Figure 7 Overall survival stratified by tumor mutational burden ( TMB ) across treatment groups in 3 , 217 patients . Samples were classified as TMB - high if the amount of non - synonymous mutations per expressed MB ( rnaMB ) was ≥ the median number of non - synonymous mutations per rnaMB across the whole SCAN - B cohort ( 0 . 082 mutations per rnaMB ) and TMB - low otherwise . In each Kaplan - Meier plot , TMB - low cases are plotted in blue , TMB - high cases are plotted in red , the log - rank P value is given , and the hazard ratio ( HR ) for TMB - high is given with a 95 % CI and after uni - variable and multi - variable ( MV ) Cox regression adjustment . Covariables included in the MV analysis were age at diagnosis , lymph node status , tumor size , and the variables denoted by the following symbols : ¶ , ER , PgR , HER2 , and NHG ; ¤ , ER , PgR , and NHG ; $ , HER2 and NHG ; # , NHG . Abbreviations : TMB , tumor mutational burden ; ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade ; HoR , hormone receptor ; TNBC , triple - negative breast cancer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.


Figure 1B RT - PCR on cytoplasmic ( C ) and nuclear ( N ) extracts showing the subcellular localization of lnc - SMaRT . RNA was isolated from C2C12 cells after two days of differentiation . GAPDH mRNA and pre - mRNA ( pre - GAPDH ) were used respectively as cytoplasmic and nuclear controls . Representative results from three independent experiments are shown . C qPCR analysis of lnc - SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) . D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs ( si - SCR ) or with two different siRNAs against lnc - SMaRT ( si - SMaRT - 1 , si - SMaRT - 2 ) and induced to differentiate for two days . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 . E Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) C2C12 murine myoblasts transfected with either control ( si - SCR ) or lnc - SMaRT siRNAs ( si - SMaRT - 1 , si - SMaRT - 2 ) fixed after at two days of differentiation . Histograms represent MHC - positive mononucleated cells / total MHC - positive cells ratio and fusion index quantification ( F . I . ) . At least 5 randomly chosen microscope fields of two independent biological samples were analyzed ( n > 600 cells for each field ) . Data are presented as the mean . of the two biological replicates ( dots ) .

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 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 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 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

Figure 1B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).


Figure 2A Gene expression heatmap showing the hierarchical clustering of protein - coding genes whose expression is altered upon lnc - SMaRT depletion . Expression values , calculated as RPKMs , were log2 - transformed and mean centered . Only genes with an average RPKM between 10 and 1000 were plotted . B Volcano plot describing the differential gene expression between si - SCR and si - SMaRT treated C2C12 samples . For each gene , log2 of fold - change versus - log10 of the unadjusted p - value is plotted . Points in red represent differentially expressed genes ( FDR adjusted p - value < 0 . 05 ) . D Western blot on protein extract from control and lnc - SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation . CASP3 antibody was used . ACTN was used as loading control . Representative results from three independent experiments are shown .

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 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

Figure 2A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.B Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.


Figure 3A Localization on the lnc - SMaRT transcript of the two sets of biotinylated probes ( Set # 1 and Set # 2 ) used for lnc - SMaRT pull - down experiment . B Left panel : qPCR analysis of lnc - SMaRT enrichment in the RNA pull - down performed in C2C12 cells at day 2 of differentiation ( DM2 ) ; Set # 1 and Set # 2 probes were used against lnc - SMaRT together with a control set of probes against LacZ mRNA ( LacZ ) . Data are expressed in percentage of input and presented as the mean ± s . e . m . of three biological replicates ( dots ) . Right panel : Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc - SMaRT pull - down ; GAPDH was used as the negative control . Representative results from three independent experiments are shown . C Upper panel : Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation . Input sample ( IN ) accounts for 2 . 5 % of the extract . Representative results from three independent experiments are shown . Lower panel : qPCR analysis of lnc - SMaRT enrichment in DHX36 RIP - derived RNA extracts . WBP4 was used as positive control [ 31 ] , while Neat1 ( a lncRNA expressed at comparable level of lnc - SMaRT according to the RNA - sequencing ) and Rps7 were used as negative controls . Data are expessed as percentage of input normalized on IgG control , and presented as the mean ± s . e . m . of three biological replicates ( dots ) . D RT - PCR validation of Mlx - α , Mlx - β and MLX - γ mRNA enrichment upon the lnc - SMaRT pull - down performed with Set # 1 and Set # 2 probes ; a control set of probes against LacZ mRNA ( LacZ ) was used as negative control . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown . E RT - PCR analysis of Mlx - α , Mlx - β and Mlx - γ mRNA enrichment in DHX36 RIP - derived RNA extracts in samples treated with control siRNA ( siSCR , upper panel ) or siRNA against lnc - SMaRT ( siSMaRT , lower panel ) . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown . F Upper panel : Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG - tagged MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . FLAG - tag has been inserted at the N - terminus of MLX protein isoforms . HPRT was used as loading control . Representative results from three independent experiments are shown . Middle panel : Quantification of FLAG - tagged protein levels normalized on HPRT signals . Data are expressed presented as the mean ± s . d . of three biological replicates ( dots ) . Statistical analysis was performed with two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * * * P < 0 . 001 . Lower panel : RT - PCR analysis of lnc - SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged - MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . GAPDH was used as loading control . Representative results from three independent experiments are shown . G Upper panel : Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector ( CTRL ) or with a plasmid overexpressing FLAG - tagged MLX - γ ( MLX - γ ) in control conditions ( si - SCR ) or in knockdown of DHX36 ( si - DHX36 ) . ACTININ was used as loading control . Representative results from three independent experiments are shown . Lower panel : RT - PCR analysis of Mlx - γ expression on the described RNA extracts . GAPDH was used as loading control . Representative results from three independent experiments are shown .

