PBMCs from four individual donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Infectivity in cell culture supernatants by plaque titration assay Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_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 O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four individual donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Viral RNA ( genome equivalents / ml ) concentrations in cell culture supernatants by Q - RT - PCR Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test comparing mock - and Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; Ruxo . = Ruxolitinib .

O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_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 O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; Ruxo. = Ruxolitinib.


PBMCs from four individual donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Viral RNA ( genome equivalents / ml ) concentrations in cell culture supernatants by Q - RT - PCR Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_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 O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four individual donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : ( E ) Relative changes of cell - associated viral genomic RNA quantities by Q - RT - PCR and normalized to RNASEP levels . Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test comparing mock - and Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant and are not shown in the figure . h . p . e . = hours post - exposure Ruxo . = Ruxolitinib .

O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: (E) Relative changes of cell-associated viral genomic RNA quantities by Q-RT-PCR and normalized to RNASEP levels. Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.


RNA extracted from Ruxolitinib - treated or mock - treated , and SARS - CoV - , SARS - CoV - 2 - PBMCs was analyzed for ( A ) IFIT1 , ( B ) IFNA1 and IFNB1 mRNA expression by Q - RT - PCR at indicated time points . Suspension and adherent cell fractions were analyzed separately , except at the four hours time point . Values were normalized to cellular RNASEP expression and are shown as fold change over mock - inoculated conditions . The dotted line indicates the expression level of mock - inoculated cell cultures and is set to 1 . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M ( A - B Statistical significance between mock - and Ruxolitinib - treated samples was tested using paired Student ' s t - testing and comparing SARS - CoV to SARS - CoV - 2 - treated samples from the same donor and time points . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) .

O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_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 B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- PBMCs was analyzed for (A) IFIT1, (B) IFNA1 and IFNB1 mRNA expression by Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours time point. Values were normalized to cellular RNASEP expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( C ) Supernatants from Ruxolitinib - or mock - treated and SARS - CoV - , SARS - CoV - 2 or mock - inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN - α2 , IP - 10 / CXCL10 , MCP - 1 / CCL2 and MCP - 3 / CCL7 were quantified using a Luminex - based immunoassay . PHA - or LPS - treated PBMCs were used as a positive control . Bars represent the results of a pool of four individual samples per condition . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , Statistical significance between mock - and Ruxolitinib - treated samples was tested using paired Student ' s t - testing and comparing SARS - CoV to SARS - CoV - 2 - treated samples from the same donor P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) . n . d . = not detectable .

O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Supernatants from Ruxolitinib- or mock-treated and SARS-CoV-, SARS-CoV-2 or mock-inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2 and MCP-3/CCL7 were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs were used as a positive control. Bars represent the results of a pool of four individual samples per condition. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.


( D ) Supernatants collected from SARS - CoV - , SARS - CoV - 2 - or mock - exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) .

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Supernatants collected from SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( A ) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock - , SARS - CoV - and SARS - CoV - 2 - challenged conditions for indicated cell types . Data information : Data shown in this figure are based on the analysis of two donors .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O 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) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock-, SARS-CoV- and SARS-CoV-2-challenged conditions for indicated cell types. Data information: Data shown in this figure are based on the analysis of two donors.


( B ) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(B) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions. Data information: Data shown in this figure are based on the analysis of two donors.


( C ) Volcano plot of all DEGs in SARS - CoV - 2 - exposed cells compared to mock - exposed cells in the indicated cell types . Known ISGs were colored in green based on their presence in the interferome database Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot of all DEGs in SARS-CoV-2-exposed cells compared to mock-exposed cells in the indicated cell types. Known ISGs were colored in green based on their presence in the interferome database Data information: Data shown in this figure are based on the analysis of two donors.


( D ) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN - module gene set . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(D) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN-module gene set. Data information: Data shown in this figure are based on the analysis of two donors.


( E ) Dot plot depicting expression of selected ISGs and cytokines . Expression levels are color - coded , the percentage of cells expressing the respective gene is coded by symbol size . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O B-EXP_ASSAY O O O O O O B-EXP_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

(E) Dot plot depicting expression of selected ISGs and cytokines. Expression levels are color-coded, the percentage of cells expressing the respective gene is coded by symbol size. Data information: Data shown in this figure are based on the analysis of two donors.


( C ) Bar graph showing the absolute number ( bars ) and relative percentage of cells that were identified as virus RNA - Positive in SARS - CoV - and SARS - CoV - 2 - inoculated cultures , respectively . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(C) Bar graph showing the absolute number (bars) and relative percentage of cells that were identified as virus RNA-Positive in SARS-CoV- and SARS-CoV-2-inoculated cultures, respectively. Data information: Data shown in this figure are based on the analysis of two donors.


( D ) Plot of Log10 average expression of genes showing a Log2 ( fold change ) > 0 . 2 in viral RNA - Positive versus viral RNA - Negative monocytes from the SARS - CoV - ( left panel ) and SARS - CoV - 2 - ( right panel ) inoculated PBMCs with genes showing the highest expression fold change between both conditions . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(D) Plot of Log10 average expression of genes showing a Log2(fold change) >0.2 in viral RNA-Positive versus viral RNA-Negative monocytes from the SARS-CoV- (left panel) and SARS-CoV-2- (right panel) inoculated PBMCs with genes showing the highest expression fold change between both conditions. Data information: Data shown in this figure are based on the analysis of two donors.


( E ) IFN Module Score of viral - RNA - negative ( grey ; 715 and 958 cells in SARS - CoV and SARS - CoV - 2 exposed cultures , respectively ) , SARS - CoV - RNA - Positive ( blue ; 56 cells ) and SARS - CoV - 2 - RNA - Positive ( magenta ; 173 cells ) monocytes . Statistical significance was tested using a Wilcoxon rank sum test with continuity correction . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant Data information : Data shown in this figure are based on the analysis of two donors .

O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) IFN Module Score of viral-RNA-negative (grey; 715 and 958 cells in SARS-CoV and SARS-CoV-2 exposed cultures, respectively), SARS-CoV-RNA-Positive (blue; 56 cells) and SARS-CoV-2-RNA-Positive (magenta; 173 cells) monocytes. Statistical significance was tested using a Wilcoxon rank sum test with continuity correction. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant Data information: Data shown in this figure are based on the analysis of two donors.


