A . DG - 75 B cells expressing CD80GFP or CD86GFP ( green ) and PD - L1mCherry ( red ) were incubated with Jurkat cells ( nuclei shown in grey ) expressing CTLA - 4 WT or mutant CTLA - 4 Del36 for the indicated durations and analysed by confocal microscopy . 2 hours prior to assay endpoint , APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was added . CD80 / CD86 and PD - L1 co - localisation at the immune synapse is shown in yellow with PD - 1 Ig binding shown in purple . Scale bar , 10µm .

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

A. DG-75 B cells expressing CD80GFP or CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat cells (nuclei shown in grey) expressing CTLA-4 WT or mutant CTLA-4 Del36 for the indicated durations and analysed by confocal microscopy. 2 hours prior to assay endpoint, APC-labelled PD-1 Ig (0.75µg/ml) was added. CD80/CD86 and PD-L1 co-localisation at the immune synapse is shown in yellow with PD-1 Ig binding shown in purple. Scale bar, 10µm.


B . Flow cytometry plots of CD80GFP levels on DG - 75 B - cells ( gated on CTFR + cells ) at 24h with graphical representation of the MFI in each condition ( RH graph ) . The Jreg cells used , and the anti - PD - 1 status is indicated above the plot . C . Flow cytometry plots of CD28 levels indicating contact between the CD80 + APC and the PD - 1 + CD28 + responder Jurkat T - cells , following CD80 transendocytosis . Graphical representation of the CD28 MFI in each condition is shown on the right . D . Flow cytometry of CD69 levels on responder Jurkat T - cells and graphical representation of CD69 MFI in each condition . E . Flow cytometry of CD69 levels on CD28KO ( PD - 1 + ) responder Jurkat T - cells and graphical representation of the CD69 + cells MFI in each condition . Cartoons on the left of B - E illustrate the cell type analysed . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 O 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. Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg cells used, and the anti-PD-1 status is indicated above the plot. C. Flow cytometry plots of CD28 levels indicating contact between the CD80+ APC and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28 MFI in each condition is shown on the right. D. Flow cytometry of CD69 levels on responder Jurkat T-cells and graphical representation of CD69 MFI in each condition. E. Flow cytometry of CD69 levels on CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.


B . Titration of Abatacept staining ( left hand panel ) and EC50 values of Abatacept ( right hand panel ) for CD80 only or PD - L1 / CD80 co - expressing DG - 75 . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : paired t - test ( B )

O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O 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. Titration of Abatacept staining (left hand panel) and EC50 values of Abatacept (right hand panel) for CD80 only or PD-L1/CD80 co-expressing DG-75. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B)


D . Comparison of PD - L1 detection using PD - 1 - Ig or anti - PD - L1 antibodies ( MIH3 or 29E . 2A3 clone ) with and without prior incubation with 10µg / ml of abatacept , based on data from ( C ) . E . Graphs showing PD - L1 detection under conditions used in ( C ) following different Abatacept incubation periods ( 30mins , 3h or 6h ) . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : RM one - way ANOVA ( D ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 O O O O O O O O O O O

D. Comparison of PD-L1 detection using PD-1-Ig or anti-PD-L1 antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C). E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept incubation periods (30mins, 3h or 6h). Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: RM one-way ANOVA (D).


A . SDS - PAGE analysis of the D391N DIS3L2 - RNA complexes . RNAs were radioactively labeled at the 5 ' end and DIS3L2 - bound RNAs were detected by autoradiography . The negative control was performed with HEK293T - Rex cells ( Control )

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A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)


B . DIS3L2 CLIP reads possess oligo ( U ) 3 ' termini . Histograms of reads with 3 ' - terminal stretches of identical nucleotides . Only oligo ( U ) tails longer than 4 nucleotides are observed .

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B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed.


C . The untemplated uridylation is mostly found in the proximity of mature 3 ' RNA ends . The heat map shows the coverage of various RNA types by uridylated reads . RNA class and number of uridylated genes / transcripts for each category are indicated on the left . The length of each mature RNA was scaled to 100 % .