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

Figure 3A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown. C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots). D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001. Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown. G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown. Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.


Figure 4A Immunofluorescence for total MLX protein detection ( red signal ) performed on C2C12 cells treated with siRNAs against Mlx - γ ( si - Mlx - γ ) , lnc - SMaRT ( si - SMaRT ) , DHX36 ( si - DHX36 ) or with a control siRNA ( si - SCR ) . Cells were fixed two days after the switch to differentiation medium . Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment . The inserts on the topside of the images sketch the MLX signal peaks in the nuclei ( white stars ) to highlight the variation of MLX staining in each condition . Dashed lines indicate the edge of the nucleus . B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear ( Nuc ) and cytoplasmic ( Cyt ) compartment in the indicated conditions . About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates ( dots ) .

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Figure 4A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).


Figure 5A Left panel : schematic representation of the luciferase constructs produced for Mlx . Mlx 5 ' - and 3 ' UTR - interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region ( RLuc - Mlx 5 ' - 3 ' ) and mutant derivatives devoid of the 5 ' interacting region ( RLuc - Mlx - 3 ' ) or the 3 ' UTR interacting region ( RLuc - Mlx - 5 ' ) were obtained . These constructs were co - transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx 5 ' - 3 ' , RLuc - Mlx - 3 ' , RLuc - Mlx - 5 ' ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * P < 0 . 05 . B The RLuc - Mlx - 5 ' construct was co - transfected in N2a cells together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) and treated with control siRNA ( + ) or siRNA against DHX36 ( - ) . Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to RLuc - Mlx - 5 ' vector set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * P < 0 . 01 , * * * P < 0 . 001 . C Left panel : schematic representation of the luciferase constructs used . The RLuc - Mlx - 5 ' construct and its derived mutants ( RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) were transfected in N2a cells in growth conditions together with lnc - SMaRT - expressing plasmids ( lnc - SMaRT ) or with its derived mutant ( lnc - SMaRTmut ) in the indicated combinations . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx - 5 ' , RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

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

Figure 5A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


Figure 6A Upper panel : schematic representation of the predicted base pairing between lnc - SMaRT ( region A ) and Mlx γ mRNA ; the predicted G - 4 structure is boxed in red while the synthetic oligos sequences are underlined in red . Lower panel : Lane 1 and 2 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA ( SYBR GOLD , indicated as SYBR in the figure ) . Lane 3 and 4 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 5 and 6 : 200 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX ( NMM ) . Lane 7 and 8 : 5 pmol of Mlx oligo alone ( 7 ) or in combination with 5pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 9 and 10 : 200 pmol of Mlx oligo alone ( 7 ) or in combination with 200 pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown . B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel . Oligos were treated as described . ° C = oligos were heated ( + ) or were not heated ( - ) at 100 ° C for 10 min and loaded after slow cooling . KCl = oligos were incubated ( + ) or were not ( - ) in a buffer containing 100mM KCl ( for G - quadruplex stabilization ) . The gel was stained with total RNA staining ( SYBR GOLD ) . Data information : Representative gels from at least three independent experiments are shown . C 200 pmol of indicated oligos treated as in ( B ) were loaded on a native polyacrylamide gel with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

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

Figure 6A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


Figure 1 ( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d . Lung sections stained with Haematoxylin and eosin ( H & E ) ( A ) and quantification of lung lesion burden from H & E - stained sections ( B ) . Data information : Data are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm . ( D - G ) H & E staining of lung sections from mice 4 weeks after intranasal infection with M . bovis BCG ( D and E ) or Mtb H37Rv ( F and G ) in the absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Quantification of lung inflamed regions shown in ( E and G ) . Data information : Data in are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test and Student ' s t test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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Figure 1(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d. Lung sections stained with Haematoxylin and eosin(H&E) (A) and quantification of lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.(D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with M. bovis BCG (D and E) or Mtb H37Rv (F and G) in the absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


Figure 2 ( B ) Quantitative polymerase chain reaction ( qPCR ) analysis of Mmps including Mmp2 , Mmp9 , Mmp10 , Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG ( 1 μg / ml ) for indicated times . ( C ) Immunoblots of cell supernatants to analyze secreted MMP2 , MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG ( 1 μg / mL ) for indicated times ; GADPH of cell lysates served as a loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . ( H ) qPCR analysis of Mmps including Mmp9 , Mmp10 , Mmp12 , and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h ( MOI = 5 ) in absence or presence of AG aptamers ( 1 μg / ml ) . ( I ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in the absence or presence of AG aptamers ( 0 . 5 μg / ml ) ; GADPH of cell lysates served as the loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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Figure 2(B) Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times. (C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times; GADPH of cell lysates served as a loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG aptamers (1 μg/ml). (I) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


Figure 3 ( G ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 , and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in absence or presence of inhibitor targeting ERK ( PD98059 ) at the concentration of 10 μM ; GADPH of cell lysates served as the loading control .