F : IMAF for the CSF , plasma and urine of the patients with samples collected at 6 months follow - up ( n = 3 ) . Matched MRI scans are added for annotation .

O O B-EXP_ASSAY O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O

F: IMAF for the CSF, plasma and urine of the patients with samples collected at 6 months follow-up (n=3). Matched MRI scans are added for annotation.


A : Mutant allele fraction ( MAF ) measured in tumor , CSF , plasma and urine samples ( on the y axis ) for the detected mutations , in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases ( on the x axis ) , for the 8 patients analysed by targeted sequencing panels . MAF from tumor are derived from exome sequencing . MAF from CSF , plasma and urine samples are derived from the capture panels sequencing .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O B-EXP_ASSAY O

A: Mutant allele fraction (MAF) measured in tumor, CSF, plasma and urine samples (on the y axis) for the detected mutations, in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases (on the x axis), for the 8 patients analysed by targeted sequencing panels. MAF from tumor are derived from exome sequencing. MAF from CSF, plasma and urine samples are derived from the capture panels sequencing.


A - C : Fragment size distributions for mutant ( blue ) and non - mutant ( red ) cfDNA reads , determined from the capture sequencing data for CSF samples ( A ) , plasma samples ( B ) and urine samples ( 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 B-EXP_ASSAY O O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O

A-C: Fragment size distributions for mutant (blue) and non-mutant (red) cfDNA reads, determined from the capture sequencing data for CSF samples (A), plasma samples (B) and urine samples (C).


A Arabidopsis seedlings grown for 7 days in LD at 20 ° C or 28 ° C . B Root lengths of seedlings from day 2 to day 12 after sowing ( grown as in A ) .

O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O

A Arabidopsis seedlings grown for 7 days in LD at 20°C or 28°C. B Root lengths of seedlings from day 2 to day 12 after sowing (grown as in A).


C Seedlings of Brassica oleracea and Solanum lycopersicum ( lycop . ) grown for 7 days at 20 ° C or 28 ° C . D Root lengths of 7 days - old seedlings grown at 20 ° C or 28 ° C ( n = 18 - 22 ) .

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C Seedlings of Brassica oleracea and Solanum lycopersicum (lycop.) grown for 7 days at 20°C or 28°C. D Root lengths of 7 days-old seedlings grown at 20°C or 28°C (n = 18-22).


A Elongation responses of detached roots . Shoots were removed from 4 days - old Arabidopsis thaliana ( n = 18 - 19 ) , 4 days - old Brassica oleracea ( n = 11 ) and 5 days - old Solanum lycopersicum ( lycop . , n = 10 - 12 ) seedlings grown at 20 ° C . Detached roots were grown for additional 4 days at 20 ° C or 28 ° C ,

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A Elongation responses of detached roots. Shoots were removed from 4 days-old Arabidopsis thaliana (n = 18-19), 4 days-old Brassica oleracea (n = 11) and 5 days-old Solanum lycopersicum (lycop., n = 10-12) seedlings grown at 20°C. Detached roots were grown for additional 4 days at 20°C or 28°C,


B - D 9 days - old seedlings were hypocotyl - grafted , recovered for 7 days and then cultivated at 20 ° C or 28 ° C for additional 7 days ( n = 16 - 26 ) . Boxplots show medians , interquartile ranges and min - max values with individual data points superimposed as colored dots . Diﬀerent letters denote statistical diﬀerences at P < 0 . 05 as assessed by one - way two - way ( A - D ) ANOVA and Tukey ' s HSD posthoc test .

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B-D 9 days-old seedlings were hypocotyl-grafted, recovered for 7 days and then cultivated at 20°C or 28°C for additional 7 days (n = 16-26). Boxplots show medians, interquartile ranges and min-max values with individual data points superimposed as colored dots. Diﬀerent letters denote statistical diﬀerences at P < 0.05 as assessed by one-way two-way (A-D) ANOVA and Tukey's HSD posthoc test.


Total length of root zones between days 2 and 7 after sowing of Arabidopsis seedlings grown in LD at 20 ° C or 28 ° C . A Meristem , B Elongation zone , C Differentiation zone . A - C Solid lines and points show mean root zone lengths with half - transparent ribbons denoting SEM ( n = 5 - 7 individual roots ) .

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Total length of root zones between days 2 and 7 after sowing of Arabidopsis seedlings grown in LD at 20°C or 28°C. A Meristem, B Elongation zone, C Differentiation zone. A-C Solid lines and points show mean root zone lengths with half-transparent ribbons denoting SEM (n = 5-7 individual roots).


E EdU staining marks cells that were in S - phase during the 1h incubation time prior to microscopic imaging . Scale bar = 50 µm , n = 12 . F DAPI staining was used to identify cells currently in mitosis ( examples are highlighted by yellow asterisks ) .

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E EdU staining marks cells that were in S-phase during the 1h incubation time prior to microscopic imaging. Scale bar = 50 µm, n= 12. F DAPI staining was used to identify cells currently in mitosis (examples are highlighted by yellow asterisks).


A Root growth assay of seedlings grown for 7 days in the presence of increasing concentrations of auxin biosynthesis inhibitors kynurenine and yucasin ( n = 14 - 26 , both inhibitors always in equal concentrations ) at the indicated temperatures .

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A Root growth assay of seedlings grown for 7 days in the presence of increasing concentrations of auxin biosynthesis inhibitors kynurenine and yucasin (n = 14-26, both inhibitors always in equal concentrations) at the indicated temperatures.


D Temperature induced root growth is unaffected in 8 days - old seedlings if 0 . 5 mM NPA is applied to the root shoot junction on day 5 ( n = 13 - 16 ) at the indicated temperatures .

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D Temperature induced root growth is unaffected in 8 days-old seedlings if 0.5 mM NPA is applied to the root shoot junction on day 5 (n = 13-16) at the indicated temperatures.


G ( Co - ) incubation of 5 days - old seedlings for 3h with 10 μM EdU only or in combination with either 100 nM NAA or 50 μM PEO - IAA ( n = 11 - 24 ) ,

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G (Co-)incubation of 5 days-old seedlings for 3h with 10 μM EdU only or in combination with either 100 nM NAA or 50 μM PEO-IAA (n = 11-24),


B Imaging and quantification of DR5 : : GFP reporter activity in root tips of 5 days - old seedlings ( scale bar = 50 µm , n = 9 - 14 ) .

