Figure 2A ) Scheme depicting the screen validation strategy ( top ) and representative density plots ( bottom ) of flow cytometry data from cells infected with VSVG - or Spike - typed lentivirus . The infected population is labeled with a darker color . B ) Susceptibility to infection with Spike - typed and VSVG - typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss - of - function of selected screen candidates . NT596 and NT764 are two non - targeting CRISPR controls . ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2 . Bars represent the average and standard error of the mean ( SEM ) of the percentage of infected cells in each condition ( three biological replicates ) , normalized to the non - targeting control cell line NT596 . The t - test p - value between the relative percentage of infection with Spike - typed and VSVG - typed for each cell line is indicated . C ) Confocal microscopy images of SPNS1 - KO1 and PLAC8 - KO1 Calu1ACE2 cells overexpressing CRISPR - resistant SPNS1 - or GFP - PLAC8 , respectively . D ) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines : bar plot showing the average ( three biological replicates ) and SEM percentage of infection ( normalized to NT596 with GFP overexpression ) of Spike - typed and VSVG - typed lentiviruses in PLAC8 - KO1 and SPNS1 - KO1 cell lines that overexpress CRISPR - resistant PLAC8 - or GFP - SPNS1 constructs . The significance above each bar represents the t - test p - value between each condition and the control cell line ( NT596 with GFP overexpression )

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Figure 2A) Scheme depicting the screen validation strategy (top) and representative density plots (bottom) of flow cytometry data from cells infected with VSVG- or Spike-typed lentivirus. The infected population is labeled with a darker color.B) Susceptibility to infection with Spike-typed and VSVG-typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss-of-function of selected screen candidates. NT596 and NT764 are two non-targeting CRISPR controls. ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2. Bars represent the average and standard error of the mean (SEM) of the percentage of infected cells in each condition (three biological replicates), normalized to the non-targeting control cell line NT596. The t-test p-value between the relative percentage of infection with Spike-typed and VSVG-typed for each cell line is indicated.C) Confocal microscopy images of SPNS1-KO1 and PLAC8-KO1 Calu1ACE2 cells overexpressing CRISPR-resistant SPNS1- or GFP-PLAC8, respectively.D) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines: bar plot showing the average (three biological replicates) and SEM percentage of infection (normalized to NT596 with GFP overexpression) of Spike-typed and VSVG-typed lentiviruses in PLAC8-KO1 and SPNS1-KO1 cell lines that overexpress CRISPR-resistant PLAC8- or GFP-SPNS1 constructs. The significance above each bar represents the t-test p-value between each condition and the control cell line (NT596 with GFP overexpression)


Figure 3A , B ) Susceptibility to infection with SARS - CoV - 2 viruses in Calu1ACE2 ( A ) and H1299ACE2 ( B ) cell lines with loss - of - function of the indicated genes . Left : representative immunofluorescence images of SARS - CoV - 2 nucleocapsid protein ( green ) and cell nuclei ( red ) in the indicated cell lines . Scale bar : 20 µm . Right : immunofluorescence quantification of the number of infected cells at 24 h post - infection in the different loss - of - function cell lines infected with SARS - CoV - 2 . Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non - targeting transduced ( NT596 ) cells in three biological replicates ( three different CRISPR KO cell lines infected independently ) . The significance above each bar represents the one - tail t - test p - value between each condition and CRISPR non - targeting transduced ( NT596 ) cells . C ) Western - blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression . Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A , and that ACE2 and Actin panels from Figure 1A were included here to allow comparison . D ) Susceptibility to Spike - typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP , GFP - PLAC8 or GFP - SPNS1 . Bars represent the average and SEM of the percentage of infected cells in three technical replicates ( independent infections of the same cell line ) relative to Calu1ACE2 - GFP cells . The significance above each bar represents the t - test between GFP - PLAC8 or GFP - SPNS1 and GFP cells for each cell line . E ) Susceptibility to infection with SARS - CoV - 2 viruses in cell lines that overexpress ACE2 and GFP - SPNS1 or GFP - PLAC8 . Bars represent the immunofluorescence quantification of the number of SARS - CoV - 2 infected cells at 24 h post - infection . Representative images of SARS - CoV - 2 nucleocapsid immunofluorescence ( green ) and cell nuclei ( red ) in GFP and GFP - PLAC8 cells are shown right to each plot .

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Figure 3A, B) Susceptibility to infection with SARS-CoV-2 viruses in Calu1ACE2 (A) and H1299ACE2 (B) cell lines with loss-of-function of the indicated genes. Left: representative immunofluorescence images of SARS-CoV-2 nucleocapsid protein (green) and cell nuclei (red) in the indicated cell lines. Scale bar: 20 µm. Right: immunofluorescence quantification of the number of infected cells at 24 h post-infection in the different loss-of-function cell lines infected with SARS-CoV-2. Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non-targeting transduced (NT596) cells in three biological replicates (three different CRISPR KO cell lines infected independently). The significance above each bar represents the one-tail t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.C) Western-blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression. Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A, and that ACE2 and Actin panels from Figure 1A were included here to allow comparison.D) Susceptibility to Spike-typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP, GFP-PLAC8 or GFP-SPNS1. Bars represent the average and SEM of the percentage of infected cells in three technical replicates (independent infections of the same cell line) relative to Calu1ACE2-GFP cells. The significance above each bar represents the t-test between GFP-PLAC8 or GFP-SPNS1 and GFP cells for each cell line.E) Susceptibility to infection with SARS-CoV-2 viruses in cell lines that overexpress ACE2 and GFP-SPNS1 or GFP-PLAC8. Bars represent the immunofluorescence quantification of the number of SARS-CoV-2 infected cells at 24 h post-infection. Representative images of SARS-CoV-2 nucleocapsid immunofluorescence (green) and cell nuclei (red) in GFP and GFP-PLAC8 cells are shown right to each plot.