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Figure 3(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.


Figure 4 ( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E ( A ) and quantification of lung lesion burden from H & E stained sections ( B ) . Data information : Data in ( A ) are representative of n = 3 independent experiments . Data in ( B ) are means + SD of indicated numbers of mice from one of n = 3 independent experiments and each symbol represents data from 1 mouse . One - way ANOVA followed by Dunnett ' s post hoc test ( B ) was used for statistical analysis . ns , not significant ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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Figure 4(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.


Figure 5 ( A ) Surface plasmon resonance ( SPR ) assay of the direct interaction of AG with galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines . ( C ) SPR assay of the interaction of AG with CRD2 of galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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Figure 5(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


Figure 6 ( I ) Immunoblot of lysates of shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for indicated times . Data are representative of n = 3 independent experiments . ( K ) qRT - PCR detection of MMP transcripts including Mmp9 , Mmp10 , and Mmp12 in shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for 24 h in absence or presence of ERK inhibitor PD98059 ( 10 μM ) . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis . ns , not significant ; * , p < 0 . 05 ; * * * , p < 0 . 001 * * * * , p < 0 . 0001 .

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Figure 6(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.


Figure 7 ( D ) Confocal microscopy of mouse peritoneal macrophages left untreated ( NC ) ( upper row ) or stimulated with AG ( 1 μg / mL ) for 2 h ( middle row ) or infected with H37Rv for 3 h ( MOI = 5 ) ( bottom row ) , staining with anti - Galectin - 9 and anti - TAK1 antibody . DAPI , nuclei , blue . Scale bar , 5 μm . Data in the right graph show mean + SD of n = 12 fields from three independent experiments . The symbols indicate the colocalization ratio of at least 10 cells in each field . ( F ) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin - 9 KO mice stimulated with AG ( 1 μg / ml ) in absence or presence of TAK1 inhibitor 5Z - 7 - OZ ( 1 μM ) for indicated times . Data are representative of n = 3 independent experiments .

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Figure 7(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments.


Figure 8 ( B , WT or Galectin - 9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments . ( D , WT or Galectin - 9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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Figure 8(B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.(D, WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


Figure 1 . ( C ) Reaction progress was measured for the Twist COVID - 19 synthetic RNA ( MT007544 . 1 ) from 1 million molecules of virus ( 106 ) , then titrated down by log10 dilutions . The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS - CoV - 2 RNA corresponding to a yellow color . The limit of detection ( LoD ) range is shown with a gradient after 30 minutes between 10 and 100 viral copies ( lower right ) . ( D ) Replicates of the titrated viral copies using LAMP , as measured by Quantifluor fluorescence . ( E ) The sensitivity and specificity of the LAMP assay from 201 patients ( 132 negative and 69 positive for SARS - CoV - 2 , as measured by qRT - PCR ) . Thresholds are DNA quantified by the Quantifluor .

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Figure 1.(C) Reaction progress was measured for the Twist COVID-19 synthetic RNA (MT007544.1) from 1 million molecules of virus (106), then titrated down by log10 dilutions. The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS-CoV-2 RNA corresponding to a yellow color. The limit of detection (LoD) range is shown with a gradient after 30 minutes between 10 and 100 viral copies (lower right).(D) Replicates of the titrated viral copies using LAMP, as measured by Quantifluor fluorescence.(E) The sensitivity and specificity of the LAMP assay from 201 patients (132 negative and 69 positive for SARS-CoV-2, as measured by qRT-PCR). Thresholds are DNA quantified by the Quantifluor.


Figure 2 . LAMP . ( a ) Clinical samples tested by qRT - PCR ( Positive , dark red or Negative , light grey ) were sequenced and run through the LAMP assay . These results were compared to the buffer blanks ( Negative Control ) , dark grey , Synthetic RNAs or Vero 6 cell extracts with SARS - CoV - 2 infection ( Positive Controls , light red ) , and Subway Samples ( Environmental , blue ) . Read proportions are shown on the y - axis . ( b ) SARS - CoV - 2 abundance , as measured with NGS and percentage of reads ( y - axis ) is compared to the Ct Threshold for qRT - PCR ( x - axis ) , with lower Ct values representing higher viral abundance , and the LAMP reaction output ( Fluorimeter values , black to yellow scale ) . ( c , d ) Read mapping to archaea ( red ) , bacteria ( green ) , fungi ( yellow ) , human ( blue ) , and SARS - CoV - 2 ( orange ) , and other viruses ( grey ) , across the clinical controls ( CN , CP ) , environmental samples , qRT - PCR negative , and qRT - PCR positive samples .