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B Imaging and quantification of DR5::GFP reporter activity in root tips of 5 days-old seedlings (scale bar = 50 µm, n= 9-14).


C EdU staining of 5 days - old seedlings at indicated temperatures ( n = 11 - 14 ) .

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C EdU staining of 5 days-old seedlings at indicated temperatures (n = 11-14).


D qRT - PCR expression analyses of PIN1 - 4 from whole root and roo tip samples , respectively , Col - 0 RNA was extracted with respective organs and first - strand cDNA was then synthesized ( n = 3 ) .

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D qRT-PCR expression analyses of PIN1-4 from whole root and roo tip samples, respectively, Col-0 RNA was extracted with respective organs and first-strand cDNA was then synthesized (n = 3).


( a , b ) Survival of wild - type ( Oregon R ) , yw , PGRP - LE112 , PGRP - LC7454 and PGRP - LE112 , PGRP - LC7454 flies after injection of wild - type L . monocytogenes ( a ) or Δhly L . monocytogenes ( b ) at 28 ° C .

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(a,b) Survival of wild-type (Oregon R), yw, PGRP-LE112, PGRP-LC7454 and PGRP-LE112,PGRP-LC7454 flies after injection of wild-type L. monocytogenes (a) or Δhly L. monocytogenes (b) at 28 °C.


( c ) Survival of wild - type , yw and PGRP - LE112 flies , as well as flies treated with RNAi targeting PGRP - LE with the hml - Gal4 driver ( hml - Gal4 > PGRP - LE RNAi ) and PGRP - LE112 flies with hml - Gal4 - driven PGRP - LE expression ( PGRP - LE112 , hml - Gal4 > PGRP - LE ) , after injection of wild - type L . monocytogenes at 28 ° C . NS , not significant ; * , P 0 . 01 ( Wilcoxon - Mann - Whitney test ) : P = 0 . 0006 , 0 . 0043 or 0 . 0043 , for yw versus PGRP - LE112 , PGRP - LC7454 or PGRP - LE112 , PGRP - LC7454 , respectively ( a ) ; P = 0 . 0095 , for yw versus RNAi targeting PGRP - LE ( c ) . Data represent the average of four independent experiments with over 30 flies of each genotype examined at the same time .

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(c) Survival of wild-type, yw and PGRP-LE112 flies, as well as flies treated with RNAi targeting PGRP-LE with the hml-Gal4 driver (hml-Gal4>PGRP-LE RNAi) and PGRP-LE112 flies with hml-Gal4-driven PGRP-LE expression (PGRP-LE112,hml-Gal4>PGRP-LE), after injection of wild-type L. monocytogenes at 28 °C. NS, not significant; *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0006, 0.0043 or 0.0043, for yw versus PGRP-LE112, PGRP-LC7454 or PGRP-LE112,PGRP-LC7454, respectively (a); P = 0.0095, for yw versus RNAi targeting PGRP-LE (c). Data represent the average of four independent experiments with over 30 flies of each genotype examined at the same time.


( a ) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild - type L . monocytogenes . Hemocyte nuclei ( filled arrowhead ) and DNA of L . monocytogenes ( open arrowhead ) are visualized by DAPI staining ( blue or white ) ; the actin cytoskeleton is stained with rhodamine - labeled phalloidin ( red ) . hml - Gal4 > Atg5IR , expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5 . Scale bars , 10 μm .

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(a) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild-type L. monocytogenes. Hemocyte nuclei (filled arrowhead) and DNA of L. monocytogenes (open arrowhead) are visualized by DAPI staining (blue or white); the actin cytoskeleton is stained with rhodamine-labeled phalloidin (red). hml-Gal4>Atg5IR, expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5. Scale bars, 10 μm.


( b ) Intracellular wild - type L . monocytogenes ( WT Lm ) or Δhly L . monocytogenes ( Δhly Lm ) counted manually in DAPI - stained infected hemocytes . LE112 , hml > LE , hml - Gal4 - driven expression of PGRP - LE in PGRP - LE112 hemocytes ; imd1 , imd ; RelE20 , RelishE20 ; hml > Atg5IR , inverted repeat sequence described in a ; w , hml > Atg1 , hml - Gal4 - driven expression of Atg1 in w hemocytes ; LE112 , hml > Atg1 , hml - Gal4 - driven expression of Atg1 in PGRP - LE112 hemocytes ; PGRP - LE112 + rap , PGRP - LE112 hemocytes plus rapamycin . * , P 0 . 001 , compared with wild type ( t - test ) . Data are representative of four experiments ( a ) or at least three independent experiments ( b ; error bars , s . d . of triplicate measurements ) .

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(b) Intracellular wild-type L. monocytogenes (WT Lm) or Δhly L. monocytogenes (Δhly Lm) counted manually in DAPI-stained infected hemocytes. LE112,hml>LE, hml-Gal4-driven expression of PGRP-LE in PGRP-LE112 hemocytes; imd1, imd; RelE20, RelishE20; hml>Atg5IR, inverted repeat sequence described in a; w, hml>Atg1, hml-Gal4-driven expression of Atg1 in w hemocytes; LE112,hml>Atg1, hml-Gal4-driven expression of Atg1 in PGRP-LE112 hemocytes; PGRP-LE112+rap, PGRP-LE112 hemocytes plus rapamycin. *, P 0.001, compared with wild type (t-test). Data are representative of four experiments (a) or at least three independent experiments (b; error bars, s.d. of triplicate measurements).


Survival of yw flies , PGRP - LE112 flies , flies treated with Atg5 - directed RNAi ( hml - Gal4 > Atg5IR ) and control flies carrying hml - Gal4 ( hml - Gal4 > GFP ) , after injection of wild - type L . monocytogenes ( a ) , Δhly L . monocytogenes ( b ) or E . carotovora ( c ) into adult flies . * , P 0 . 01 ( Wilcoxon - Mann - Whitney test ) : P = 0 . 0024 , Atg5 - directed RNAi versus control ( a ) , P = 0 . 0007 , PGRP - LE112 versus control ( a ) ; P = 0 . 0055 , PGRP - LC7454 versus yw ( c ) . Data represent the average of four independent experiments .