Figure 5A ) Binding and endocytosis studies with Alexa Fluor 647 - conjugated S - RBD or transferrin in Calu1 - ACE2 - RFP cells deficient in PLAC8 and SPNS1 . Left : scatterplots of flow cytometry data showing the cutoffs used to define ACE2 - RFP and Alexa Fluor 647 positive cells . Right : bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2 - RFP positive ( red ) or negative ( blue ) cells in binding and endocytosis experiments with S - RBD or transferrin . Bars represent the average and SEM of three biological replicates ( three different CRISPR KO cell lines ) . The significance above each bar represents the t - test p - value between each condition and CRISPR non - targeting transduced ( NT596 ) cells . B - E ) Endocytosis experiments with S - RBD in PLAC8 - or SPNS1 - KO Calu1 - ACE2 - RFP cells with overexpression of GFP - PLAC8 or GFP - SPNS1 , respectively . Cells were incubated with S - RBD for 1 h at 37 ºC and fixed with 4 % p - formaldehyde after an acid wash to remove non - internalized S - RBD . Representative wide - field fluorescence images ( B ) and quantification of mean fluorescence intensities of each fluorophore ( C ) in each cell show that S - RBD signal correlates with ACE2 - RFP expression but not with GFP - PLAC8 or GFP - SPNS1 . The Pearson correlation coefficient ( r ) and correlation test p - value ( p ) is indicated within each scatterplot . Each dot represents a cell measurement from different microphotographs of a single replicate . Scale bar : 20 µm . Representative fluorescence confocal microphotographs ( D ) and spot colocalization analyses using ComDet plugin ( E ) demonstrate colocalization of S - RBD endosomes with ACE2 - RFP and GFP - PLAC8 or GFP - SPNS1 . ( D ) Red ovals defining the detected S - RBD dots are overlaid in each channel to assess the colocalization with ACE2 - RFP , GFP - PLAC8 or GFP - SPNS1 . Inlets show a magnification of the region defined by the white squares . Scale bar : 20 µm . ( E ) Boxplots representing the percentage of internalized S - RBD that colocalize with ACE2 - RFP , GFP - PLAC8 , GFP - SPNS1 or their combination ( ACE2 - RFP and GFP - PLAC8 or ACE2 - RFP and GFP - SPNS1 ) . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate .

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Figure 5A) Binding and endocytosis studies with Alexa Fluor 647-conjugated S-RBD or transferrin in Calu1-ACE2-RFP cells deficient in PLAC8 and SPNS1. Left: scatterplots of flow cytometry data showing the cutoffs used to define ACE2-RFP and Alexa Fluor 647 positive cells. Right: bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2-RFP positive (red) or negative (blue) cells in binding and endocytosis experiments with S-RBD or transferrin. Bars represent the average and SEM of three biological replicates (three different CRISPR KO cell lines). The significance above each bar represents the t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.B-E) Endocytosis experiments with S-RBD in PLAC8- or SPNS1-KO Calu1-ACE2-RFP cells with overexpression of GFP-PLAC8 or GFP-SPNS1, respectively. Cells were incubated with S-RBD for 1 h at 37 ºC and fixed with 4% p-formaldehyde after an acid wash to remove non-internalized S-RBD. Representative wide-field fluorescence images (B) and quantification of mean fluorescence intensities of each fluorophore (C) in each cell show that S-RBD signal correlates with ACE2-RFP expression but not with GFP-PLAC8 or GFP-SPNS1. The Pearson correlation coefficient (r) and correlation test p-value (p) is indicated within each scatterplot. Each dot represents a cell measurement from different microphotographs of a single replicate. Scale bar: 20 µm. Representative fluorescence confocal microphotographs (D) and spot colocalization analyses using ComDet plugin (E) demonstrate colocalization of S-RBD endosomes with ACE2-RFP and GFP-PLAC8 or GFP-SPNS1. (D) Red ovals defining the detected S-RBD dots are overlaid in each channel to assess the colocalization with ACE2-RFP, GFP-PLAC8 or GFP-SPNS1. Inlets show a magnification of the region defined by the white squares. Scale bar: 20 µm. (E) Boxplots representing the percentage of internalized S-RBD that colocalize with ACE2-RFP, GFP-PLAC8, GFP-SPNS1 or their combination (ACE2-RFP and GFP-PLAC8 or ACE2-RFP and GFP-SPNS1). The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate.


Figure 6C ) Representative images ( left ) and quantification ( right ) of the number of acidic particles in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) using live - cell lysotracker staining . The two samples Wilcoxon Test p - value of the pooled observations compared to NT596 cells is indicated above each condition . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate . Three biological replicates per condition were used . D ) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ - Red - BSA . Left : density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ - Red - BSA under growth or serum starvation conditions . Right : bar plot representing the mean and SEM of the median fluorescence intensity values ( MFI ) of three biological replicates of PLAC8 - and SPNS1 - KO and CRISPR non - targeting cells in growth and serum starvation conditions . E ) Representative images ( left ) and quantification ( right ) of the number of LC3B particles in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) using LC3B immunofluorescence staining . The two samples Wilcoxon Test p - value of the pooled observations compared to NT596 cells is indicated above each condition . The central band represents the median , the box represents the interquartile range ( IQR ) between the 25th and 75th percentile , and the whiskers extend from the box to 1 . 5 * IQR . Each dot represents a cell measurement from different microphotographs of a single replicate . Three biological replicates per condition were used . F ) Autophagic flux analyses using the fluorescence reporter GFP - LC3 - RFP - LC3ΔG in PLAC8 - and SPNS1 - KO Calu1ACE2 cells compared to CRISPR non - targeting control cells ( NT596 ) . Left : density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells . Right : bar plots showing the mean and SEM of the median GFP / RFP intensity ratio of three biological replicates in the different cell lines and conditions .