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Figure 2.LAMP.(a) Clinical samples tested by qRT-PCR (Positive, dark red or Negative, light grey) were sequenced and run through the LAMP assay. These results were compared to the buffer blanks (Negative Control), dark grey, Synthetic RNAs or Vero 6 cell extracts with SARS-CoV-2 infection (Positive Controls, light red), and Subway Samples (Environmental, blue). Read proportions are shown on the y-axis.(b) SARS-CoV-2 abundance, as measured with NGS and percentage of reads (y-axis) is compared to the Ct Threshold for qRT-PCR (x-axis), with lower Ct values representing higher viral abundance, and the LAMP reaction output (Fluorimeter values, black to yellow scale).(c,d) Read mapping to archaea (red), bacteria (green), fungi (yellow), human (blue), and SARS-CoV-2 (orange), and other viruses (grey), across the clinical controls (CN, CP), environmental samples, qRT-PCR negative, and qRT-PCR positive samples.


Figure 3 : coverage ( a ) The coverage plot across the SARS - CoV - 2 genomes ( viral coordinate on bottom , colored by sample ) from a representative set of clinical positive samples . Sample names with the suffixes CN and N are clinical negative ( buffer ) , P are qRT - PCR positive , and CP are Vero 6 cells with virus .

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Figure 3:coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples. Sample names with the suffixes CN and N are clinical negative (buffer), P are qRT-PCR positive, and CP are Vero 6 cells with virus.


Figure 4 : The phylogenetic placement of these COVID samples is shown on the tree ( left ) and the global map of known COVID genomes ( right ) . Genetic variants called from the RNA - seq data show a range of variants that are distinct from the Wuhan reference strain , and the samples from this study , highlighted in blue , show enrichment for European and Asian alleles . ( b ) Proportion of the L ( green ) and S strain ( yellow ) are shown for the NYC viruses . Phylogeny of samples from this study on the left and total GISAID samples on the right , with a map of variants in this study ' s samples in the middle , colored by event type and sized by number of nucleotides impacted . Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database . ( c ) The 9 - bp deletion in ORRF1b ( NSP1 ) that was detected in samples from two patients was confirmed in the GISAID database ( below ) . ( d ) Comparison of the ORF1b of the SARS - CoV - 2 and SARS - CoV genomes with the 9 - bp deletion region shown enlarged .

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Figure 4:The phylogenetic placement of these COVID samples is shown on the tree (left) and the global map of known COVID genomes (right). Genetic variants called from the RNA-seq data show a range of variants that are distinct from the Wuhan reference strain, and the samples from this study, highlighted in blue, show enrichment for European and Asian alleles.(b) Proportion of the L (green) and S strain (yellow) are shown for the NYC viruses. Phylogeny of samples from this study on the left and total GISAID samples on the right, with a map of variants in this study's samples in the middle, colored by event type and sized by number of nucleotides impacted. Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database.(c) The 9-bp deletion in ORRF1b (NSP1) that was detected in samples from two patients was confirmed in the GISAID database (below).(d) Comparison of the ORF1b of the SARS-CoV-2 and SARS-CoV genomes with the 9-bp deletion region shown enlarged.


Figure 5 : ( a ) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM / NYP cases as compared with non - NYC strains of Nextstrain clade A2a ( 1059 : C > T P = 6 . 26e - 22 , 11916 : C > T P = 3 . 09e - 9 , 18998 : C > T P = 2 . 51e - 7 , 20755 : A > C P = 1 . 32e - 4 , 25563 : G > T P = 9 . 6e - 23 , 29540 : G > A P = 2 . 51e - 7 ) . These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non - NYC clade A2a ( 1059 : C > T P = 4 . 35e - 84 , 11916 : C > T P = 2 . 16e - 43 , 18998 : C > T P = 1 . 34e - 25 , 20755 : A > C P = 1 . 67e - 4 , 25563 : G > T P = 2 . 60e - 83 , 29540 : G > A P = 1 . 34e - 46 ) . ( b ) ( left ) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM / NYP in red and other NYC cases in green . ( right ) occurrence of the six NYC - enriched alleles and the 9 nucleotide deletion across genomes .

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Figure 5:(a) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM/NYP cases as compared with non-NYC strains of Nextstrain clade A2a (1059:C>T P = 6.26e-22, 11916:C>T P = 3.09e-9, 18998:C>T P= 2.51e-7, 20755:A>C P = 1.32e-4, 25563:G>T P = 9.6e-23, 29540:G>A P = 2.51e-7). These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non-NYC clade A2a (1059:C>T P = 4.35e-84, 11916:C>T P = 2.16e-43, 18998:C>T P= 1.34e-25, 20755:A>C P = 1.67e-4, 25563:G>T P = 2.60e-83, 29540:G>A P = 1.34e-46).(b) (left) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM/NYP in red and other NYC cases in green. (right) occurrence of the six NYC-enriched alleles and the 9 nucleotide deletion across genomes.