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Survival of yw flies, PGRP-LE112 flies, flies treated with Atg5-directed RNAi (hml-Gal4>Atg5IR) and control flies carrying hml-Gal4 (hml-Gal4>GFP), after injection of wild-type L. monocytogenes (a), Δhly L. monocytogenes (b) or E. carotovora (c) into adult flies. *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0024, Atg5-directed RNAi versus control (a), P = 0.0007, PGRP-LE112 versus control (a); P = 0.0055, PGRP-LC7454 versus yw (c). Data represent the average of four independent experiments.


( a ) Growth of L . monocytogenes in S2 cells ( S2 ) or S2 cells expressing PGRP - LE ( S2 - LE ) transfected with double - stranded RNA ( RNAi ) specific for Atg5 , Rel or Dif and dl ( Dif - dl ) and infected for 1 . 5 h with wild - type or Δhly L . monocytogenes , followed by 6 h of incubation in medium containing CuSO4 and gentamicin , quantified by plate assay of colony - forming units . * , P 0 . 001 ( t - test ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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(a) Growth of L. monocytogenes in S2 cells (S2) or S2 cells expressing PGRP-LE (S2-LE) transfected with double-stranded RNA (RNAi) specific for Atg5, Rel or Dif and dl (Dif-dl) and infected for 1.5 h with wild-type or Δhly L. monocytogenes, followed by 6 h of incubation in medium containing CuSO4 and gentamicin, quantified by plate assay of colony-forming units. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


( b , c ) Fluorescence confocal microscopy of the localization of PGRP - LE together with wild - type L . monocytogenes in S2 cells engineered to express YFP - PGRP - LE ( YFP - LE ) and infected for 0 . 5 h with wild - type L . monocytogenes ( b ) or Δhly L . monocytogenes ( c ) , followed by 1 h of incubation in gentamicin - containing medium , then DAPI staining . Merge , YFP - PGRP - LE ( green ) and DAPI ( magenta ) . Filled arrowheads indicate L . monocytogenes ; open arrowhead indicates accumulation of YFP - PGRP - LE around the bacteria . Scale bars , 5 μm . Data are representative of three experiments .

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(b,c) Fluorescence confocal microscopy of the localization of PGRP-LE together with wild-type L. monocytogenes in S2 cells engineered to express YFP-PGRP-LE (YFP-LE) and infected for 0.5 h with wild-type L. monocytogenes (b) or Δhly L. monocytogenes (c), followed by 1 h of incubation in gentamicin-containing medium, then DAPI staining. Merge, YFP-PGRP-LE (green) and DAPI (magenta). Filled arrowheads indicate L. monocytogenes; open arrowhead indicates accumulation of YFP-PGRP-LE around the bacteria. Scale bars, 5 μm. Data are representative of three experiments.


( a ) Dot - or ring - shaped GFP - LC3 ( LC3 dot ) signals in S2 cells expressing both PGRP - LE and GFP - LC3 under the control of an actin promoter ( PGRP - LE GFP - LC3 ) or GFP - LC3 alone ( GFP - LC3 ) after infection with wild - type or Δhly L . monocytogenes or after 1 . 5 h of incubation with 5 μM rapamycin ( rap ) . - , no infection .

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(a) Dot- or ring-shaped GFP-LC3 (LC3 dot) signals in S2 cells expressing both PGRP-LE and GFP-LC3 under the control of an actin promoter (PGRP-LE GFP-LC3) or GFP-LC3 alone (GFP-LC3) after infection with wild-type or Δhly L. monocytogenes or after 1.5 h of incubation with 5 μM rapamycin (rap). -, no infection.


( b ) Colocalization of GFP - LC3 dots and wild - type L . monocytogenes in S2 cells , quantified by confocal microscopy .

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(b) Colocalization of GFP-LC3 dots and wild-type L. monocytogenes in S2 cells, quantified by confocal microscopy.


( c ) Confocal microscopy of S2 cells expressing PGRP - LE and GFP - LC3 ( green ) infected with wild - type L . monocytogenes and stained with DAPI ( magenta ) . Arrow indicates colocalization of GFP - LC3 and L . monocytogenes .

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(c) Confocal microscopy of S2 cells expressing PGRP-LE and GFP-LC3 (green) infected with wild-type L. monocytogenes and stained with DAPI (magenta). Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( d ) Immunoblot analysis of lysates of S2 cells left untreated ( - ) or infected with wild - type L . monocytogenes or treated with 5 μM rapamycin .

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(d) Immunoblot analysis of lysates of S2 cells left untreated (-) or infected with wild-type L. monocytogenes or treated with 5 μM rapamycin.


( e - i ) Ultrastructural analysis of S2 cells expressing PGRP - LE and GFP - LC3 and infected with wild - type L . monocytogenes . ( e , f ) Fluorescence microscopy ( e ) and electron microscopy ( f ) of cells expressing GFP - LC3 ( green ) and stained with DAPI ( magenta ) . ( g ) Enlargement of a bacteria - containing vacuole from f . ( h , i ) Enlargement of the fields outlined in g . Arrows indicate double - membrane structure that surrounds the bacteria ; arrowheads indicate an endoplasmic reticulum - like membrane .

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(e-i) Ultrastructural analysis of S2 cells expressing PGRP-LE and GFP-LC3 and infected with wild-type L. monocytogenes. (e,f) Fluorescence microscopy (e) and electron microscopy (f) of cells expressing GFP-LC3 (green) and stained with DAPI (magenta). (g) Enlargement of a bacteria-containing vacuole from f. (h,i) Enlargement of the fields outlined in g. Arrows indicate double-membrane structure that surrounds the bacteria; arrowheads indicate an endoplasmic reticulum-like membrane.


( j ) Confocal microscopy of hemocytes infected with wild - type L . monocytogenes . Green , GFP - LC3 ; magenta , DAPI . Arrow indicates colocalization of GFP - LC3 and L . monocytogenes .