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Figure 6C) Representative images (left) and quantification (right) of the number of acidic particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using live-cell lysotracker staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.D) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ-Red-BSA. Left: density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ-Red-BSA under growth or serum starvation conditions. Right: bar plot representing the mean and SEM of the median fluorescence intensity values (MFI) of three biological replicates of PLAC8- and SPNS1-KO and CRISPR non-targeting cells in growth and serum starvation conditions.E) Representative images (left) and quantification (right) of the number of LC3B particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using LC3B immunofluorescence staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.F) Autophagic flux analyses using the fluorescence reporter GFP-LC3-RFP-LC3ΔG in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596). Left: density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells. Right: bar plots showing the mean and SEM of the median GFP/RFP intensity ratio of three biological replicates in the different cell lines and conditions.


Figure 1A Flow cytometric quantification of lymphocytes , monocytes , B - cells , natural killer ( NK ) cells , dendritic cells ( DCs ) and granulocytes in peripheral venous blood samples from non - diabetic ( ND ) and type - 2 diabetic ( T2D ) patients with COVID - 19 . B Quantification of monocyte subpopulation phenotypes as classical ( CD14Hi CD16 - ) , intermediate ( CD14Hi CD16 + ) or non - classical ( CD14Lo CD16 + ) . C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID - 19 patients and in T2D patients without COVID - 19 .

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Figure 1A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.


Figure 2A Quantification of CD14 expression and size ( FSC ) in monocyte populations from non diabetic ( ND ) and type - 2 diabetic ( T2D ) COVID - 19 patients . B Frequency of conventional ( FSCLo ) and large ( FSCHi ) monocytes in ND and T2D COVID - 19 patients . C Expression of CD16 , CD14 and HLA - DR in FSCLo and FSCHi monocytes from COVID - 19 patients . D Proportions of classical , intermediate and non - classical monocytes within FSCLo and FSCHi monocytes from COVID - 19 patients . E Correlative analyses of IL8 expression in peripheral blood mononuclear cells ( PBMCs ) to monocyte FSC . F IL8 , IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID - 19 patients . G Monocyte size quantified in ND and T2D COVID - 19 patients treated in general wards ( Gen ) or in intensive care unit ( ICU ) .

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Figure 2A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).


Figure 3A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non - diabetic ( ND ) and type 2 diabetic ( T2D ) COVID - 19 patients ( A ) . B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples . C IRF5 median fluorescence intensity ( MFI ) in monocytes of ND and T2D patients with COVID - 19 . D IRF5 expression overlaid onto monocyte phenotypic gating . E IRF5 expression ( MFI ) in monocyte subtypes from ND and T2D COVID - 19 patients . F HLA - DR and IRF5 expression in monocytes from COVID - 19 patients . G IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted to intensive care unit ( ICU ) or treated exclusively in general wards ( Gen ) . H IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted ICU or treated exclusively in Gen .

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Figure 3A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.D IRF5 expression overlaid onto monocyte phenotypic gating.E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.


Figure 4B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID - 19 either treated in Gen or requiring ICU admission . C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID - 19 patients treated in Gen or requiring ICU treatment .

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Figure 4B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.


Figure 4 ( B ) Quantification of SARS - CoV - 2 infection . After the siRNA knockdown of each target , viral infection was quantified as mean log2 fold - change ( log2FC ) of the percentage of SARS - CoV - 2 + cells relative to the mean of scrambled non - targeting siRNAs ( " SCRAMBLED " , green dots ; accordingly the mean log2FC value of scrambled non - targeting siRNAs was normalized to zero ) . Predicted positive targets ( " TARGETING " , grey dots ) , predicted negative targets ( " NEG " , blue dots ) and positive controls ( " POS " , red dots , including ACE2 and TMPRSS2 ) are all shown . Wilcoxon rank - sum test P value comparing the predicted positive targets to scrambled non - targeting siRNAs is given . ( C ) Quantification of cell number after each target knockdown , calculated as the mean fraction of DAPI + cells relative to the scrambled non - targeting siRNAs ( i . e . the latter was normalized to 1 ) . Colors of dots and P value are interpreted in the same way as in ( B ) . ( D ) Representative fluorescence images from the siRNA assay showing SARS - CoV - 2 infection ( green channel , top row ) , cell number ( blue channel , middle row ) and merged ( bottom row ) . Results for scrambled non - targeting siRNA as negative control ( left column ) , knockdown of three predicted top metabolic targets ( PIKFYVE , SLC16A10 , and PIP5K1C , middle columns ) , and knockdown of positive control ( ACE2 , right column ) are shown . Scale bar = 10 μm .

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 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 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

Figure 4(B) Quantification of SARS-CoV-2 infection. After the siRNA knockdown of each target, viral infection was quantified as mean log2 fold-change (log2FC) of the percentage of SARS-CoV-2+ cells relative to the mean of scrambled non-targeting siRNAs ("SCRAMBLED", green dots; accordingly the mean log2FC value of scrambled non-targeting siRNAs was normalized to zero). Predicted positive targets ("TARGETING", grey dots), predicted negative targets ("NEG", blue dots) and positive controls ("POS", red dots, including ACE2 and TMPRSS2) are all shown. Wilcoxon rank-sum test P value comparing the predicted positive targets to scrambled non-targeting siRNAs is given.(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).(D) Representative fluorescence images from the siRNA assay showing SARS-CoV-2 infection (green channel, top row), cell number (blue channel, middle row) and merged (bottom row). Results for scrambled non-targeting siRNA as negative control (left column), knockdown of three predicted top metabolic targets (PIKFYVE, SLC16A10, and PIP5K1C, middle columns), and knockdown of positive control (ACE2, right column) are shown. Scale bar=10 μm.


Figure 5 ( A ) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups : control ( no virus or remdesivir treatment ) , virus ( SARS - CoV - 2 - infected ) , virus + remdesivir ( SARS - CoV - 2 - infected treated by remdesivir ) , remdesivir ( remdesivir treatment alone without virus ) . There are 3 replicates in each group . ( C ) PCA plot of the average metabolic flux profile computed using the iMAT algorithm ( Shlomi et al . 2008 ; Methods ) representative of each of the experimental groups , the labels are the same as ( A ) .