Figure 6 : Samples were quantified by a range of viral detection methods , including LAMP ( Quantifluor ) , RNA - seq ( log10 SARS - CoV - 2 % of reads ) , and qRT - PCR ( Ct values ) to create a three - tier range of viral load for the positive samples ( right ) compared to the clinically - annotated negative samples ( class , red or grey ) . ( bottom ) The differentially expressed genes of COVID + patients compared to COVID - patients showed up - regulated ( orange ) genes as well as down - regulated ( purple ) genes . ( b ) Up - regulated genes , with boxplots across all samples , include IFI6 , ACE2 , SHFL , HERC6 , IFI27 , and IFIT1 , based on data from ( c ) , which is the total set of DEGs . The full set is shown in an intersecting heat map , with a core set of up - regulated genes ( orange ) distinct form the set of down - regulated genes ( purple ) , compared to genes that are not significantly differently expressed ( grey ) in any comparison ( DESeq2 , q - value < 0 . 01 , | logFC | > 0 . 58 ) . ( e ) Survival curves for requiring mechanical respiration ( identified by intubation procedure notes ) ( e ) and mortality ( f ) . Patients with a history of ACE inhibitor exposure were more likely to require intubation ( HR = 2 . 63 95 % CI 2 . 01 - 3 . 43 , p = 1 . 22E - 12 ; ( e ) and less likely to survive ( HR = 1 . 68 95 % CI : 1 . 22 - 2 . 31 , p = 1 . 42E - 03 ; ( f ) . Because several individuals were intubated shortly before they first tested positive for SARS - CoV - 2 infection , each first positive test was set back by seven days to account for testing delays .

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Figure 6:Samples were quantified by a range of viral detection methods, including LAMP (Quantifluor), RNA-seq (log10 SARS-CoV-2 % of reads), and qRT-PCR (Ct values) to create a three-tier range of viral load for the positive samples (right) compared to the clinically-annotated negative samples (class, red or grey). (bottom) The differentially expressed genes of COVID+ patients compared to COVID-patients showed up-regulated (orange) genes as well as down-regulated (purple) genes.(b) Up-regulated genes, with boxplots across all samples, include IFI6, ACE2, SHFL, HERC6, IFI27, and IFIT1, based on data from (c), which is the total set of DEGs. The full set is shown in an intersecting heat map, with a core set of up-regulated genes (orange) distinct form the set of down-regulated genes (purple), compared to genes that are not significantly differently expressed (grey) in any comparison (DESeq2, q-value <0.01, |logFC| >0.58).(e)Survival curves for requiring mechanical respiration (identified by intubation procedure notes) (e) and mortality (f). Patients with a history of ACE inhibitor exposure were more likely to require intubation (HR=2.63 95%CI 2.01-3.43, p=1.22E-12; (e) and less likely to survive (HR=1.68 95%CI: 1.22-2.31, p=1.42E-03; (f). Because several individuals were intubated shortly before they first tested positive for SARS-CoV-2 infection, each first positive test was set back by seven days to account for testing delays.


Figure 1A . Mutations in the kinase motif abolished the yeast toxicity of MavQ . S . cerevisiae was transformed with plasmids expressing Flag - tagged wild - type MavQ or the indicated MavQ mutants under the galactose - inducible promoter . Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition . B . The expression of MavQ in the yeast was detected by Flag antibody . The PGK ( 3 - phosphoglyceric phosphokinase ) was used as a loading control .

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Figure 1A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition.B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.


Figure 2A . Biochemical assays for the kinase activity of MavQ . Purified MavQ was incubated with or without specific PI substrates , the conversion of ATP to ADP was measured by the ADP - Glo assay . RLU , relative luminescence units . B . H149 , N152 and D160 in MavQ are critical for its kinase activity . Purified wild - type and mutant MavQ were reacted with PtdIns and ATP . The kinase activity was determined by the ADP - Glo assay . C . The PI product produced by MavQ determined by TLC assays . di - C8 - Bodipy - FL - PtdIns was used to react with MavQ ( lane 4 ) and samples were further incubated with the 3 - phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns ( 3 , 5 ) P2 ( lane 5 ) or with the PI4K LepB _ NTD which phosphorylates PtdIns3P into PtdIns ( 3 , 4 ) P2 ( lane 6 ) . A schematic illustration of the enzymatic reactions is shown in the lower panel . D . Mutations in H149 , N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P .

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Figure 2A. Biochemical assays for the kinase activity of MavQ. Purified MavQ was incubated with or without specific PI substrates, the conversion of ATP to ADP was measured by the ADP-Glo assay. RLU, relative luminescence units.B. H149, N152 and D160 in MavQ are critical for its kinase activity. Purified wild-type and mutant MavQ were reacted with PtdIns and ATP. The kinase activity was determined by the ADP-Glo assay.C. The PI product produced by MavQ determined by TLC assays. di-C8-Bodipy-FL-PtdIns was used to react with MavQ (lane 4) and samples were further incubated with the 3-phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns(3,5)P2 (lane 5) or with the PI4K LepB_NTD which phosphorylates PtdIns3P into PtdIns (3,4)P2 (lane 6). A schematic illustration of the enzymatic reactions is shown in the lower panel.D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.


Figure 3A . Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP - 2xFYVEHrs and MavQ with or without wortmannin treatment . Scale bars , 5 μm . B . Percentage of HeLa cells transfected with the indicated plasmids in which GFP - 2xFYVEHrs localizes to vesicle - like structures . At least 100 cells ( n ≥ 100 ) were counted for each sample . Wort , wortmannin . C . Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin . Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP . The wortamannin - sensitive PI3K p110α was used as a control . The ADP - Glo assay was used to measure the kinase activity . D . Wortmannin does not affect the production of PtdIns3P by MavQ . Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature . di - C8 - Bodipy - FL - PtdIns and ATP were then added to the reaction mixtures . The TLC assay was employed to detect the generation of PtdIns3P by MavQ . The wortamannin - sensitive PI3K p110α was included as a control .

