85α) (B) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


Heatmap of the differentially expressed cholesterol - related genes between normal brain tissues ( n = 28 ) and glioblastomas ( n = 217 ) from the Rembrandt dataset . Gene expression values are z - transformed and colored red for high expression and blue for low expression , as indicated in the scale bar . Volcano plot showing the fold - change ( log2 ) in cholesterol - related gene levels based on GBM vs normal brain tissue . Data were obtained from the Rembrandt dataset .

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Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar. Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset.


Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas ( n = 64 ) . Scale bar = 30 μm . Quantification of CYP46A1 IHC staining in normal brain ( n = 6 ) and different pathological grades of gliomas ( n = 58 ) .

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Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 μm. Quantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58).


Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case . Scale bar = 30 μm .

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Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 μm.


CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification . Data are shown as the mean ± the standard error of the mean ( SEM ; n = 667 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA .

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CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.


CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset . Shown are means and SEM ( n = 245 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA .

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CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.


Kaplan - Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM . Data were obtained from the CGGA dataset . P - values were obtained from the log - rank test .

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Kaplan-Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.


qRT - PCR analysis of CYP46A1 mRNA levels in LN229 and GBM # P3 cells transfected with lentivirus expressing CYP46A1 ( lenti - CYP46A1 ) or control sequence ( lenti - Ctrl ) . GAPDH was used as an internal control . Shown are means and SEM ( n = 3 ) . LN229 : * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 . Statistical significance was determined by two - sided Student ' s t ‐ test .

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qRT-PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test.


Western blot analysis to confirm CYP46A1 overexpression in GBM cells .

Western blot analysis to confirm CYP46A1 overexpression in GBM cells.


Growth curves for GBM cells in vitro infected with lenti - Ctrl or lenti - CYP46A1 derived from trypan blue staining . Shown are means and SEM ( n = 3 ) . LN229 : * * P = 0 . 003 ; GBM # P3 : * * P = 0 . 002 . Statistical significance was determined by two - sided Student ' s t ‐ test .

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Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test.


Colony forming assay for GBM cells infected with lenti - Ctrl or lenti - CYP46A1 . Shown are means and SEM ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * P = 0 . 012 . Statistical significance was determined by two - sided Student ' s t ‐ test .

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Colony forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; GBM#P3: *P = 0.012. Statistical significance was determined by two-sided Student's t‐test.


Representative H & E staining of orthotopic tumours derived from GBM cells infected with lenti - Ctrl or lenti - CYP46A1 . Scale bar = 2 mm .

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Representative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm.


Kaplan - Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti - Ctrl or lenti - CYP46A1 ( n = 5 per group ) . A log - rank test was used to assess statistical significance .

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Kaplan-Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance.


IHC for CYP46A1 , PCNA and cleaved caspase - 3 protein levels in the intracranial tumours . Scale bar = 100 μm .

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IHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 μm.


24OHC levels in spent media from LN229 ( left ) and GBM # P3 ( right ) cells transduced with lenti - Ctrl or lenti - CYP46A1 measured using targeted LC - MS /

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24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS


IC50 curves for 24OHC in LN229 and GBM # P3 cells using the CCK - 8 assay .

IC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.


Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of LN229 and GBM # P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h . Data are shown as the mean ± SEM ( n = 3 ) .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3).


Colony forming ability of GBM cell lines treated with 24OHC ( 0 - 20 μM ) for 14 days .

Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days.


Western blot analysis of the apoptosis marker c - PARP , c - caspase 3 and PCNA in lysates ( 20 µg ) from GBM cells treated with 24OHC ( 0 - 20 μM ) for 72 h .

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Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h.


Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of the quantification of tumoursphere formation . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * P = 0 . 0192 , * * * P = 0 . 0006 , * * * P = 0 . 0002 ; GBM # BG7 : * * P = 0 . 0017 , * * * P < 0 . 0001 , * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0027 , * * * P = 0 . 0001 , * * * P < 0 . 0001 .

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Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001.


Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen ™ Amplex ™ Red Cholesterol Assay Kit and normalized to total protein . Shown are means and SEM ( n = 3 ) . LN229 : * * P = 0 . 002 ; GBM # P3 : * * P = 0 . 011 . Statistical significance was determined by two - sided Student ' s t ‐ test .

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Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test.


Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC ( 0 - 20 μM ) for 72 h . Scale bar = 30 μm .

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Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm.


Growth curves for LN229 cells treated with DMSO , 0 . 5 μg / mL cholesterol or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol using trypan blue staining .

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Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining.


Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0021 , * * P = 0 . 0046 ; GBM # P3 : * * P = 0 . 0012 , * P = 0 . 0193 .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193.


Western blot analysis of c - PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol for 72 h .