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(j) Confocal microscopy of hemocytes infected with wild-type L. monocytogenes. Green, GFP-LC3; magenta, DAPI. Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( k ) Dot - or ring - shaped GFP - LC3 signals in ex vivo - cultured hemocytes expressing GFP - LC3 ( infection and treatment , horizontal axis ) .

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(k) Dot- or ring-shaped GFP-LC3 signals in ex vivo-cultured hemocytes expressing GFP-LC3 (infection and treatment, horizontal axis).


( l ) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L . monocytogenes or treated with rapamycin . Arrowhead indicates processed form of GFP - LC3 .

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(l) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L. monocytogenes or treated with rapamycin. Arrowhead indicates processed form of GFP-LC3.


( m ) GFP - LC3 dots in S2 cells expressing PGRP - LE and GFP - LC3 and treated with RNAi ( below graph ) and infected for 0 . 5 h with L . monocytogenes then incubated for 1 h in gentamicin - containing medium ; dots quantified by confocal microscopy . Scale bars , 5 μm ( c , j ) , 1 μm ( e , f ) or 500 nm ( g ) . * , P 0 . 001 ( t - test ) . Data are representative of three ( a , k , m ) , two ( b , e - i ) , six ( c ) , four ( d ) or five ( j , l ) experiments ( error bars , s . d . of triplicate measurements ( a , b , l ) or at least triplicate measurements ( m ) ) .

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(m) GFP-LC3 dots in S2 cells expressing PGRP-LE and GFP-LC3 and treated with RNAi (below graph) and infected for 0.5 h with L. monocytogenes then incubated for 1 h in gentamicin-containing medium; dots quantified by confocal microscopy. Scale bars, 5 μm (c,j), 1 μm (e,f) or 500 nm (g). *, P 0.001 (t-test). Data are representative of three (a,k,m), two (b,e-i), six (c), four (d) or five (j,l) experiments (error bars, s.d. of triplicate measurements (a,b,l) or at least triplicate measurements (m)).


( a ) GFP - LC3 dots in S2 cells expressing GFP - LC3 alone or PGRP - LE and GFP - LC3 , treated for 2 h with TCT ( 100 nM ) , highly purified DAP - type PGN from L . plantarum ( DAP ; 100 μg / ml ) or lysine - type PGN from S . epidermidis ( Lys ) .

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(a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L. plantarum (DAP; 100 μg/ml) or lysine-type PGN from S. epidermidis (Lys).


( b ) Dot - or ring - shaped GFP - LC3 signals in hemocytes after infection with wild - type or Δhly L . monocytogenes or treatment with rapamycin ( 5 μM ) or with TCT , DAP - type PGN or lysine - type PGN . * , P 0 . 001 ( t - test ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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(b) Dot- or ring-shaped GFP-LC3 signals in hemocytes after infection with wild-type or Δhly L. monocytogenes or treatment with rapamycin (5 μM) or with TCT, DAP-type PGN or lysine-type PGN. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


molecular detection of nCoV - 2019 in seven patients during two times of sampling . Patient information can be found in Extended Data Table 1 and 2 . Details on detection method can be found in material and methods . BALF , bronchoalveolar lavage fluid ; OS , oral swab ; AS , anal swab .

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molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.


dynamics of nCoV - 2019 antibodies in one patient who showed sign of disease on 2019 . 12 . 23 ( ICU - 06 ) . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


serological test of nCoV - 2019 antibodies in five patients ( more information can be found in Extended Data Table 2 ) . Star indicates data collected from patient ICU - 06 on 2020 . 01 . 10 . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


viral particles in the ultrathin sections under electron microscope at 200 kV , sample from viral infected Vero E6

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viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells


usage . Determination of virus infectivity in HeLa cells with or without the expression of ACE2 . h , human ; b , Rhinolophus sinicus bat ; c , civet ; s , swine ( pig ) ; m , mouse . ACE2 protein ( green ) , viral protein ( red ) and nuclei ( blue ) was shown . Scale bar = 10 um .

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usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.


A . Measurement of oxygen consumption rate ( OCR ) , extracellular acidification rate ( ECAR ) and oxidative burst of monocytes from COVID - 19 patients ( COVID ) and healthy controls ( CTR ) . OCR was measured in real time , under basal condition and in response to mitochondria inhibitors : oligomycin ( Oligo , 2 μM ) , cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 0 . 5 μM ) , and antimycin A plus rotenone ( Rot / AA , 0 . 5 μM ) . Oxidative burst was measured in response to PMA / ionomycin ( PMA / Iono , 1 μg / mL ) . Scatter plots show the quantification of basal respiration ( indicated as Basal OCR ) , ATP - linked respiration ( ATP - linked ) , maximal respiration ( Max Resp ) , proton - leak , spare respiratory capacity ( Spare ) , oxidative burst immediate response and basal ECAR . The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve ( AUC ) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement , respectively . Data represent individual values , mean and standard error of the mean ( CTR , n = 12 ; COVID , n = 13 ) . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure . Representative traces of OCR of monocytes from COVID - 19 patients ( COVID ) and healthy controls ( CTR ) are also reported .

O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_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 O O O O O O O O O O O B-DISEASE O O O O 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-EXP_ASSAY O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O O O O

A. Measurement of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and oxidative burst of monocytes from COVID-19 patients (COVID) and healthy controls (CTR). OCR was measured in real time, under basal condition and in response to mitochondria inhibitors: oligomycin (Oligo, 2 μM), cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 μM), and antimycin A plus rotenone (Rot/AA, 0.5 μM). Oxidative burst was measured in response to PMA/ionomycin (PMA/Iono, 1 μg/mL). Scatter plots show the quantification of basal respiration (indicated as Basal OCR), ATP-linked respiration (ATP-linked), maximal respiration (Max Resp), proton-leak, spare respiratory capacity (Spare), oxidative burst immediate response and basal ECAR. The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve (AUC) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement, respectively. Data represent individual values, mean and standard error of the mean (CTR, n=12; COVID, n=13). Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure. Representative traces of OCR of monocytes from COVID-19 patients (COVID) and healthy controls (CTR) are also reported.