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Figure 3A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm.B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin.C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.


Figure 4A . Representative immunofluorescence images of the association of MavQ with the LCV . BMDM cells infected with wild - type or Dot / Icm - deficient ( dotA - ) L . pneumophila were sequentially stained with antibodies against L . pneumophila and MavQ . Images were taken under a fluorescence microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm . B . The rates of of LCVs positively stained for MavQ 2 h post - infection . At least 100 LCVs ( n ≥ 100 ) were scored for each sample . C . Dynamics of MavQ association with LCV . BMDMs were infected with the wild - type L . pneumophila strain at an MOI of 2 for the indicated experimental durations . At least 100 phagosomes ( n ≥ 100 ) were counted for each sample to obtain the percentage of MavQ - positive LCVs . D . Intracellular growth of L . pneumophila in macrophages . BMDMs were infected with indicated L . pneumophila strains at an MOI of 0 . 05 . At the indicated time points , CFUs were determined by plating the saponin - solubilized lysates of infected cells on CYE plates . E . Expression of mavQ in L . pneumophila strains used for infection . Total proteins of bacteria cultured to the post - exponential phase separated by SDS - PAGE were probed with MavQ - specific antibodies . ICDH was detected as a loading control .

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Figure 4A. Representative immunofluorescence images of the association of MavQ with the LCV. BMDM cells infected with wild-type or Dot/Icm-deficient (dotA-) L. pneumophila were sequentially stained with antibodies against L. pneumophila and MavQ. Images were taken under a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.B. The rates of of LCVs positively stained for MavQ 2 h post-infection. At least 100 LCVs (n≥100) were scored for each sample.C. Dynamics of MavQ association with LCV. BMDMs were infected with the wild-type L. pneumophila strain at an MOI of 2 for the indicated experimental durations. At least 100 phagosomes (n≥100) were counted for each sample to obtain the percentage of MavQ-positive LCVs.D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates.E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.


Figure 5A . Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ , LepB _ NTD and SidF . MavQ was reacted with di - C8 - Bodipy - FL - PtdIns , resulting in the production of PtdIns3P ( lane 6 ) . The PtdIns3P - specific PI4K LepB - NTD was then added into the reaction , yielding PtdIns ( 3 , 4 ) P2 ( lane 7 ) . The product of the sample receiving LepB _ NTD was further incubated with the 3 - phosphatase SidF to yield PtdIns4P ( lane 8 ) . The lipid products were detected and visualized by TLC . A schematic illustration of the enzymatic reactions is shown in the lower panel . B . Representative images of SidC anchoring on LCVs . BMDMs infected with the indicated L . pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC . Images were acquired using a fluorescence microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm . C . Quantitation of SidC - positive LCVs . At least 100 phagosomes ( n ≥ 100 ) were scored for each sample . D . Expression and translocation of SidC . The lysates of L . pneumophila strains used for infection were probed for protein expression , ICDH was detected as a loading control ( top panel ) . SidC translocation was detected by probing the saponin - soluble fractions of infected U937 cells with SidC - specific antibodies . Tubulin was probed as a loading control ( bottom panel ) .

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Figure 5A. Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ, LepB_NTD and SidF. MavQ was reacted with di-C8-Bodipy-FL-PtdIns, resulting in the production of PtdIns3P (lane 6). The PtdIns3P-specific PI4K LepB-NTD was then added into the reaction, yielding PtdIns(3,4)P2 (lane 7). The product of the sample receiving LepB_NTD was further incubated with the 3-phosphatase SidF to yield PtdIns4P (lane 8). The lipid products were detected and visualized by TLC. A schematic illustration of the enzymatic reactions is shown in the lower panel.B. Representative images of SidC anchoring on LCVs. BMDMs infected with the indicated L. pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC. Images were acquired using a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.C. Quantitation of SidC-positive LCVs. At least 100 phagosomes (n≥100) were scored for each sample.D. Expression and translocation of SidC. The lysates of L. pneumophila strains used for infection were probed for protein expression, ICDH was detected as a loading control (top panel). SidC translocation was detected by probing the saponin-soluble fractions of infected U937 cells with SidC-specific antibodies. Tubulin was probed as a loading control (bottom panel).


Figure 6A . Direct interactions between SidP and MavQ in the GST pull - down assay . GST beads coated with GST - SidP or GST were incubated with His6 - MavQ . After washing the beads with GST binding buffer , the proteins resolved by SDS - PAGE were detected by Coomassie brilliant blue staining . B , SidP does not impair the kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , and the kinase activity of MavQ was measured by the ADP - Glo assay ( B ) C . SidP does not impair the kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , and the kinase activity of MavQ was measured by TLC assay ( C ) . D . Co - expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ . The association of the fluorescence probe GFP - 2xFYVEHrs with vesicle - like structures was used to indicate the distribution of PtdIns3P in cells . At least 100 cells were scored in each sample ( n ≥ 100 ) ( left panel ) . Wort , wortmannin . Expression of MavQ and SidP in the samples were shown in the right panel . EV represents empty vector . E . Deletion of sidP does not impact the association of SidC with the LCV . BMDMs infected with the indicated L . pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined ( n ≥ 100 ) ( left panel ) . Representative images of infected cells harboring bacterial phagosomes were also shown ( right panel ) . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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