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C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%.


D . List of DIS3L2 - bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs . Members of the let - 7 family are shown in bold .

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D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold.


F . Secondary structure of pre - hsa - let - 7i , with both 5p and 3p mature products highlighted in bold . The intramolecular ligation position is indicated in dash line between nt 28 and 61 . It corresponds to cleavage positions of RNaseT1 , which leaves 3 ' - terminal guanosine residues .

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F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues.


G . D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs . Scatter plot of transcript abundances estimated from RNA - seq from the cell line overexpressing D391N DIS3L2 and control HEK293T - Rex cells . CLIPed transcripts are marked in white .

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G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.


A . Types of uridylated ncRNAs identified by DIS3L2 CLIP - seq .

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A. Types of uridylated ncRNAs identified by DIS3L2 CLIP-seq.


B . U + read coverage in downstream regions of mature U12 snRNA , Vault1 and 7SL ncRNAs . The full dark box is representing mature ncRNA , the thin line indicates the region downstream of the mature 3 ' end ( extension ) .

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B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).


C . Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature ( Mat ) , extended ( ext ) or trimmed ( Trim ) 3 ' end . + U is number of clones with untemplated 3 ' oligo ( U ) ends . For more details see Figure S2 . ( D ) An example of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) for RNU5D snRNA .

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C. Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature (Mat), extended (ext) or trimmed (Trim) 3' end. +U is number of clones with untemplated 3' oligo(U) ends. For more details see Figure S2. (D) An example of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) for RNU5D snRNA.


E . U + reads mapped to the rDNA locus by using the artificial chromosome U13369 . 1 .

E. U+ reads mapped to the rDNA locus by using the artificial chromosome U13369.1.


B . Uridylated tRNA reads overlap mainly with 3 ' trailers , T - loop and anticodon loop regions . Heat map showing the coverage of uridylated reads on pre - tRNAs . The pre - tRNA sub - regions ( as indicated in ( A ) ) are in small letters on the x - axis .

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B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.


C . Northern blot analysis of RNAs immunoprecipitated with Flag - tagged WT ( DIS3L2 ) and D391N ( MUT ) DIS3L2 , AGO2 and DIS3 , respectively . The blots were probed with radioactively labeled probes for 5 ' and 3 ' ends of mature tRNAVal , respectively .

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C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.


D . Sequencing of MUT DIS3L2 - bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP - seq . Genomic sequence for each tRNA is indicated on top with the 3 ' trailer region separated by a vertical line . Non - templated uridine residues are in red .

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D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.


E . Identification of hybrid reads ( produced by intra - strand ligation of RNaseT1 products during CLIP protocol ) mapping to tRNAPhe ( GAA ) and tRNAAsn ( GTT ) genes . To the right : the schematic representation of selected hybrid reads with corresponding color - code .

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E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.


A . DIS3L2 - bound uridylated mRNA reads map to the 5 ' and 3 ' UTRs .

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A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs.


B . Most 3 ' UTR U + reads match the stem - loop structure of histone mRNAs . Heat map representation of the uridylation position .

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B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position.


C . The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2 . Small RNAs were modified with 3 ' end linker , and linker - specific primer was used for cDNA synthesis . Uridylated RNAs were PCR amplified in a semi - quantitative way with the same amount of input RNA used for RT reaction . We used a combination of the gene - and linker - specific primers , - RT ctrl is RT - PCR control , where no reverse transcriptase was added ; PCR control is a reaction with no cDNA added . The graph on the right summarizes the percentage of uridylated and nonuridylated 5 ' fragments of OAT mRNA in HEK293T - Rex cells ( Ctrl ) and cells overexpressing mutant DIS3L2 ( D391N ) .

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 O 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 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 B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O

C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N).


