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 .

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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 .

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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.


( 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 .

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(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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(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.


( 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 .

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(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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(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.


( 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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(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.


( 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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(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.


( 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 .

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(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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(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.


( 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 .

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(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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(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.


( 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 .

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(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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(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.


( 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 .

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(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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(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.


( 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 ) .

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(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 .

(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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(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.


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 .

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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 ) .

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(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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(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.


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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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.


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 .

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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 .

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(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 ) .

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(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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(d) Comparison of the ORF1b of the SARS-CoV-2 and SARS-CoV genomes with the 9-bp deletion region shown enlarged.


( 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 ) .

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(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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(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.


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 .

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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 ) .

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(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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(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.


A . 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 .

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A. 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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B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.


A . 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 .

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A. 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 .

O O O O O O O O O O O O O O O O O O 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. 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 .

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

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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D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.


A . Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP - 2xFYVEHrs and MavQ with or without wortmannin treatment . Scale bars , 5 μm .

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A. 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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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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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).


A . 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 .

A. 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 )

O O O O O O O O O O O O O O O O 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, 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 ) .

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

C. 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 .

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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 .

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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.


( 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 .

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

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

(C) 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).


( 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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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).


( 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 ) .

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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).


Left , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left, 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).


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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.


( A ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days . GLT - 1a levels are significantly increased in mice treated with Cef ( Cef WT , N = 4 ) compared to control saline - injected mice ( Ctr WT , N = 4 ) ( Mann - Withney U test : P = 0 . 028 ) .

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

(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).


( B ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in sections from SI of P45 - 46 WT mice that were treated with saline ( Ctr WT ) or Cef ( Cef WT ) . Arrows point to some GLT - 1a / VGLUT1 related puncta . Framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment increased significantly the size of GLT - 1a + puncta overlaying VGLUT1 ( 142 and 186 GLT - 1a + puncta analyzed from 4 Ctr WT and 4 Cef WT mice , respectively ; 3 sections / animal ) ( Mann - Withney U test : P < 0 . 0001 ) . 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 .

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

(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). 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.


( C ) CSD threshold and CSD velocity in cortical slices from P30 - 33 KI mice that were injected with saline ( Ctr KI , n = 38 ; N = 7 ) or Cef ( Cef KI , n = 31 ; N = 6 ) . CSD threshold is 12 % higher ( Mann - Withney U test : P = 0 . 02 ) in Cef - treated compared to saline - treated KI mice . CSD velocity is not altered by Cef treatment ( P = 0 . 90 ) .

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

(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).


( A ) Distribution of GLT - 1agold particles in PAP of saline - injected ( Ctr KI , N = 4 ) and Cef - treated KI mice ( Cef KI , N = 4 ) ( P45 - 46 ) . Cef treatment does not modify the density of total , cytoplasmic ( arrows ) and membrane - associated ( arrowheads ) gold particles in PAP of KI mice ( cf Table II ) .

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

(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).


( B ) Distribution of GLT - 1agold particles in AxT in SI of Ctr KI and Cef KI KI mice . The density of the membrane - associated gold particles ( arrowheads ) is increased in Cef - treated KI mice ( cf Table II ) .

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

(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).


( C ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment . GLT - 1a levels are similar in Cef - treated ( Cef KI , N = 4 ) and and saline - injected ( Ctr KI , N = 4 ) ( Mann - Withney U test : P = 0 . 83 ) .

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

(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).


( D ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in KI mice that received saline ( Ctr KI ) and in Cef - treated KI mice ( Cef KI ) ( P45 - 46 ) . Arrows point to some GLT - 1a / VGLUT1 related puncta ; framed regions are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment does not increase the size of GLT - 1a + puncta overlaying with VGLUT1 ( 160 and 175 GLT - 1a + puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post - embedding electron microscopy analysis ; 3 sections / animal ) ( Mann - Withney U test : P = 0 . 31 ) . Scale bar : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . All microscopic fields are from layers II / III .