Figure 6A. Direct interactions between SidP and MavQ in the GST pull-down assay. GST beads coated with GST-SidP or GST were incubated with His6-MavQ. After washing the beads with GST binding buffer, the proteins resolved by SDS-PAGE were detected by Coomassie brilliant blue staining.B, SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by the ADP-Glo assay (B)C. SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by TLC assay (C).D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector.E. Deletion of sidP does not impact the association of SidC with the LCV. BMDMs infected with the indicated L. pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined (n≥100) (left panel). Representative images of infected cells harboring bacterial phagosomes were also shown (right panel). Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


Figure 1 ( B ) Time constants of decay , τdecay , of the STC isolated pharmacologically in WT and FHM2 KI mice . Top , superimposed representative traces of the inward current evoked in an astrocyte ( held at - 90 mV ) by a single pulse stimulation ( indicated by the arrow ) in a WT slice , before ( trace a ) and after ( trace b ) application of a saturating concentration of the GluT inhibitor TFB - TBOA ( TBOA ) . The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current ( trace a - b ) ; the decay of the STC was best fitted by a single exponential function with τdecay = 6 . 53 ms ( in red ) . The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices ( n = 13 ) from P22 - 23 WT ( N = 7 ) and KI mice ( n = 9 ; N = 3 ) . STC τdecay is 17 % higher in KI compared to WT astrocytes ( unpaired t - test : P = 0 . 003 ) . Hereafter , n indicates the number of slices and N indicates the number of mice . ( C ) τdecay of the STC isolated using an exponential waveform approximating the average TBOA - insensitive current in WT and KI mice . Top trace , average normalized TBOA - insensitive current obtained by pooling the normalized TBOA - insensitive currents recorded in 16 WT and KI cells . This current was best fitted by an exponentially rising function ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms ( in green ) . The STC was obtained by subtracting from the total current elicited in the astrocyte ( a : same representative trace as in ( B ) ) the exponential function A ( 1 - exp ( - t / τrise ) ) with τrise = 2 . 35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation ( trace c ) ; the decay of the STC ( trace a - c ) was best fitted by a single exponential function with τdecay = 6 . 49 ms ( in red ) . The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22 - 23 WT ( n = 28 ; N = 11 ) and KI mice ( n = 27 ; N = 9 ) . STC τdecay is 21 % higher in KI compared to WT astrocytes ( unpaired t - test : P < 0 . 0001 ) .

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 O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice.(C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).


Figure 2 ( A ) Isolation of the STC elicited by the last pulse of a high - frequency train of action potentials . Left : superimposed representative traces of the inward current evoked in an astrocyte ( held at - 90 mV ) by extracellular stimulation with a train of 10 pulses ( trace a : black ) and a train of 9 pulses ( trace b : brown ) at 50 Hz in a WT cortical slice . The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses ( trace a - b ) . Right : The STC elicited by the 10th pulse ( trace a - b - c ) was obtained by subtracting the exponential function that simulates the TBOA - insensitive current elicited by a single pulse ( trace c , obtained as in Figure 1C ) to the 10 - 9 pulses difference current ( trace a - b ) . The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8 . 04 ms ( in red ) . ( B ) τdecay of the STC elicited by the 10th pulse of 50 Hz ( τ10 ( 50 Hz ) , left panel ) and 100 Hz ( τ10 ( 100 Hz ) , right panel ) trains in layer 1 astrocytes in acute cortical slices from P22 - 23 WT ( n = 23 ; N = 10 for 50 Hz ; n = 18 ; N = 8 for 100 Hz ) and KI ( n = 21 ; N = 9 for 50 Hz and n = 14 ; N = 7 for 100 Hz ) mice . The STC elicited by the 10th pulse was obtained as described in ( A ) . STC τ10 ( 50 Hz ) and τ10 ( 100 Hz ) are 30 % and 37 % higher in KI compared to WT mice , respectively ( unpaired t - test : P < 0 . 0001 in both cases ) .

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 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

Figure 2(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).(B) τdecay of the STC elicited by the 10th pulse of 50 Hz (τ10 (50 Hz), left panel) and 100 Hz (τ10 (100 Hz), right panel) trains in layer 1 astrocytes in acute cortical slices from P22-23 WT (n = 23; N=10 for 50 Hz; n = 18; N=8 for 100 Hz) and KI (n = 21; N=9 for 50 Hz and n = 14; N=7 for 100 Hz) mice. The STC elicited by the 10th pulse was obtained as described in (A). STC τ10 (50 Hz) and τ10 (100 Hz) are 30% and 37% higher in KI compared to WT mice, respectively (unpaired t-test: P < 0.0001 in both cases).