D . DIS3L2 - bound uridylated RNAs are degraded by DIS3L2 in vivo . RT - PCR amplification of 5 ' mRfs of OAT and RPL12 genes and 3 ' extended forms of U12 snRNA ( RNU12 ext ) from HEK293T cell lines with modified expression of DIS3L2 ( as marked on top ) was performed as described in C . Regions of mature 5S rRNA and 7SL RNA were used as loading controls . DIS3L2 KO is a DIS3L2 knock - out cell line . DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant ( D391N ) and wild type ( WT ) DIS3L2 expressing constructs , respectively . Bands marked with * are unspecific primer - dimer PCR products .

O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD 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

D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products.


E . Metagene analysis of the position of DIS3L2 D391N clipped U + reads peak maximum around mRNA transcription start sites ( TSS ) and of the ChIP - seq data for RNAP II .

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E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II.


F . U + 5 ' mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm . RT - PCR detection of 5 ' mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions , respectively . The RT - PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions . The identity and uridylation status of cytoplasmic OAT U + 5 ' mRfs was confirmed by sequencing ( shown on the bottom ) . In red are untemplated nucleotides , in green region corresponding to the first intron of OAT pre - mRNA . The efficiency of the subcellular fractionation was monitored by western blot ( right panel , WB ) . DDX5 is nucleoplasmic , ACO2 is mitochondrial , and DIS3L2 and TUBa are cytoplasmic markers .

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F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBa are cytoplasmic markers.


( A ) SARS - CoV - 2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco - 2 and A549 - ACE2 cells at 24 and 48 hpi . The most abundant small RNAs are marked by the red and blue boxes . For all the experiments shown n = 2 .

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(A) SARS-CoV-2 genomic view showing the distribution of normalized 22nt small RNA reads from Caco-2 and A549-ACE2 cells at 24 and 48 hpi. The most abundant small RNAs are marked by the red and blue boxes. For all the experiments shown n=2.


( C ) Percentage of conservation along the nucleotide positions in the ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a . Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71 - 366 nt of ORF7a among 4 , 055 , 609 sequenced SARS - CoV - 2 genomes . Box plots display median ( line ) , first and third quartiles ( box ) , and 5th / 95th percentile value ( whiskers ) . Each dot represents the outliers . Two - tailed P values were calculated using the student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD 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 O O O O

(C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.


( A ) Log2 fold changes of the levels of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 in DICER1 knockdown SARS - CoV - 2 - infected A549 - ACE2 cells compared to control cells analyzed by stem - loop RT - qPCR . Results from three independent replicates are shown .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(A) Log2 fold changes of the levels of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 in DICER1 knockdown SARS-CoV-2-infected A549-ACE2 cells compared to control cells analyzed by stem-loop RT-qPCR. Results from three independent replicates are shown.


( B ) In vitro kinetics of DICER1 mediated cleavage of pre - CoV2 - miR - O7a stem - loop precursor , pre - CoV2 - miR - O7a with mutations that prevent the formation of stem - loop structure and pre - hsa - miR21 . Folded conformations for the three precursor molecules are shown . Fold change in the production of mature CoV2 - miR - O7a . 2 is shown in presence of DICER1 compared to control reaction ( no DICER1 ) as measure by stem - loop RT - qPCR . Bars and error bars represent the average and standard deviation from three independent experiments .

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

(B) In vitro kinetics of DICER1 mediated cleavage of pre-CoV2-miR-O7a stem-loop precursor, pre-CoV2-miR-O7a with mutations that prevent the formation of stem-loop structure and pre-hsa-miR21. Folded conformations for the three precursor molecules are shown. Fold change in the production of mature CoV2-miR-O7a.2 is shown in presence of DICER1 compared to control reaction (no DICER1) as measure by stem-loop RT-qPCR. Bars and error bars represent the average and standard deviation from three independent experiments.


( C ) Expression levels of the virus - derived miRNAs as a percentage of the viral genome . Absolute quantification of virus - derived miRNAs and viral genome from infected A549 - ACE2 cells was performed using two spike - in ( see methods ) . Line represents the average and individual dots represent data from two independent experiments .