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

Figure 5(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.(C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A).


figf1 ( a ) Survival rate of wild - type mice ( WT ) and indicated knockout mice after intranasal infection with 750 plaque forming units ( PFU ) of the PR8 virus . ( b ) Hematoxylin and eosin ( H & amp ; amp ; E ) - stained lung sections from mice infected with PR8 virus on day 2 . Arrows indicate cellular infiltrates and necrotic debris occluding the airways . Original magnification , × 40 . ( c ) Disease severity ( ' lung score ' ) and extent of cell death ( necrosis score ) on day 2 after infection based on analysis of H & amp ; amp ; E - stained lung sections . ( d ) Necrosis score on day 7 , estimated visually from H & amp ; amp ; E - stained lung sections , and flow cytometry analysis for annexin V - stained lung cell populations . ( e ) Immunohistochemistry staining of neutrophils occluding the airways of PR8 - infected mice . Original magnification , × 20 . ( f , g ) Flow cytometry analysis of neutrophil , dendritic cell ( DC ) and macrophage cell numbers on days 2 and 7 . ( h ) Pulmonary viral titers on days 2 and 7 after infection in Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( log - rank test ( a ) and unpaired two - sided Student ' s t - test ( c , d , f , g ) . Data are pooled from three experiments ( a , f - h ; mean ± s . e . m . in f - h ; n = 10 - 12 mice total ) or are representative of three experiments ( b - e ; mean ± s . e . m . ; n = 3 - 5 mice per experiment ) .

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 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 B-PANEL_START 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 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

figf1(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus.(b) Hematoxylin and eosin (H&amp;E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&amp;E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&amp;E-stained lung sections, and flow cytometry analysis for annexin V-stained lung cell populations.(e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20.(f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7.(h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f-h; mean ± s.e.m. in f-h; n = 10-12 mice total) or are representative of three experiments (b-e; mean ± s.e.m.; n = 3-5 mice per experiment).


figf2 ( a - c ) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild - type mice ( WT ) and Ripk2 − / − mice with the PR8 virus were analyzed for IL - 6 and TNF ( day 2 ; a ) , IL - 18 and IFN - γ ( b ) , and indicated chemokines ( c ) . * P 0 . 05 , * * P 0 . 01 ( unpaired two - sided Student ' s t - test ) . Data were normalized for total protein ( t . p . ) from lung homogenates . Data are pooled from three experiments ( mean ± s . e . m . ; n = 10 - 12 mice total ) .

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

figf2(a-c) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild-type mice (WT) and Ripk2 −/− mice with the PR8 virus were analyzed for IL-6 and TNF (day 2; a), IL-18 and IFN-γ (b), and indicated chemokines (c). *P 0.05, **P 0.01 (unpaired two-sided Student's t-test). Data were normalized for total protein (t.p.) from lung homogenates. Data are pooled from three experiments (mean ± s.e.m.; n = 10-12 mice total).


figf3 ( a - c ) Analysis on day 2 after infection with PR8 virus of wild - type mice ( WT ) or Ripk2 − / − mice injected intraperitoneally with control rabbit serum and Ripk2 − / − mice injected intraperitoneally with anti - IL - 18 neutralizing antiserum ( αIL - 18Ab ) 3 h before infection , for IL - 18 , IFN - γ and CCL2 amounts in whole lung homogenates ( a ; t . p . , total protein ) , and for neutrophil numbers by immunohistochemistry ( b ) and by flow cytometry ( c ) . Original magnification , × 20 . ( d ) Analysis of survival of indicated mice . ( e ) Neutrophil numbers in WT , Ripk2 − / − and Ripk2 − / − Il18 − / − mice on day 2 after PR8 infection . ( f ) Survival of Ripk2 − / − Il18 − / − , Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ( a , c , e ) and log - rank test ( d , f ) ) . Data are representative of two independent experiments ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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 B-PANEL_START 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 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

figf3(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20.(d) Analysis of survival of indicated mice.(e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection.(f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).


figf4 ( a , b ) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL - 18 production by using IL - 18 immunohistochemistry ( a ) and IL - 18 ELISA ( b ) of whole lung homogenates . WT , wild type ; t . p . , total protein . In a , blue arrows denote IL - 18 + macrophages , and red arrows denote IL - 18 + bronchial epithelium . Original magnification , × 200 . ( c ) Flow cytometry analysis of NK cell counts in Ripk2 − / − and wild - type mice . ( d ) Analysis of wild - type and Ripk2 − / − NK cells for IFN - γ production on a per cell basis . ( e ) Accumulation of T and B cells in lungs . ( f ) Analysis of CD8 + T cells for IFN - γ and CD44 expression in wild - type and Ripk2 − / − mice . * P 0 . 05 , * * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( b ) and unpaired two - sided Student ' s t - test ( d , f ) ) . Data are representative of two experiments ( a , b , f ) or three experiments ( c - e ) ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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 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 B-PANEL_START 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

figf4(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200.(c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice.(d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis.(e) Accumulation of T and B cells in lungs.(f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).


figf5 ( a ) Immunoblots showing active caspase - 1 ( casp - 1 ) p20 compared to GAPDH loading controls in wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs after PR8 infection . ( b ) IL - 18 release in PR8 - infected WT , Ripk2 − / − and Nod2 − / − BMDCs . ( c , d ) Immunoblot analysis showing active casp - 1 p20 ( c ) and IL - 18 production ( d ) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10 . ( e , f ) IL - 18 and casp - 1 activation in Ripk2 − / − BMDCs treated with the NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection . ( e , f ) IL - 18 and casp - 1 activation in Ripk2 − / − BMDCs treated with the NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection . ( g ) Immunoblot analysis of casp - 1 p20 during Listeria infection of WT and Aim2 − / − BMDCs . * P 0 . 01 , * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ) . Data are representative of six experiments ( a , b ) or three experiments ( c - g ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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 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 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 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

figf5(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection.(b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs.(c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10.(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.(g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c-g) (mean ± s.d.; n = 2-3 replicates per experiment).