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

(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.


( A ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 2 . 5 µM DL - TBOA ( TBOA 2 . 5 ) ( n = 4 ; N = 2 ) ( right panel ) . The traces on the left are the corresponding average normalized STCs , isolated as in Figure 1C . The STC τdecay in TBOA 2 . 5 is 32 ± 2 % higher than in Ctr WT ( paired t - test : P = 0 . 002 ) . The left top panel shows the time course of τdecay of the transient component ( due to the STC ) of the current recorded in an astrocyte during a representative experiment in which TBOA 2 . 5 was applied at the time indicated by the horizontal bar . The steady - state effect was reached within 10 - 15 minutes from the beginning of the drug perfusion .

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

(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion.


( B ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 2 . 5 µM DL - TBOA ( TBOA 2 . 5 : n = 25 ; N = 8 ) . CSD threshold in TBOA 2 . 5 is 36 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 20 % higher ( unpaired t - test : P < 0 . 0001 ) .

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

(B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001).


( C ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 1 . 5 µM DL - TBOA ( TBOA 1 . 5 ) ( n = 4 ; N = 4 ) . The STC τdecay in TBOA 1 . 5 is 22 ± 3 % higher than in Ctr WT ( paired t - test : P = 0 . 004 ) .

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(C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004).


( D ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 1 . 5 µM DL - TBOA ( TBOA 1 . 5 : n = 18 ; N = 6 ) . CSD threshold in TBOA 1 . 5 is 23 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 13 % higher ( unpaired t - test : P = 0 . 003 ) .

(D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).


A . Relative mRNA expression in skeletal muscle from SBMA patients ( white bars , n = 3 ) and controls ( black bars , n = 3 ) ( mean + / − SEM ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test .

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A. Relative mRNA expression in skeletal muscle from SBMA patients (white bars, n = 3) and controls (black bars, n = 3) (mean +/− SEM). * p<0.05, ** p<0.01 by Student's t test.


B . Splicing of XBP1 mRNA was assessed by RT - PCR . Products from unspliced ( XBP1u ) and spliced ( XBP1s ) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green .

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B. Splicing of XBP1 mRNA was assessed by RT-PCR. Products from unspliced (XBP1u) and spliced (XBP1s) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green.


C . Relative mRNA expression in proximal hind limb muscle ( mean + / − SEM ) . Mice evaluated were littermate WT ( n = 6 ) , AR113Q ( n = 6 ) , castrated WT ( C - WT , n = 6 ) and castrated AR113Q males ( C - AR113Q , n = 5 ) on a mixed C57BL / 6J - 129 genetic background . * p < 0 . 05 by ANOVA .

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C. Relative mRNA expression in proximal hind limb muscle (mean +/− SEM). Mice evaluated were littermate WT (n = 6), AR113Q (n = 6), castrated WT (C-WT, n = 6) and castrated AR113Q males (C-AR113Q, n = 5) on a mixed C57BL/6J-129 genetic background. *p<0.05 by ANOVA.


D . Western blot of BiP and PDI expression in proximal hind limb muscle . Right panels show quantification of signal relative to loading control ( mean + / − SEM ) . * p < 0 . 05 by Student ' s t test .

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D. Western blot of BiP and PDI expression in proximal hind limb muscle. Right panels show quantification of signal relative to loading control (mean +/− SEM). * p<0.05 by Student's t test.


E . Relative mRNA expression in proximal hind limb muscle of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males backcrossed to C57BL / 6J . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . F . Relative mRNA expression in spinal cord of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males ( mean + / − SEM ) . n . s . = not significant by Student ' s t test .

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E. Relative mRNA expression in proximal hind limb muscle of AR21Q (n = 5) and AR113Q (n = 3) males backcrossed to C57BL/6J. ** p<0.01, ***p<0.001 by Student's t test. F. Relative mRNA expression in spinal cord of AR21Q (n = 5) and AR113Q (n = 3) males (mean +/− SEM). n. s. = not significant by Student's t test.