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Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.


Tumoursphere formation assays for GBM # P3 and GBM # BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0 . 5 μg / mL cholesterol . Scale bar = 100 μm . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * * P = 0 . 0032 , * P = 0 . 031 ; GBM # BG7 : * * * P = 0 . 0008 , * P = 0 . 0242 .

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Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242.


CCK - 8 assay to determine relative cell survival in LN229 or GBM # P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0051 , * * * P = 0 . 0002 ; GBM # P3 : * * P = 0 . 0018 , * * * P < 0 . 0001 .

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CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001.


Heatmap of differentially expressed transcripts in RNA - seq data from GBM # P3 cells treated with DMSO or 20 μM 24OHC for 72 h . Gene expression data was mean z - transformed for display , and colored red for high expression and blue for low expression .

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Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression.


GSEA plot showing normalized enrichment scores ( NES ) for cholesterol homeostasis , positive regulation of cholesterol efflux , SREBP targets and stem cell proliferation signatures using RNA - seq data from 24OHC and DMSO treated cells .

GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells.


Volcano plot highlighting up - regulated ( red ) and down - regulated ( blue ) genes based on RNA - seq data from GBM # P3 cells after 24OHC treatment . LXR targets , SREBP targets and stemness transcription factors are specifically included .

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Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.


qRT - PCR analysis of 24OHC - regulated genes in GBM # P3 cells after treatment with 20 μM 24OHC for 72 h . GAPDH was used as an internal control . Data are shown as the mean ± SEM ( n = 3 ) .

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qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3).


Cell viability assay performed on GBM # P3 cells after 3 days of treatment with DMSO or 5 - 10 μM LXR - 623 in the presence or absence of 10 μM 24OHC . Data are shown as the mean ± SEM ( n = 3 ) .

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Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3).


Western blot analysis of cholesterol metabolism - related proteins in LN229 or GBM # P3 after treatment with 24OHC ( 0 - 20 μM ) .

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Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM).


Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC ( 0 - 40 μM ) as assessed by a trypan blue test . Data are shown as the mean ± SEM ( n = 3 ) .

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Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3).


Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days .

Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days.


Representative images of filipin staining in LN229 ( up ) or LN18 ( down ) cells treated with different concentration of fatostatin ( 0 - 20 μM ) . Scale bar = 30 μm .

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Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.


Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * P = 0 . 013 ; GBM # P3 : * * P = 0 . 0023 .

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Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023.


Filipin staining of GBM cells after treatment with EFV ( 0 - 20 μM ) . Scale bar = 30 μm .

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Filipin staining of GBM cells after treatment with EFV (0 - 20 μM). Scale bar = 30 μm.


IC50 curves for EFV in GBM cells determined using OD readings ( 450nm ) from the CCK - 8 assay .

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IC50 curves for EFV in GBM cells determined using OD readings (450nm) from the CCK-8 assay.


24OHC levels in spent media from LN229 ( left ) or GBM # P3 ( right ) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC - MS / MS .

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24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS.


Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV ( 0 - 20 μM ) . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * P = 0 . 0056 , * * * P < 0 . 0001 ; LN18 : * * * P < 0 . 0001 .

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Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001.


Flow cytometry to detect annexin V - FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV ( 0 - 20 μM ) for 72 h . Data are shown as the mean ± SEM ( n = 3 ) . LN229 : * * * P = 0 . 0006 , * * * P < 0 . 0001 ; GBM # P3 : * * * P < 0 . 0001 .

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Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0 - 20 μM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001.


Tumoursphere formation assays for GSCs treated with different concentrations of EFV ( 0 - 20 μM ) . Scale bar = 100 μm . Graphic representation of the quantification of tumoursphere formation ( right ) . Data are shown as the mean ± SEM ( n = 3 ) . GBM # P3 : * * P = 0 . 0011 , * * * P = 0 . 0003 , * * * P < 0 . 0001 ; GBM # BG7 : * * * P < 0 . 0001 ; GBM # BG5 : * * P = 0 . 0037 , * * P = 0 . 0015 , * * * P = 0 . 0002 .

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Tumoursphere formation assays for GSCs treated with different concentrations of EFV (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002.


Western blot analysis of cholesterol homeostasis related proteins ( LDLR , ABCA1 , P - SREBP1 and N - SREBP1 ) , c - PARP , PCNA and SOX2 in GBM cells treated with EFV ( 0 - 20 μM ) for 72 h .

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Western blot analysis of cholesterol homeostasis related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0 - 20 μM) for 72 h.


The survival curves of tumour - bearing mice implanted with LN229 or GBM # P3 cells after EFV or PBS treatment ( n = 5 per group ) . A log - rank test was used to assess statistical significance .

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The survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.


Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM # P3 cells at days 7 ( n = 5 per group , P = 0 . 58 ) and 21 ( n = 5 per group , P = 0 . 005 ) . Statistical significance was determined by two - sided Student ' s t ‐ test . Body weight of tumour - bearing mice after 3 weeks of EFV or PBS treatment . Data are shown as the mean ± SEM . Statistical significance was determined by two - sided Student ' s t ‐ test .

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Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Statistical significance was determined by two-sided Student's t‐test. Body weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM. Statistical significance was determined by two-sided Student's t‐test.


( A to D ) 10 - to 11 - week - old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks ( N = 7 or 8 per cohort ) . ( A ) Vector copy number , ( B ) GAA levels , ( C ) GAA activity , and ( D ) glycogen content in cardiac and skeletal muscles . ( C to D ) Statistical analysis : two - way ANOVA , Dunnett ' s test . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice ( black ) or wild - type mice ( red ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . See also Appendix Table S1 .

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(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.


Representative images of H & E ( top ) and PAS ( bottom ) staining of the quadriceps in wild - type and Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg ) . Scale bar , 80 µm . ( See also Appendix Table S1 )

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Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). Scale bar, 80 µm. (See also Appendix Table S1)


A - D ( A , B ) Body weight curves and ( C , D ) grip response - ability to grip onto an inverted wire screen for a 60 - second period - in wild - type and Gaa - / - mice untreated or treated with escalating doses of AT845 ( 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg ) . All groups had n = 9 at all timepoints , except the 6 week timepoint ( females ) and the 12 week timepoint ( males and females ) for the Gaa - / - mice , which had n = 8 per group . Female mice dosed with 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg had a 22 % , 11 % , and 0 % failure rate at week 12 , respectively ; in comparison , the female control WT mice had a 22 % failure rate and the control Gaa - / - mice had a 38 % failure . Male mice dosed with 3 × 1013 , 1 × 1014 , and 3 × 1014 vg / kg had a 56 % , 33 % , and 11 % failure rate at week 12 , respectively ; in comparison , the male control WT mice had a 11 % failure rate and the control Gaa - / - mice had a 100 % failure . Bars represent median . Data show a dose - dependent improvement in grip response following treatment with AT845 . Statistical analysis : two - way ANOVA , Dunnett ' s test . Asterisks ( * ) indicate significant differences compared with untreated Gaa - / - mice at each respective time point . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

A-D (A, B) Body weight curves and (C, D) grip response - ability to grip onto an inverted wire screen for a 60-second period - in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). All groups had n=9 at all timepoints, except the 6 week timepoint (females) and the 12 week timepoint (males and females) for the Gaa-/- mice, which had n=8 per group. Female mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 22%, 11%, and 0% failure rate at week 12, respectively; in comparison, the female control WT mice had a 22% failure rate and the control Gaa-/- mice had a 38% failure. Male mice dosed with 3×1013, 1×1014, and 3×1014 vg/kg had a 56%, 33%, and 11% failure rate at week 12, respectively; in comparison, the male control WT mice had a 11% failure rate and the control Gaa-/- mice had a 100% failure. Bars represent median. Data show a dose-dependent improvement in grip response following treatment with AT845. Statistical analysis: two-way ANOVA, Dunnett's test. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice at each respective time point. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


A , B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control ( n = 4 ) or escalating doses AT845 ( n = 6 ) and followed for approximately 12 weeks . ( A ) Vector copy number and ( B ) GAA activity in heart , quadriceps , triceps , diaphragm , and liver . Statistical analysis : mixed - effects analysis with Dunnett ' s test . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum values . Asterisks ( * ) indicate significant differences compared with vehicle control NHPs . * , P < 0 . 05 ; * * , P < 0 . 01 .

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

A, B Equal numbers of male and female cynomolgus monkeys were treated with vehicle control (n=4) or escalating doses AT845 (n=6) and followed for approximately 12 weeks. (A) Vector copy number and (B) GAA activity in heart, quadriceps, triceps, diaphragm, and liver. Statistical analysis: mixed-effects analysis with Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values. Asterisks (*) indicate significant differences compared with vehicle control NHPs. *, P<0.05; **, P<0.01.


A - C Male cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) at 6 × 1013 , 2 × 1014 , or 5 × 1014 vg / kg , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg and followed for approximately 12 weeks . ( A ) Anti - GAA antibody titer , ( B ) cardiac troponin I ( cTnI ) , and ( C ) N - terminal pro b - type natriuretic peptide ( BNP ) . Bars show mean and SEM .

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A-C Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3) at 6×1013, 2×1014, or 5×1014 vg/kg, or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. (A) Anti-GAA antibody titer, (B) cardiac troponin I (cTnI), and (C) N-terminal pro b-type natriuretic peptide (BNP). Bars show mean and SEM.