O O O B-EXP_ASSAY 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 O B-CELL_LINE 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

(C) Expression levels of the virus-derived miRNAs as a percentage of the viral genome. Absolute quantification of virus-derived miRNAs and viral genome from infected A549-ACE2 cells was performed using two spike-in (see methods). Line represents the average and individual dots represent data from two independent experiments.


( D ) copy number per cell of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and CoV2 - miR - O7a . 2 in infected A549 - ACE2 cells quantified using a synthetic small RNA spike - in standard curve by stem - loop RT - qPCR . Levels of hsa - miR - let - 7a were normalized for the percentage of infected cells . Bars and error bars represent the average and standard deviation from at least three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD O 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 I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-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

(D) copy number per cell of hsa-miR-let-7a, CoV2-miR-O7a.1 and CoV2-miR-O7a.2 in infected A549-ACE2 cells quantified using a synthetic small RNA spike-in standard curve by stem-loop RT-qPCR. Levels of hsa-miR-let-7a were normalized for the percentage of infected cells. Bars and error bars represent the average and standard deviation from at least three independent experiments.


( E ) Loading of hsa - miR - let - 7a , CoV2 - miR - O7a . 1 and COV2 - miR - O7a . 2 into AGOs as measured by stem - loop RT - qPCR and analyzed as a percentage of input from the immunoprecipitates ( IPs ) of either pan - AGO IP or control IgG IP from infected A549 - ACE2 cells . Levels of miRNA were normalized for the percentage of infected cells .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O

(E) Loading of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 into AGOs as measured by stem-loop RT-qPCR and analyzed as a percentage of input from the immunoprecipitates (IPs) of either pan-AGO IP or control IgG IP from infected A549-ACE2 cells. Levels of miRNA were normalized for the percentage of infected cells.


( B ) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual - Luciferase miRNA Target Expression Vector with either CoV2 - miR - O7a . 2 target site or CoV2 - miR - O7a . 2 site with mutations in seed region at 24 h on co - transfection with CoV2 - miR - O7a . 2 or control miRNA mimics . Activity on transfecting control miRNA mimic was considered 100 % . Bars and error bars represent the average and standard deviation from three independent experiments .

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

(B) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual-Luciferase miRNA Target Expression Vector with either CoV2-miR-O7a.2 target site or CoV2-miR-O7a.2 site with mutations in seed region at 24 h on co-transfection with CoV2-miR-O7a.2 or control miRNA mimics. Activity on transfecting control miRNA mimic was considered 100 %. Bars and error bars represent the average and standard deviation from three independent experiments.


( D ) Kinetics of BATF2 and LAMP3 mRNA levels in non - induced and interferon IFN - α - induced ( for 8 , 16 , and 24 h ) A549 - ACE2 cells transfected with either control or CoV2 - miR - O7a . 2 mimics . Normalized read abundances in transcript per million ( TPM ) are shown for two individual experiments ( n = 2 ) .

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(D) Kinetics of BATF2 and LAMP3 mRNA levels in non-induced and interferon IFN-α-induced (for 8, 16, and 24 h) A549-ACE2 cells transfected with either control or CoV2-miR-O7a.2 mimics. Normalized read abundances in transcript per million (TPM) are shown for two individual experiments (n=2).


( E ) Genomic view of the human BATF2 gene showing normalized RNA - seq reads from non - induced and IFN - α - induced ( for 8 h ) A549 - ACE2 cells transfected with either control , CoV2 - miR - O7a . 1 or CoV2 - miR - O7a . 2 mimics . The complementary site of CoV2 - miR - O7a to BATF2 3 ´ UTR is shown .

O O O O O O O B-ORGANISM B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O

(E) Genomic view of the human BATF2 gene showing normalized RNA-seq reads from non-induced and IFN-α-induced (for 8 h) A549-ACE2 cells transfected with either control, CoV2-miR-O7a.1 or CoV2-miR-O7a.2 mimics. The complementary site of CoV2-miR-O7a to BATF2 3´UTR is shown.