figf6 ( a ) Immunoblot analysis of wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs for LC3 - II ( marker for autophagy ) after 18 h of PR8 infection . ( b , c ) Numbers of LC3 - II + puncta ( b ) , counted in at least 100 cells from five random fields from confocal images ( c ) of BMDCs after PR8 infection and / or chloroquine treatment . Original magnification , × 400 . ( d , e ) Immunoblots of LC3 - II and caspase - 1 ( casp - 1 ; e ) and IL - 18 production ( d ) in wild - type and Ripk2 − / − BMDCs after treatment with rapamycin ( Rap ) . ( f ) Immunoblot analysis for LC3 - II in lung lysates of PR8 - infected wild - type and Ripk2 − / − mice ( 1 - 5 indicate individual mice ) . ( g , h ) Lungneutrophil infiltration ( g ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) . ( g , h ) IL - 18 and IFN - γ amounts ( h ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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 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 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 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 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

figf6(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).(f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice).(g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).(g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


figf7 ( a - c ) Flow cytometry analysis of wild - type ( WT ) and Ripk2 − / − BMDCs stained with the mitochondrial superoxide - specific stain MitoSOX during IAV or Listeria infection ( a , b ) and with the general mitochondrial stain MitoTracker Green during IAV infection ( c ) . ( d , e ) Analysis of IL - 18 production and immunoblotting to analyze caspase - 1 ( casp - 1 ) p20 activation in IAV - infected BMDCs that had been pretreated with the ROS inhibitor NAC . ( f ) Immunoblot of casp - 1 p20 in cell lysates of IAV - infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO . ( g ) Confocal microscopy analysis of BMDCs stained for mitochondria ( red ) and LC3 ( green ) for colocalization as an indicator of mitophagy . Original magnification , × 400 ; inset ( red boxed region ) was digitally enlarged × 10 . * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( d ) and unpaired two - sided Student ' s t - test ( g ) ) . Data are representative of nine experiments ( a ) , three experiments ( b , d - g ) or six experiments ( c ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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 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 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

figf7(a-c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide-specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c).(d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC.(f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO.(g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d-g) or six experiments (c) (mean ± s.d.; n = 2-3 replicates per experiment).


figf8 ( a , b ) IL - 18 amounts ( a ) , and caspase - 1 ( casp - 1 ) p20 and LC3 - II immunoblots ( b ) in PR8 - infected wild - type ( WT ) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580 . ( c ) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2 − / − BMDCs after PR8 infection . ( d ) Immunoblots for casp - 1 p20 in untreated or NAC - treated WT and Ulk1 − / − BMDCs after PR8 infection . ( e ) Flow cytometry analysis of MitoSOX staining in PR8 - infected WT and Ulk1 − / − BMDCs . ( f ) MitoTracker Green staining of PR8 - infected WT and Ulk1 − / − BMDCs .

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figf8(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580.(c) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2−/− BMDCs after PR8 infection.(d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection.(e) Flow cytometry analysis of MitoSOX staining in PR8-infected WT and Ulk1−/− BMDCs.(f) MitoTracker Green staining of PR8-infected WT and Ulk1−/− BMDCs.


Figure 1 ( G ) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration . The NDST3 KO cells with stable expression of Flag - NDST3 or Flag - EGFP ( control ) were treated with 25 nM Baf A1 for 72 h , and cell survival was measured using calcein AM staining . The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells ( n = 4 independent cultures , * * * * P < 0 . 0001 ) . Data information : Error bars represent ± standard deviation . Scale bar , 200 μm .

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Figure 1(G) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration. The NDST3 KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001). Data information: Error bars represent ± standard deviation. Scale bar, 200 μm.


Figure 2 ( C ) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow / Blue dextran staining against the pH calibration curve in ( B ) ( n = 3 independent cultures , * P = 0 . 0242 ) . ( D ) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 ( 100 nM , 1h ) ( n = 3 independent cultures , * P = 0 . 0305 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm . ( F ) Magic Red assay for Cat B proteolytic activity . WT and NDST3 KO RPE1 cells were treated with vehicle ( VEH ) or Baf A1 ( 100 nM , 1 h ) and then stained with MR - ( RR ) 2 to measure the Cathepsin B activity ( n = 48 cells in the VEH - treated WT group , n = 37 cells in the VEH - treated NDST3 KO group , n = 64 cells in the Baf A1 - treated WT group , n = 42 cells in the Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) . ( G ) Alexa Fluor ( AF ) 488 - dextran degradation assay . WT and NDST3 KO RPE1 cells were loaded with AF488 - dextran in culture medium and observed for fluorescence . The AF488 fluorescence signals without a chase period , with a 4 - h chase period , and with the 4 - h chase period in the presence of Baf A1 ( 100 nM , 1h ) were recorded as fluorescence of uptake , chase , and chase with Baf A1 , respectively . The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods ( n = 62 cells in the VEH - treated WT group , n = 36 cells in the VEH - treated NDST3 KO group , n = 65 cells in the Baf A1 - treated WT group , n = 78 cells in the Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) . ( J ) The LC3 turnover assay after a 48 - h Baf A1 treatment . WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B - II fold change as in ( I ) ( n = 6 independent experiments , * * P = 0 . 0014 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

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 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 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 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

Figure 2(C) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.(F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).(G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488 fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated NDST3 KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).(J) The LC3 turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


Figure 3 ( A ) To examine the levels of the V - ATPase V1 - V0 holoenzyme in WT and NDST3 KO RPE1 cells , cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V - ATPase subunits V1A ( ATP6V1A ) , V1C1 ( ATP6V1C1 ) , and V0D ( ATP6V0D ) . LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins , respectively . ( B ) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from ( A ) ( n = 3 independent experiments , P = 0 . 7786 ) . Data information : Error bars represent ± standard deviation .