B . Representative electron micrographs of mitochondrial ultrastructural changes observed in COVID - 19 monocytes . Panels " a " and " c " are representative electron micrographs of monocytes from healthy controls , whereas panels " b " , " d " and " e " are representative electron micrographs of monocytes from COVID - 19 patients . In panel " f " the quantification of mitochondrial area , external perimeter , Feret ' s diameter , aspect ratio circularity and roundness are reported ( n = 225 mitochondria for each group ) . Black arrows in panel " d " indicate mitochondria ; N refers to nucleus . Exact p values are reported in panel " f " .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE I-DISEASE I-DISEASE B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O

B. Representative electron micrographs of mitochondrial ultrastructural changes observed in COVID-19 monocytes. Panels "a" and "c" are representative electron micrographs of monocytes from healthy controls, whereas panels "b", "d" and "e" are representative electron micrographs of monocytes from COVID-19 patients. In panel "f" the quantification of mitochondrial area, external perimeter, Feret's diameter, aspect ratio circularity and roundness are reported (n=225 mitochondria for each group). Black arrows in panel "d" indicate mitochondria; N refers to nucleus. Exact p values are reported in panel "f".


B . Heatmaps representing different monocyte clusters identified by FlowSOM , with percentages in healthy donors ( CTR ) and COVID - 19 patients . The colors in the heat map represent the median of the arcsinh , 0 - 1 transformed marker expression calculated over cells from all the samples , varying from blue for lower expression to red for higher expression . Each cluster has a unique color assigned ( bar on the left which colors correspond to those used in panel A ) .

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B. Heatmaps representing different monocyte clusters identified by FlowSOM, with percentages in healthy donors (CTR) and COVID-19 patients. The colors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over cells from all the samples, varying from blue for lower expression to red for higher expression. Each cluster has a unique color assigned (bar on the left which colors correspond to those used in panel A).


C . Differential analysis between healthy donors ( CTR , n = 8 ) and COVID - 19 ( COVID , n = 7 ) . The heatmap represents arcsine - square - root transformed cell frequencies that were subsequently normalized per cluster ( rows ) to mean of zero and standard deviation of one . The color of the heat varies from blue , indicating relative under - representation , to red indicating relative over - representation . Bar and numbers at the right indicate significant differentially abundant clusters ( green ) and adjusted p values ( Differential analysis performed by diffcyt package present in the Catalyst package ) . The statistical analyses was done using GLMM ( according to the actual guidelines ) and all p - values were Bonferroni corrected . Clusters were sorted according to the adjusted p values , so that the cluster at the top shows the most significant abundance changes between the two conditions . D . Boxes and whiskers plots representing maximum , minimum , median value , 25 and 75 percentile of different subpopulations of monocytes in healthy donors ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) .

O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O

C. Differential analysis between healthy donors (CTR, n=8) and COVID-19 (COVID, n=7). The heatmap represents arcsine-square-root transformed cell frequencies that were subsequently normalized per cluster (rows) to mean of zero and standard deviation of one. The color of the heat varies from blue, indicating relative under-representation, to red indicating relative over-representation. Bar and numbers at the right indicate significant differentially abundant clusters (green) and adjusted p values (Differential analysis performed by diffcyt package present in the Catalyst package). The statistical analyses was done using GLMM (according to the actual guidelines) and all p-values were Bonferroni corrected. Clusters were sorted according to the adjusted p values, so that the cluster at the top shows the most significant abundance changes between the two conditions. D. Boxes and whiskers plots representing maximum, minimum, median value, 25 and 75 percentile of different subpopulations of monocytes in healthy donors (CTR, n=8) and COVID-19 patients (COVID, n=7).


E . Scatter plots showing the frequency of PD - 1 + cells , PD - L1 + cells among total monocytes in healthy controls ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) . A scatter plot reporting the median fluorescence intensity ( MFI ) of PD - L1 in total monocytes is also shown . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Scatter plots showing the frequency of PD-1+ cells, PD-L1+ cells among total monocytes in healthy controls (CTR, n=8) and COVID-19 patients (COVID, n=7). A scatter plot reporting the median fluorescence intensity (MFI) of PD-L1 in total monocytes is also shown. Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


A . Quantification of cytokines and other mediators in plasma obtained from COVID - 19 patients ( COVID , n = 15 ) and healthy controls ( CTR , n = 15 ) . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

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A. Quantification of cytokines and other mediators in plasma obtained from COVID-19 patients (COVID, n=15) and healthy controls (CTR, n=15). Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


B . Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID - 19 patients . Scatter plots showing the quantification of immature and mature monocytes in healthy controls ( CTR , n = 6 ) and COVID - 19 patients ( COVID , n = 7 ) are reported . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

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B. Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID-19 patients. Scatter plots showing the quantification of immature and mature monocytes in healthy controls (CTR, n=6) and COVID-19 patients (COVID, n=7) are reported. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


( A ) Bar chart showing the relative abundance ( signal intensity ) of the phosphorylated tau species identified using an exploratory IP - MS approach using HT7 antibody on TBS - soluble control and Alzheimer ' s disease ( AD ) pools . Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP - MS : mono - phosphorylated p - tau231 and double phosphorylated p - tau ( 231 + 235 ) .

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(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235).


( A ) Box - and - whiskers plot showing the levels of CSF p - tau235 in neurological controls ( n = 21 ) and biologically defined Alzheimer ' s disease ( AD ) cases ( n = 19 ) .

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(A) Box-and-whiskers plot showing the levels of CSF p-tau235 in neurological controls (n=21) and biologically defined Alzheimer's disease (AD) cases (n=19).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in the different groups : amyloid - negative cognitively unimpaired ( CU - ) participants ( n = 50 ) , participants across the Alzheimer ' s disease ( AD ) continuum ( amyloid - positive cognitively unimpaired ( CU + , n = 32 ) ; amyloid - positive mild cognitive impairment ( MCI + , n = 20 ) ; AD ( n = 20 ) and non - AD cases ( n = 19 ) . CSF p - tau235 is highly specific for AD pathology . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-DISEASE O O O O O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in the different groups: amyloid-negative cognitively unimpaired (CU-) participants (n=50), participants across the Alzheimer's disease (AD) continuum (amyloid-positive cognitively unimpaired (CU+, n=32); amyloid-positive mild cognitive impairment (MCI+, n=20); AD (n=20) and non-AD cases (n=19). CSF p-tau235 is highly specific for AD pathology. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in A - T - participants and across preclinical Alzheimer ' s disease ( AD , dichotomised as A + T - and A + T + ) . CSF p - tau235 was increased already in A + T - , which represents the early cases within preclinical AD . A prominent increase was observed from A + T - to A + T + cases , the latter representing late preclinical AD cases . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method ( Loess curve ) . Changes on biomarkers levels in CSF are represented as z - scores using Aβ PET in centiloid scale ( CL ) as a proxy of disease progression . Abnormal biomarker levels were determined as two standard deviations ( SD ) above the mean . Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 ( Mila - Aloma et al . , 2020 ) . All samples were run in singlicates .