Figure 3 ( A ) Simultaneous visualization of GLT - 1a + ( green ) and VGLUT1 + puncta ( red ) in first somatic sensory cortex ( SI ) of a WT and a KI mouse ( P35 ) . Arrows point to some GLT - 1a + puncta overlaying with VGLUT1 + puncta ( i . e . , GLT - 1a / VGLUT1 related puncta ) ; framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . All microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . ( B ) Percentage and size of GLT - 1a + puncta overlaying with VGLUT1 in P35 WT and KI mice . Left , percentage of GLT - 1a + puncta overlaying with VGLUT1 is comparable in WT and KI mice ( data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice ( 4 sections / animal ) , respectively ) ( unpaired t - test : P = 0 . 52 ) . Right , size of GLT - 1a / VGLUT1 related puncta is 19 % reduced in KI mice ( 169 GLT - 1a + puncta analyzed from 2 mice ) compared to WT ( 174 GLT - 1a + puncta analyzed from 2 mice ) ( Mann - Withney U test : P = 0 . 016 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e., GLT-1a/VGLUT1 related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.(B) Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 µm x 20 µm from 2 WT and 2 KI mice (4 sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1 related puncta is 19 % reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann-Withney U test: P = 0.016).


Figure 4 ( A ) Distribution of GLT - 1agold particles in astrocytic processes contacting asymmetric synapses ( perisynaptic astrocytic processes : PAP ) in SI of 5 WT and 5 KI ( 2 male , 3 female ) mice ( P34 - 35 ) . In cortical PAP of KI mice the density ( particles / µm2 ) of total and membrane - associated gold particles ( arrowheads ) are reduced compared to WT mice , whereas the density of cytoplasmicgold particles ( arrows ) is comparable to that in WT ( cf Table I for numerical values and statistics ) . ( B ) Distribution of GLT - 1agold particles in excitatory axon terminals forming asymmetric synaptic contact ( AxT ) in SI of P34 - 35 WT and KI mice . In cortical AxT of KI and WT mice , GLT - 1a density is comparable ( arrowheads : membrane - associated gold particles ; arrows : cytoplasmicgold particles ) ( cf Table I ) .

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 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

Figure 4(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).(B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).


Figure 5Left , superimposed representative traces of the normalized inward current evoked in a WT ( black trace ) and a KI corticalastrocyte ( blue trace ) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices . The inset shows in an expanded time scale the portion of the traces indicated by the dotted line . The slowly decaying current ( τdecay = 2 . 00 s and 3 . 04 s for the WT and KI trace , respectively ) is largely a [ K + ] e - dependent K + current ( IK ) whose decay kinetics provide a measure of the rate of K + clearance by astrocytes ( see text ) . Right , time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22 - 23 WT ( n = 21 ; N = 12 ) and KI mice ( n = 20 ; N = 8 ) . IK τdecay is 22 % higher in KI compared to WT astrocytes ( unpaired t - test : P = 0 . 001 ) .

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

Figure 5Left, superimposed representative traces of the normalized inward current evoked in a WT (black trace) and a KI corticalastrocyte (blue trace) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices. The inset shows in an expanded time scale the portion of the traces indicated by the dotted line. The slowly decaying current (τdecay = 2.00 s and 3.04 s for the WT and KI trace, respectively) is largely a [K+]e-dependent K+current (IK) whose decay kinetics provide a measure of the rate of K+ clearance by astrocytes (see text). Right, time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22-23 WT (n = 21; N=12) and KI mice (n = 20; N=8). IK τdecay is 22% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.001).


Figure 6 ( A ) Images of a cortical slice before ( t = 0 ) and after ( t = 15 . 6 s ) pressure ejection of a high KCl pulse that elicits a CSD ( top panels ) , showing that the propagating CSD is associated with a change in light transmittance . Scale bar : 500 µm . The traces below show representative changes in intrinsic optical signal ( IOS ) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff , as indicated in color code in the right image on the top . The velocity of CSD propagation , obtained from the rate of horizontal spread of the change in IOS , in these two representative WT and KI slices is 3 . 11 and 4 . 14 mm / min , respectively . ( B ) Stimulation threshold for CSD induction ( CSD threshold ) and rate of CSD propagation ( CSD velocity ) in WT ( n = 24 ; N = 3 ) and KI ( n = 27 ; N = 8 ) cortical slices from P22 - 23 mice . CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD . CSD threshold is 28 % lower ( Mann Whitney U test , P < 0 . 0001 ) and CSD velocity 21 % higher ( unpaired t - test : P < 0 . 0001 ) in KI compared to WT mice .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of a cortical slice before (t = 0) and after (t = 15.6 s) pressure ejection of a high KCl pulse that elicits a CSD (top panels), showing that the propagating CSD is associated with a change in light transmittance. Scale bar: 500 µm. The traces below show representative changes in intrinsic optical signal (IOS) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff, as indicated in color code in the right image on the top. The velocity of CSD propagation, obtained from the rate of horizontal spread of the change in IOS, in these two representative WT and KI slices is 3.11 and 4.14 mm/min, respectively.(B) Stimulation threshold for CSD induction (CSD threshold) and rate of CSD propagation (CSD velocity) in WT (n = 24; N=3) and KI (n = 27; N=8) cortical slices from P22-23 mice. CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD. CSD threshold is 28% lower (Mann Whitney U test, P < 0.0001) and CSD velocity 21% higher (unpaired t-test: P< 0.0001) in KI compared to WT mice.