A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limb muscle of AR113Q ( black ) or AR113Q , CHOP − / − mice ( white ) at 12 wks . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test .

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A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


B . Representative image of muscle fibers following NADH stain . Bar = 20 µM .

B. Representative image of muscle fibers following NADH stain. Bar = 20 µM.


C . Distribution of proximal hind limbmuscle fiber size from wt ( black ) and CHOP − / − ( white ) mice at 12 wks . Difference not significant by Mann - Whitney test .

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C. Distribution of proximal hind limbmuscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.


A . Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice ( n = 5 - 6 / genotype ) . * p < 0 . 05 by ANOVA , n . s . = not significant .

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A. Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice (n = 5-6/genotype). *p<0.05 by ANOVA, n. s. = not significant.


B . LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * p < 0 . 01 by Student ' s t test .

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B. LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot. Right panels show quantification of signal relative to GAPDH. **p<0.01 by Student's t test.


C . Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle . * p < 0 . 05 by Student ' s t test .

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C. Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle. *p<0.05 by Student's t test.


Androgen receptor protein expression in skeletal muscle of 12 wk mice . Hsp90 serves as a loading control . Right panel shows quantification of relative signal intensity ( n = 3 / genotype ) .

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Androgen receptor protein expression in skeletal muscle of 12 wk mice. Hsp90 serves as a loading control. Right panel shows quantification of relative signal intensity (n = 3/genotype).


D . Proximal hind limb muscles stained for the androgen receptor ( in red ) exhibit intranuclear inclusions . Nuclei are stained by DAPI ( in blue ) .

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D. Proximal hind limb muscles stained for the androgen receptor (in red) exhibit intranuclear inclusions. Nuclei are stained by DAPI (in blue).


E . P - JNK and P - eIF2 alpha expression ( top , middle ) and XBP1 mRNA splicing ( bottom ) in proximal hind limbmuscle of 12 wk mice . Right panels show quantification of signal relative to loading control . * * p < 0 . 01 by Student ' s t test . n . s . = not significant .

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E. P-JNK and P-eIF2 alpha expression (top, middle) and XBP1 mRNA splicing (bottom) in proximal hind limbmuscle of 12 wk mice. Right panels show quantification of signal relative to loading control. **p<0.01 by Student's t test. n. s. = not significant.


Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice . A . Western blot shows enhanced eIF2 alpha phosphorylation ( top ) and RT - PCR demonstrates increased XBP1 mRNA splicing ( bottom ) in denervated muscle . Right panels show relative quantification of signal intensity . * p < 0 . 05 by Student ' s t test .

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Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test.


. B . Relative expression of BiP , ATF4 and CHOP mRNA ( n = 3 ) . * p < 0 . 05 by Student ' s t test .

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. B. Relative expression of BiP, ATF4 and CHOP mRNA (n = 3). *p<0.05 by Student's t test.


C . Following surgical denervation of wild type or CHOP − / − mice , LC3 and p62 expression was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * * p < 0 . 001 by ANOVA , n . s . = not significant .

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C. Following surgical denervation of wild type or CHOP−/− mice, LC3 and p62 expression was assessed by western blot. Right panels show quantification of signal relative to GAPDH. ***p<0.001 by ANOVA, n. s. = not significant.


D . Muscle fiber size ( 100 fibers / mouse ) was quantified from wild type ( black , n = 5 ) , CHOP − / − ( white , n = 3 ) and Beclin - 1 + / − ( grey , n = 3 ) mice 7 days post sciatic nerve transection . Shown is relative fiber cross sectional area ( mean + / − SEM ) . * * * p < 0 . 001 by ANOVA .

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D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA.


E . Muscle fiber size ( 100 fibers / mouse ) was quantified from CHOP − / − ( black , n = 6 ) or CHOP − / − , Beclin - 1 + / − mice ( white , n = 6 ) 7 days post sciatic nerve transection . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test .