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Figure 3(A) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively. (B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786). Data information: Error bars represent ± standard deviation.


Figure 4 ( A , B ) Perinuclear enrichment and colocalization of acetylated microtubules , lysosomes , and V - ATPase V1 subunits . RPE1 cells stably expressing LAMP1 - GFP were treated with vehicle ( VEH ) or 10 μg / mL nocodazole ( Noco ) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated - α - tubulin ( Ac - α - tubulin ) . The nucleus was stained with DAPI . Representative images of LAMP1 - GFP , ATP6V1C1 , and Ac - α - tubulin immunostaining are shown in ( A ) . Representative distribution profiles of Ac - α - tubulin , LAMP1 - GFP and ATP6V1C1 were plotted by Fiji software in ( B ) . The assembly of V - ATPase V1 - V0 holoenzymes in WT RPE1 cells over - expressing the α - tubulin deacetylase HDAC6 or EGFP as a control . Representative immunoblots for ATP6V0D , ATP6V1A , ATP6V1C1 , LAMP1 and GAPDH are shown in ( H ) . ( M - O ) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V - ATPase V1 subunits . WT and NDST3 KO PRE1 cells stably expressing LAMP1 - GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac - α - tubulin . The relative levels of Ac - α - tubulin ( N ) ( n = 75 cells in the WT group , n = 79 cells in the NDST3 KO group , * * * * P < 0 . 0001 ) and the colocalization of LAMP1 - GFP with ATP6V1C1 ( O ) ( n = 75 cells in the WT group , n = 79 cells in the NDST3 KO group , * * * * P < 0 . 0001 ) are quantified .

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Figure 4(A, B) Perinuclear enrichment and colocalization of acetylated microtubules, lysosomes, and V-ATPase V1 subunits. RPE1 cells stably expressing LAMP1-GFP were treated with vehicle (VEH) or 10 μg/mL nocodazole (Noco) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated-α-tubulin (Ac-α-tubulin). The nucleus was stained with DAPI. Representative images of LAMP1-GFP, ATP6V1C1, and Ac-α-tubulin immunostaining are shown in (A). Representative distribution profiles of Ac-α-tubulin, LAMP1-GFP and ATP6V1C1 were plotted by Fiji software in (B).The assembly of V-ATPase V1-V0 holoenzymes in WT RPE1 cells over-expressing the α-tubulin deacetylase HDAC6 or EGFP as a control. Representative immunoblots for ATP6V0D, ATP6V1A, ATP6V1C1, LAMP1 and GAPDH are shown in (H).(M-O) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V-ATPase V1 subunits. WT and NDST3 KO PRE1 cells stably expressing LAMP1-GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac-α-tubulin. The relative levels of Ac-α-tubulin (N) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) and the colocalization of LAMP1-GFP with ATP6V1C1 (O) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) are quantified.


Figure 5 ( C ) In vitro enzymatic assay quantitating the effect of NDST3 on α - tubulin acetylation . Flag - NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti - Flag beads . Flag - EGFP was used as a control . The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro , with or without 1 mM NAD + , 400 nM TSA , or 5 mM nicotinamide ( NAM ) for 2 h at room temperature . The reaction products were examined by immunoblotting against Ac - α - tubulin or total α - tubulin ( n = 3 independent experiments , * P = 0 . 0185 , * * P < 0 . 01 , * * * * P < 0 . 0001 ) . ( D ) The same in vitro enzymatic assay as in ( C ) , except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP ( n = 3 independent experiments , * * P < 0 . 01 , * * * P = 0 . 0001 ) . Data information : Error bars represent ± standard deviation . ns means non - significant .

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Figure 5(C) In vitro enzymatic assay quantitating the effect of NDST3 on α-tubulin acetylation. Flag-NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti-Flag beads. Flag-EGFP was used as a control. The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro, with or without 1 mM NAD+, 400 nM TSA, or 5 mM nicotinamide (NAM) for 2 h at room temperature. The reaction products were examined by immunoblotting against Ac-α-tubulin or total α-tubulin (n = 3 independent experiments, *P = 0.0185, **P < 0.01, ****P < 0.0001). (D) The same in vitro enzymatic assay as in (C), except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP (n = 3 independent experiments, **P < 0.01, ***P = 0.0001). Data information: Error bars represent ± standard deviation. ns means non-significant.


Figure 6 ( A , B ) NDST3 protein levels relative to β - tubulin in B lymphocytes derived from healthy controls and C9orf72 - linked ALS patients , as examined by immunoblotting ( n = 3 biologically independent samples , * * P = 0 . 0026 ) . Data information : Error bars represent ± standard deviation . ( C - F ) The protein levels of NDST3 relative to GAPDH ( D ) ( * * P = 0 . 0013 ) , Ac - α - tubulin relative to total α - tubulin ( E ) ( * P = 0 . 0379 ) , and total α - tubulin relative to GAPDH ( F ) ( P = 0 . 5700 ) , as examined by immunoblotting , in spinal cord tissues from healthy controls and C9orf72 - linked ALS patients ( n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group ) . Data information : Error bars represent ± standard deviation . ( G - I ) Immunoblotting assays showing down - regulation of NDST3 in C9orf72 shRNA ( shC9orf72 ) - treated RPE1 cells , as compared to control shRNA ( shCTRL ) - treated cells . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( H ) ( n = 4 independent experiments , * * * P = 0 . 0004 ) and ( I ) ( n = 4 independent experiments , * * * P = 0 . 0001 ) , respectively . ( J - L ) Immunoblotting assays showing up - regulation of NDST3 in RPE1 cells over - expressing Flag - C9orf72 compared to those expressing Flag - EGFP as control . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( K ) ( n = 3 independent experiments , * * P = 0 . 0011 ) and ( L ) ( n = 3 independent experiments , * * P = 0 . 0033 ) , respectively . Data information : Error bars represent ± standard deviation . ( M , N ) Baf A1 sensitivity assay for WT , NDST3 KO , and Noco - treated NDST3 KO RPE1 cells expressing proline - arginine poly - dipeptide ( PR82 ) . 24 h after the transfection of PR82 , WT and NDST3 KO cells were treated with vehicle or 10 μg / mL Noco overnight . The cell survival measured using calcein AM staining at 48 h post - transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells ( n = 3 independent cultures , * P = 0 . 0369 , * * * P = 0 . 0007 ) . Scale bar , 200 μm . Data information : Error bars represent ± standard deviation .