O O O O B-GENEPROD O O O B-DISEASE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-GENEPROD B-EXP_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 B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.


( A ) Stacked bar chart depicting the percentages of [ 231 + / 235 + ] , [ 231 + / 235 - ] , [ 231 - / 235 - ] and [ 231 - / 235 + ] cases in each preclinical group . Table below indicates the exact percentages of participants in each group ( exact number of participants in parenthesis ) . Discordant cases with the sequential phosphorylation hypothesis [ 231 - / + 235 ] shown in grey .

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(A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey.


( B ) Correlation between CSF p - tau235 and p - tau231 in the whole ALFA + cohort ( Spearman ' s rank correlation : rS = 0 . 80 , P ˂ 0 . 0001 ) . Assay cut - offs were determined as the mean + 2 SD of the A - T - group ( defining p - tau231 and p - tau235 positivity or negativity in each participant ) . Cut - off values are indicated in red ( 19 . 92 and 9 . 59 pg / mL for CSF p - tau235 and p - tau231 respectively ) and displayed with black dashed lines , resulting in four quadrants , each of them representing the four different positive or negatively status for each biomarker . Discordant cases with the sequential phosphorylation hypothesis ( [ 231 - / + 235 ] ) showed on the lower right quadrant ( 2 . 35 % of the total , 9 participants out 383 ) . All samples were run in singlicates .

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(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.


( A , B ) Two - dimensional tryptic mapping of YFP - p27 , YFP - p27T198A and a mixture of both ( A ) . Phosphoamino - acid analysis of spots a and b ( B ) . The migration of phosphoserine ( S ) , phosphothreonine ( T ) and phosphotyrosine ( Y ) is marked with circles in B . The asterisk indicates the origin of migration .

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(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration.


( C - E ) MCF - 7 transfected with YFP - p27 or YFP - p27T198A mutant ( C , D ) or cotransfected with YFP - p27 and an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ratio ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot analysis ( C ) and fluorescence microscopy ( D , E ) .

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(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).


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( F ) Western blots of protein extracts from MCF - 7 transfected with YFP - p27 , YFP - p27T198A or YFP - p27T198D mutants following incubation with cycloheximide ( CHX ) for the indicated times . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

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(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


( a ) Crystal violet staining of MCF - 7 cell transfected with YFP ( C1 ) , YFP - p27 , YFP - p27T198A or YFP - p27T198D and selected with G418 for three weeks . Data are presented as mean ± s . d . ( n = 3 ) .

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(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3).


( b ) Flow cytometry analysis of MCF - 7 cells transfected with YFP ( C1 ) or YFP - p27 , YFP - p27T198A and YFP - p27T198D for 48 h . Data are presented as mean ± s . d . ( n = 3 ) .

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(b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP-p27, YFP-p27T198A and YFP-p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3).


( c ) GFP - LC3 U2OS cells 48 h post - transfection with RFP , RFP - p27 , RFP - p27T198A or RFP - p27T198D . Data represent results of three independent experiments , from which a total of ∼ 200 cells were counted ( s . d . , 10 ∼ 15 % ) . The scale bars represent 50 μm in c .

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(c) GFP-LC3 U2OS cells 48 h post-transfection with RFP, RFP-p27, RFP-p27T198A or RFP-p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.


( a , b ) MCF - 7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis ( a ) and cell - viability assays ( b ) .

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(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis (a) and cell-viability assays (b).


( c ) MCF - 7 cells were transfected with vector alone ( C1 ) , YFP - p27 ( WT ) , or the siRNA - resistant constructs WT - R and T198A - R 24 h before RNA interference ( RNAi ) . PARP cleavage was determined by western blotting and densitometric scanning . Data represent three independent experiments ( mean ± s . d . ) .

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(c) MCF-7 cells were transfected with vector alone (C1), YFP-p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.).


( d ) Western blots of lysates of MCF - 7 cells transfected with siRNA before serum - starvation or treatment with LY294002 ( LY , 10 μM ) or rapamycin ( Rap , 100 nM ) for 48 h with or without methylpyruvate ( MP , 10 mM ) .

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(d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM).


( e ) MCF - 7 cells treated with or without LY294002 for 48 h following RNAi . p27 siRNA pool - 1 , siRNA1 , 2 and 3 ; arrows , membrane blebbing ; triangles , apoptotic bodies . The scale bar represents 50 μm .

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(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.


( f ) Western blots of protein extracts from MCF - 7 cultured with ( + ) or without ( − ) glucose for 24 h following RNAi . Fr , fragment ; percentage PARP cleavage = 100 × PARPFr / ( PARP + PARPFr ) .

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(f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).


( a ) Western blots of lysates from the indicated cell lines .

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(a) Western blots of lysates from the indicated cell lines.


( b ) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors ( MDACC ) .

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(b) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors (MDACC).


( c ) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild - type LKB1 cultured with or without glucose for 24 h .

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(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.


( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e ) ,

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),


( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e )

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)


( d - f ) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min ( f ) .

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(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).


( g ) Western blot analysis of 3T3L1 cells 72 h post - transfection RNAi with or without glucose starvation for 1 h before analysis . Densitometric quantification of western blots is shown . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

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(g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


( A ) FACS analysis of TUNEL positive p27 + / + and p27 − / − cells treated with AICAR for 20 h . Data represent mean ± s . d . of three independent experiments .

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(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments.