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E. Muscle fiber size (100 fibers/mouse) was quantified from CHOP −/− (black, n = 6) or CHOP −/−, Beclin-1 +/− mice (white, n = 6) 7 days post sciatic nerve transection. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limbmuscle of AR113Q ( red , n = 6 ) or AR113Q , Beclin - 1 + / − ( blue , n = 6 ) mice at 16 wks . Left panel shows fiber size distribution , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left panel , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 0001 by Student ' s t test .

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A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limbmuscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test.


B . AR expression in skeletal muscle of 16 wk mice was assessed by western blot . GAPDH controls for loading . Right panel shows quantification of signal relative to GAPDH ( mean + / − SEM ) . Differences not significant ( n . s . ) .

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B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).


A . Left panel , Kaplan - Meyer survival curve of AR113Q males ( red line , n = 12 ) and AR113Q , Beclin - 1 + / − males ( blue line , n = 15 ) . * p < 0 . 05 by log - rank analysis . Right panel , mean survival + / − SEM . * p < 0 . 05 by Student ' s t - test .

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A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test.


B , C . Body weight ( panel B ) and grip strength ( panel C ) at different ages for wild type ( wt , green line , n = 7 ) , Beclin - 1 + / − ( yellow line n = 9 ) , AR113Q ( red line , n = 12 ) , and AR113Q , Beclin - 1 + / − ( blue line , n = 15 ) male mice .

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B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.


D . Age of disease onset as measured by a decrease of 5 % or more in forelimb grip strength ( not significant by log rank analysis ) .

D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).


( A ) The expression of AR was determined using Western blots ( overexpressing AR in J82 cells and knocking down AR in UMUC3 cells ) .

(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).


( B ) Relative expression of the top 20 up - regulated circRNAs from the GEO dataset GSE92675 ( BCa tumor tissues compared to para - tumor tissues ) in response to AR modulation in J82 and UMUC3 cells . The # indicates an RNA with a relatively low expression ( qRT - PCR cycle number > 35 ) , thus the circRNA was excluded for further analysis .

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(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.


( E ) Real - time quantitative PCR ( qRT - PCR ) results show the knock down efficiency for these 4 circRNAs in J82 oe - AR ( J82 - AR ) cells .

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(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.


( F ) Transwell invasion assays showed that knock down of hsa _ circ _ 0084171 ( circ _ oo84171 ) , but not the other circRNAs , can partly reverse oeAR - increased invasion in J82 cells . Scale bar , 100 μm .

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(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.


( G ) MTT assays showed that shcirc _ 0084171 can partly reverse oeAR - increased cisplatin chemo - resistance ( 2 μg / ml ) in J82 cells . To calculate the mean , the value of 0 µg / ml cisplatin groups were set as 1 .

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(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.


( H ) The qRT - PCR results showed that the overexpression ( oe ) efficacy for hsa _ circ _ 0084171 ( oecirc _ 0084171 ) in UMUC3 - shAR cells .

(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.


( I ) Transwell invasion assays showed that oecirc _ 0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells . Scale bar , 100 μm .

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(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.


( J ) MTT assays showed that oecirc _ 0084171 can partly overcome shAR effect for cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 cells .

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(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


B ) The relative expression of circFNTA was elevated in multiple BCa cell lines ( T24 , J82 , UMUC3 , and 5637 ) compared to normal bladder epithelial cell line SVHUC as shown by qRT - PCR .

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B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.


C ) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT - PCR .

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C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.


D ) RNA Fluorescence in situ hybridization ( FISH ) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells . DAPI was mainly localized to the cytoplasm . Scale bar , 20 μm .

D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.


E ) Two shcircFNTA plasmids using PLKO . 1 vector were transfected into UMUC3 cells . The knock down efficacy was measured by qRT - PCR .

E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.


G ) The qRT - PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector ( left panel ) and oecircFNTA or oelinearFNTA in J82 cells ( right panel ) .

G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).


Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion ( H ) more profoundly than oelinearFNTA as compared with the oevector group . Scale bar , 100 μm .

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Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.