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

Figure 6(A, B) NDST3 protein levels relative to β-tubulin in B lymphocytes derived from healthy controls and C9orf72-linked ALS patients, as examined by immunoblotting (n = 3 biologically independent samples, **P = 0.0026). Data information: Error bars represent ± standard deviation.(C-F) The protein levels of NDST3 relative to GAPDH (D) (**P = 0.0013), Ac-α-tubulin relative to total α-tubulin (E) (*P = 0.0379), and total α-tubulin relative to GAPDH (F) (P = 0.5700), as examined by immunoblotting, in spinal cord tissues from healthy controls and C9orf72-linked ALS patients (n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group). Data information: Error bars represent ± standard deviation.(G-I) Immunoblotting assays showing down-regulation of NDST3 in C9orf72 shRNA (shC9orf72)-treated RPE1 cells, as compared to control shRNA (shCTRL)-treated cells. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (H) (n = 4 independent experiments, ***P = 0.0004) and (I) (n = 4 independent experiments, ***P = 0.0001), respectively. (J-L) Immunoblotting assays showing up-regulation of NDST3 in RPE1 cells over-expressing Flag-C9orf72 compared to those expressing Flag-EGFP as control. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (K) (n = 3 independent experiments, **P = 0.0011) and (L) (n = 3 independent experiments, **P = 0.0033), respectively. Data information: Error bars represent ± standard deviation.(M, N) Baf A1 sensitivity assay for WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of PR82, WT and NDST3 KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm. Data information: Error bars represent ± standard deviation.


Figure 2Tap - 1 is required for the secretion of TseL . Pellet ( P ) and supernatant ( S ) of bacterial cultures were analyzed by SDS - PAGE . The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown . The asterisk marks an unspecific band , detected by the antiserum against TseL , present in the pre - immunized serum . One representative experiment of three independent experiments is shown . Tap - 1 is required for TseL - mediated killing . Wild - type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay . The y - axis indicates the number of surviving C6706 . Tap - 1 is required for TseL function . Killing assays in which V52Δtap - 1 was mixed with wild - type C6706 or mutants lacking tsiV1 , tsiV2 , or tsiV3 . The y - axis indicates surviving C6706 .

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Figure 2Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS-PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown.Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706.Tap-1 is required for TseL function. Killing assays in which V52Δtap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706.


Figure 3TseL secretion depends on VgrG - 1 . Western blot analysis of pellet ( P ) and supernatant ( S ) samples of the indicated strains . Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK . The unspecific band detected by rabbit serum is marked with an asterisk . One representative of three experiments is shown . VgrG - 1 is required for TseL - mediated killing . Killing assay with wild - type V52 or indicated mutants and wild - type C6706 or a mutant lacking the immunity gene tsiV1 . The y - axis indicates surviving C6706 . VasX - and VgrG - 3 - mediated killing but not TseL - mediated killing occurs in the absence of VgrG - 1 . V52ΔvgrG - 1 was mixed in a killing assay with wild - type C6706 or mutants lacking immunity genes tsiV1 , tsiV2 , or tsiV3 ( specific for TseL , VasX , and VgrG - 3 , respectively ) . The y - axis indicates surviving C6706 .

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Figure 3TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown.VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706.VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52ΔvgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706.


Figure 4A , B Interactions between Tap - 1 and TseL ( A ) or VgrG - 1 ( B ) . Shown are immunoblots of lysates ( total ) and immunoprecipitates with anti - FLAG affinity beads ( IP : FLAG ) of V52Δtap - 1 ( A ) or V52 vgrG - 1 : : myc ( B ) transformed with empty vector or a plasmid encoding His - tagged or FLAG - tagged Tap - 1 . C Interaction between TseL and VgrG - 1 . Shown are immunoblots of lysates ( total ) and immunoprecipitates with an anti - FLAG affinity beads ( IP : FLAG ) of V52 or V52Δtap - 1 transformed with empty vector or a plasmid encoding HIS - tagged or FLAG - tagged VgrG - 1 .

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Figure 4A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.


Figure 5Specificity of the C - terminal Tap - 1 segment . Killing assay in which the lack of tap - 1 in V52 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to kill in a TseL - dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1 . Specificity of N - terminal segment of Tap - 1 . Killing assay in which the lack of tap - 1 in 1587 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . The ability to facilitate killing by the effector A55 _ 1502 is indicated by the logarithm of surviving 1587ΔA55 _ 1501 - 03 .

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Figure 5Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1.Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.


Figure 1Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in pilocarpine ( A , B ) models . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F . Representative western - blots of the cortical ( Ctx ) or hippocampal ( Hip ) extracts from mice at the indicated time after seizure activity in kindling ( D , E ) models . For D and E , samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG . ( C , F ) Statistics of Gli1 or Shh expression levels shown in A , B or D , E respectively . n = 8 - 14 mice in C and n = 8 - 23 mice in F . ( G ) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus ( SE ) induced by pilocarpine ( n = 7 - 10 ) . ( H , I ) Shh levels assayed by ELISA in the medium of slices ( H , n = 9 ) or hippocampal neurons ( I , n = 6 ) incubated with picrotoxin ( Pic ) or Mg2 + - free ( 0Mg ) for the indicated times . ( J ) Representative western - blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times ( n = 13 - 19 ) . a - tubulin ( a - Tub ) , a loading control . Data were mean + SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Control ( Ctrl ) with student ' s t test .