A , B Male cynomolgus monkeys were treated with vehicle control ( n = 2 ) , AT845 ( n = 3 ) , or Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg and followed for approximately 12 weeks . GAA activity in ( A ) heart , quadriceps , triceps , and diaphragm , and ( B ) liver , brain , spinal cord , and testes . Data are presented as box - and - whisker plots with Tukey whiskers that show minimum , median , and maximum values .

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

A, B Male cynomolgus monkeys were treated with vehicle control (n=2), AT845 (n=3), or Cyno-AT845 (n=3) at 2×1014 vg/kg and followed for approximately 12 weeks. GAA activity in (A) heart, quadriceps, triceps, and diaphragm, and (B) liver, brain, spinal cord, and testes. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum values.


A , B Western blot analysis of heart and quadriceps lysates using anti - GAA detects both the precursor GAA ( 110 kDa ) and the mature lysosomal GAA ( 70 to 76 kDa ) forms in muscle of NHPs treated with ( A ) AT845 ( n = 3 ) or ( B ) Cyno - AT845 ( n = 3 ) at 2 × 1014 vg / kg . Each tissue was analyzed in triplicate , and each respective symbol indicates use of a vehicle control heart tissue ( ^ ) or a vehicle control quadriceps tissue ( * ) . ( See also Appendix Table S3 )

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A, B Western blot analysis of heart and quadriceps lysates using anti-GAA detects both the precursor GAA (110 kDa) and the mature lysosomal GAA (70 to 76 kDa) forms in muscle of NHPs treated with (A) AT845 (n=3) or (B) Cyno-AT845 (n=3) at 2×1014 vg/kg. Each tissue was analyzed in triplicate, and each respective symbol indicates use of a vehicle control heart tissue (^) or a vehicle control quadriceps tissue (*). (See also Appendix Table S3)


( D ) At 48h after transfection of H7 plasmid , the transfectants were incubated with TPCK - trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage .

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(D) At 48h after transfection of H7 plasmid, the transfectants were incubated with TPCK-trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage.


( E ) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate , A549 cells were inoculated with H7N9 / ah virus . At the indicated hours post infection ( hpi ) , culture media ( supernatant ) and cell lysate were harvested for viral load detection . Data are presented as mean and SD in a representative experiment performed 3 times . * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(E) At 24h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed 3 times. **, P<0.01; ***, P<0.001. Student's t test.


( A ) At 48 hours post transfection of the indicated protease plasmids in triplicate , BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss - of - function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay . Data represent mean and SD of triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t test .

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(A) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21 cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6 or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 48h after co - transfection of the indicated protease plasmids and H7 or H1 plasmid , BHK21 cells were lysed to examine HA cleavage with WB .

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(B) At 48h after co-transfection of the indicated protease plasmids and H7 or H1 plasmid, BHK21 cells were lysed to examine HA cleavage with WB.


( E ) At 48h after triple - transfection of H1 , the indicated proteases and wtSPINK6 or mutSPINK6 , BHK21 cells were lysed for the detection of HA cleavage .

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(E) At 48h after triple-transfection of H1, the indicated proteases and wtSPINK6 or mutSPINK6, BHK21 cells were lysed for the detection of HA cleavage.


( A ) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate , BHK21 cells were inoculated with H7N9 / ah at a MOI of 0 . 5 . Cell - free culture media were harvested at 24 hpi for viral titration . Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times . * * , P < 0 . 01 . Student ' s t test .

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(A) At 36h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell-free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.


( B ) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate , A549 cells were inoculated with H1N1 / pdm at MOI of 0 . 25 . The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h . Culture media in each well were stored in two aliquots , one was applied to viral titration with conventional plaque assay ( black bars ) , the other was pre - treated with TPCK - trypsin for 1 hour prior to plaque assay ( grey bars ) . Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(B) At 24h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars), the other was pre-treated with TPCK-trypsin for 1 hour prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( A ) Confocal imaging ( left ) shows intrinsic SPINK6 ( green ) expression in the epithelial cells in 2D human airway organoids . Nuclei and actin filaments are counterstained and shown as blue and purple respectively . Scale , 10 µm . Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids . The plots ( middle ) show the representative result of one experiment performed 3 times . The percentages of HAT and SPINK6 positive cells ( right ) are shown as mean and SD of 3 independent experiments . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(A) Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple respectively. Scale, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed 3 times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of 3 independent experiments. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( B ) At 24h after inoculation of H1N1 / pdm or mock inoculation in triplicate , 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH . Data represent the mean and SD of a representative experiment performed three times . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.