( A , B ) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN - α treatment in A549 - ACE2 cells transfected with CoV2 - miR - O7a . 1 ( A ) or CoV2 - miR - O7a . 2 ( B ) compared to control mimic . Significantly downregulated genes are marked in red , and upregulated genes in blue . The orange horizontal line indicates two - tailed P = 0 . 05 . n = 2 , dashed lines represent + 1 and - 1 log2 fold change .

O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-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

(A, B) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.1 (A) or CoV2-miR-O7a.2 (B) compared to control mimic. Significantly downregulated genes are marked in red, and upregulated genes in blue. The orange horizontal line indicates two-tailed P = 0.05. n=2, dashed lines represent +1 and -1 log2 fold change.


( C ) Log2 fold change of ISGs at 8 , 16 , 24 hours of IFN - α treatment and categorized based on CoV2 - miR - O7a . 2 target sites 8mer , 7mer - m8 , 7mer - A1 , and no seed as shown in the schematic . The mean and standard error of the mean is shown . Number of ISGs with 8mer site n = 8 , 7mer - m8 n = 32 , 7mer - A1 n = 26 and no seed n = 92 . The two - tailed p values were calculated using the Mann - Whitney - Wilcoxon test . ISGs were calculated as all the upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in two replicates of IFN - induced versus non - induced conditions in all time points .

O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-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 O O O O O O O O O O O O O O O O O O O O O O O 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

(C) Log2 fold change of ISGs at 8, 16, 24 hours of IFN-α treatment and categorized based on CoV2-miR-O7a.2 target sites 8mer, 7mer-m8, 7mer-A1, and no seed as shown in the schematic. The mean and standard error of the mean is shown. Number of ISGs with 8mer site n=8, 7mer-m8 n= 32, 7mer-A1 n=26 and no seed n= 92. The two-tailed p values were calculated using the Mann-Whitney-Wilcoxon test. ISGs were calculated as all the upregulated genes (≥ 3-fold; padj < 0.05) in two replicates of IFN-induced versus non-induced conditions in all time points.


( A ) Heat map showing log2 fold change of expression of ISGs in A549 - ACE2 cells across 8 , 16 and 24 h time points upon IFN - α treatment compared to non - treated controls . The common upregulated genes ( ≥ 3 - fold ; padj < 0 . 05 ) in IFN - induced versus non - induced conditions at all time points were categorized as ISGs .

O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-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 B-GENEPROD O O O O O O O O O O O O O O O O

(A) Heat map showing log2 fold change of expression of ISGs in A549-ACE2 cells across 8, 16 and 24 h time points upon IFN-α treatment compared to non-treated controls. The common upregulated genes (≥ 3-fold; padj < 0.05) in IFN-induced versus non-induced conditions at all time points were categorized as ISGs.


( A ) Expression levels of CoV2 - miR - O7a . 1 , CoV2 - miR - O7a . 2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs ( Control ORF6 ) analyzed by stem - loop RT - qPCR from nasopharyngeal swabs of patients tested positive for COVID - 19 or another seasonal human coronavirus ( HCoV ) . Relative expression to hsa - miR - let - 7a is shown . Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples .

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

(A) Expression levels of CoV2-miR-O7a.1, CoV2-miR-O7a.2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs (Control ORF6) analyzed by stem-loop RT-qPCR from nasopharyngeal swabs of patients tested positive for COVID-19 or another seasonal human coronavirus (HCoV). Relative expression to hsa-miR-let-7a is shown. Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Body weight curve .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O 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 O

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Body weight curve.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Cumulative body weight .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O 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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Cumulative body weight.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Fasting and fed blood glucose .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Fasting and fed blood glucose.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Rectal temperature .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Rectal temperature.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . Locomotor tolerance .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O 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

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Locomotor tolerance.