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 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 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 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

Figure 1Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.(G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10).(H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times.(J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.


Figure 2 ( A - C ) Whole - cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons . A , representative traces showing the neuronal activity under the indicated treatments . Ctrl : extracellular solution . Cyclo : cyclopamine . B , the expanded view of a single burst ( arrow ) from A ; C , statistics of the percentage of neurons showing epileptiform activity ( left , n = 6 - 7 ) and the burst frequency ( right , n = 18 - 19 ) shown in A . Cells were incubated in the presence of Cyclo in extracellular solution for 30 min , then transferred to 0Mg plus Cyclo ( 0Mg + Cyclo ) and incubated for another 30 min . ( D , E ) The effects of Sant - 1 , robotnikinin ( Robot ) or 5E1 on the percentage of neurons showing epileptiform activity ( D , n = 6 - 10 ) and the burst frequency ( E , n = 15 - 22 ) . ( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) . ( F - H ) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices . F , representative traces under the indicated treatments . Ctrl : artificial cerebrospinal fluid ( aCSF ) . G , the expanded view of a single burst ( arrow ) in F . Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min , then transferred to picrotoxin in aCSF ( Pic ) or with Cyclo ( Pic + Cyclo ) and recorded for 30 min . H , statistics of the spontaneous burst frequency of F from 10 slices ( 7 rats ) . ( I , J ) Left are representative traces showing the D - CPP - sensitive currents recorded from cultured hippocampal neurons ( I ) or slices ( J ) incubated in 0Mg or Pic , respectively , with or without Cyclo . Black lines : application of D - CPP . Right are statistics of D - CPP - sensitive current density from 18 - 26 cells ( I ) or 16 - 18 slices ( 15 rats ) ( J ) . Unless stated , 10 µM cyclopamine was used . Data were mean + SEM . * p < 0 . 05 , * * < 0 . 01 , * * * p < 0 . 001 vs . Ctrl ; # p < 0 . 05 , # # < 0 . 01 , # # # p < 0 . 001 vs . 0Mg or Pic ; & amp ; amp ; p < 0 . 05 vs . 0Mg + IgG . C - E used one - way ANOVA . H - J used student ' s t test .

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 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 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 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

Figure 2(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min.(D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF).(F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats).(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.


Figure 3 ( A ) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents . n = 4 - 6 . ( B ) The paired - pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle ( Ctrl ) or Shh ( 13 - 14 slices , 6 rats ) . Insets : representative traces recorded in response to paired pulse stimuli with different intervals . Statistics of 3H - glutamate uptake by neurons treated with indicated agents ( C , n = 10 ) Statistics of 3H - glutamate uptake by neurons transfected with two lentivirus - based RNAi against EAAC1 ( R2 and R3 ) or nonsense RNAi ( Non ) in response to vehicle ( Ctrl ) or Shh ( D , n = 3 - 6 ) . No difference between R2 or R3 in Ctrl and those in Shh . Inset of ( D ) : representative western - blots for EAAC1 . Aspartate ( Asp ) - evoked currents at - 70 mV from hippocampal neurons transfected with RNAi or Non ( E , n = 17 - 23 ) or treated with indicated agents ( F , n = 16 - 18 ) . ( G ) Upper , representative western - blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors ( Ctrl ) , constitutively active form of Smoothened ( SmoA1 ) , EAAC1 , or EAAC1 plus SmoA1 . Lower , 3H - glutamate uptake by cells transfected with the indicated vectors . n = 9 . ( H , I ) Shh effects on 3H - glutamate uptake ( H , n = 6 ) or Asp - evoked currents ( I , n = 17 - 23 ) of neurons with or without pertussis toxin ( PTX ) pretreatment . For E , F , I , the uppers : representative traces . Shh : 500 ng / mL . Data were mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl or Non ( Ctrl ) with student ' s t test .

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 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 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 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 O O O O O O O O

Figure 3(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.(B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals.Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.


Figure 4Effects of Cyclo ( A - C , n = 14 ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice . Effects of 5E1 ( D - F , n = 16 - 20 ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice . Effects of ablation of Smo in CaMKIIα - positive neurons ( G - I ) , on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In G - I , Ctrl : Smofl / fl induced by tamoxifen ; CaMK : Smofl / flCaMKIIα - CreERT2 induced by tamoxifen , n = 14 - 20 . Effects of ablation of Smo in Aldh1l1 - positive astrocytes ( J - L ) on the progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) and the number of stimulations required to reach equivalent seizure intensity ( C , F , I , L ) . In J - L , Ctrl : Smo + / fl ; Aldh1l1 : Smo + / flAldh1l1 - Cre , n = 19 - 20 . ( M ) Frequency of spontaneous seizures ( class 4 - 5 ) in mice administrated with Cyclo or vehicle ( Ctrl ) at 4th , 6th and 8th week after pilocarpine SE induction . n = 22 - 23 . ( N ) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction . n = 22 - 23 . Cyclo : 10 mg / kg . 5E1 : 900 ng / mouse . Data were mean ± SEM from at least three independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl with student ' s t test .

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 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 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 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 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

Figure 4Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.Effects of 5E1 (D-F, n=16-20), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.Effects of ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20.(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.


Figure 1 ( B ) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit . For quantification , TAG levels were normalized to protein . Error bars represent ± SEM . * * * : P < 0 . 001 ; NS : non - significant . ( G ) Quantification of the lipid droplet size in ( C and F ) . ND : normal diet ; HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison test . Each point represents data from one fat body , and at least 30 cells were analyzed for each fat body . Error bars represent ± SEM . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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 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

Figure 1(B) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.(G) Quantification of the lipid droplet size in (C and F). ND: normal diet; HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were analyzed for each fat body. Error bars represent ±SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.