( B , C ) Electron microscopy of p27 + / + ( a , c and e ) and p27 − / − ( b , d and f ) MEFs treated with ( c and d ) , or without ( a and b ) AICAR ( 1 mM ) , or without glucose ( e and f ) for 14 h . Magnified images of the boxed areas were shown on the left ( a ′ , b ′ , c ′ , d ′ , e ′ and f ′ ) with arrowheads indicating autophagic vacuoles including autolysosomes ( black arrowheads ) . The scale bars represent 10 μm in a - f and 0 . 5 μm in a ′ - f ′ . N , nucleus . Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s . d . ( C ) .

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(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).


B . Left , representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl / + ( WT ) , cHet , and cKO mice using the indicated antibodies . Right , quantification of protein levels normalized to β - actin ( n = 3 mice per group ) .

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B. Left, representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl/+(WT), cHet, and cKO mice using the indicated antibodies. Right, quantification of protein levels normalized to β-actin (n = 3 mice per group).


C . Left , representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl / + , cHet , and cKO mice . Right , quantification of protein levels which were normalized to β - actin ( n = 4 mice per group ) .

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C. Left, representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl/+, cHet, and cKO mice. Right, quantification of protein levels which were normalized to β-actin (n = 4 mice per group).


D . Immunostaining against ARID1A ( red ) in the dentate gyrus ( DG ) , cortex , CA1 , and CA3 regions of Arid1afl / + and cHet mice .

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D. Immunostaining against ARID1A (red) in the dentate gyrus (DG), cortex, CA1, and CA3 regions of Arid1afl/+and cHet mice.


E . cHet mice spent more time reaching the platform during 5 - day training in Morris water maze assay compared to the control group mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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E. cHet mice spent more time reaching the platform during 5-day training in Morris water maze assay compared to the control group mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


F . In the probe trial , cHet mice displayed spatial learning disabilities compared with Arid1afl / + mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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F. In the probe trial, cHet mice displayed spatial learning disabilities compared with Arid1afl/+ mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


G . cHet mice undertook fewer platform crossings than Arid1afl / + mice in the Morris water maze assay . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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G. cHet mice undertook fewer platform crossings than Arid1afl/+ mice in the Morris water maze assay. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


H - J . cHet mice were evaluated using the Barnes maze test . During the training phase , the cHet mice ( n = 12 mice ) failed to diminish the latency of the first entrance to the hiding box ( H ) , indicating that Arid1a haploinsufficiency results in impaired spatial memory . Probe trials demonstrated that cHet mice also had spatial memory deficits ( I ) , as they visited the target hole less often than the Arid1afl / + mice ( n = 12 mice ) ( J ) .

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H-J. cHet mice were evaluated using the Barnes maze test. During the training phase, the cHet mice (n = 12 mice) failed to diminish the latency of the first entrance to the hiding box (H), indicating that Arid1a haploinsufficiency results in impaired spatial memory. Probe trials demonstrated that cHet mice also had spatial memory deficits (I), as they visited the target hole less often than the Arid1afl/+ mice (n = 12 mice) (J).


A . Representative traces of spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in CA1 hippocampal acute slices from Arid1afl / + and cHet mice . B . Quantification of amplitude ( B , left ) and frequency ( B , right ) spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in Arid1afl / + and cHet hippocampal CA1 neurons . Arid1afl / + ( 17 neurons from n = 3 mice ) and cHet ( 15 neurons from n = 4 mice ) .

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A. Representative traces of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in CA1 hippocampal acute slices from Arid1afl/+ and cHet mice. B. Quantification of amplitude (B, left) and frequency (B, right) spontaneous miniature excitatory postsynaptic currents (mEPSCs) in Arid1afl/+ and cHet hippocampal CA1 neurons. Arid1afl/+ (17 neurons from n =3 mice) and cHet (15 neurons from n = 4 mice).


C . Representative recording of the paired - pulse ratio at the 50 - ms interpulse interval ms from slices prepared from Arid1afl / + ( n = 4 ) and cHet ( n = 4 ) mice . D . Paired - pulse facilitation ( PPF ) studies across different interpulse intervals ( 20 ms , 40 ms , 50 ms , 100 ms , 200 ms , 300ms , and 500 ms ) revealed a significant difference in paired - pulse ratios at 50 - ms intervals .

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C. Representative recording of the paired-pulse ratio at the 50-ms interpulse interval ms from slices prepared from Arid1afl/+ (n = 4) and cHet (n = 4) mice. D. Paired-pulse facilitation (PPF) studies across different interpulse intervals (20 ms, 40 ms, 50 ms, 100 ms, 200 ms, 300ms, and 500 ms) revealed a significant difference in paired-pulse ratios at 50-ms intervals.


E . Summary plots showing the time course of long - term potentiation ( LTP ) induced by frequency stimulation in the CA1 region from Arid1afl / + or cHet mice . Field excitatory postsynaptic potential ( fEPSP ) traces before ( black ) and after ( black or red ) are shown in the inset above . F . Average amplitude of LTP measured at 55 - 60 min postinduction ( n = 8 slices from 4 mice per group ; 10 fEPSP slope ( % ) values were collected from 1 slice ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_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-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O

E. Summary plots showing the time course of long-term potentiation (LTP) induced by frequency stimulation in the CA1 region from Arid1afl/+or cHet mice. Field excitatory postsynaptic potential (fEPSP) traces before (black) and after (black or red) are shown in the inset above. F. Average amplitude of LTP measured at 55-60 min postinduction (n = 8 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice).


G . Representative Western blot images for the expression of several LTP - related proteins in the Arid1afl / + and cHet hippocampi . H . Quantification of the expression of each protein normalized to the β - actin level .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

G. Representative Western blot images for the expression of several LTP-related proteins in the Arid1afl/+ and cHet hippocampi. H. Quantification of the expression of each protein normalized to the β-actin level.


A . Golgi - stained coronal section in the hippocampal CA1 region of 2 - month - old Arid1afl / + and cHet mice . Scale bar , 100µm . B - D . Partial loss of Arid1a significantly decreased dendritic length ( B ) , dendritic nodes ( C ) , and dendritic ends ( D ) in cHet neurons .

O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_TYPE O

A. Golgi-stained coronal section in the hippocampal CA1 region of 2-month-old Arid1afl/+ and cHet mice. Scale bar, 100µm. B-D. Partial loss of Arid1a significantly decreased dendritic length (B), dendritic nodes (C), and dendritic ends (D) in cHet neurons. E