Eight - week - old male WT and DOCK5 - / - mice were fed a standard diet ( SD ) or high - fat diet ( HFD ) for 3 months . 24 - h oxygen consumption . Respiratory exchange ratio ( RER : VCO2 / VO2 ) . Energy expenditure .

O O O O O O O O B-GENEPROD O O O B-ORGANISM 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 I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. 24-h oxygen consumption. Respiratory exchange ratio (RER: VCO2/VO2). Energy expenditure.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Blood glucose levels and area under curve ( AUC ) during the glucose tolerance test . Blood glucose levels and AUC during the insulin tolerance test .

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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Blood glucose levels and area under curve (AUC) during the glucose tolerance test. Blood glucose levels and AUC during the insulin tolerance test.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) .

O O O O O O O O B-GENEPROD O O O B-ORGANISM 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-EXP_ASSAY I-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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP).


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or HFD for 3 months . Percentage of suppression of hepatic glucose production ( HGP ) .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O 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

Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Percentage of suppression of hepatic glucose production (HGP).


Eight - week - old male WT and DOCK5 - / - mice Glucose infusion rates ( GIR ) during lipid infusion .

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

Eight-week-old male WT and DOCK5-/- mice Glucose infusion rates (GIR) during lipid infusion.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . The expression of expression of phosphoenolpyruvate carboxykinase ( PEPCK ) and glucose - 6 - phosphatase ( G6Pase ) mRNA and protein .

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

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. The expression of expression of phosphoenolpyruvate carboxykinase(PEPCK) and glucose-6- phosphatase(G6Pase) mRNA and protein.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated insulin receptor ( InsR ) , IRS - 1ser1101 and Akt .

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

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt.


Eight - week - old male WT and DOCK5 - / - mice were fed a SD or a HFD for 3 months . Mice were treated with insulin ( 1 U / kg ) or control ( PBS ) by intraperitoneal injection for 10 minutes ; animals were killed and livers were collected . Total and phosphorylated mTOR , S6K1 , and the levels of Raptor and Rictor protein .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated mTOR, S6K1, and the levels of Raptor and Rictor protein.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in Hep1 - 6 cells .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in Hep1-6 cells.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in Hep1 - 6 cells .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. - mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in Hep1-6 cells.


Hep1 - 6 cells were infected with pCDNA3 . 1 - DOCK5 ( DOCK5 + ) or pCDNA3 . 1 ( DOCK5 - ) for 48 h . mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in Hep1 - 6 cells .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in Hep1-6 cells.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . The mRNA and protein expression of PEPCK and G6Pase in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in MPHs.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in MPHs.


MPHs isolated from WT and DOCK5 - / - mice were incubated in control ( GlcN - ) or GlcN ( GlcN + ) medium for indicated times , followed with ( Ins + ) or without ( Ins - ) 100 nmol / L insulin stimulation for 20 min . Total and phosphorylated mTOR and S6K1 , and the levels of Raptor and Rictor protein in MPHs .

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

MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in MPHs.


Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Fasting and fed blood glucose 14 days post - infection .

O O O O O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O

Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Fasting and fed blood glucose 14 days post-infection.


Eight - week - old male Raptor flox / flox mice ( Raptorflox / flox Cre + ) were fed a HFD for 12 weeks and injected with AAV8 - shDOCK5 ± AAV8 - Cre or AAV8 - GFP ( at a dose of 3 × 1011 vg / 200 μL / mouse ) via the tail - vein 14 days prior to the in vivo study . Blood glucose levels and AUC during glucose tolerance tests . Blood glucose levels and AUC during insulin tolerance tests . Glucose infusion rates ( GIR ) . Glucose disposal rate ( GDR ) . Hepatic glucose production ( HGP ) . Percentage of suppression of hepatic glucose production ( HGP ) .

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

Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The mRNA and protein expression of PEPCK and G6Pase in the liver .

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

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The mRNA and protein expression of PEPCK and G6Pase in the liver.


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt in the liver .

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

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.


Male Raptor flox / flox mice ( 8 weeks ) were fed a HFD for 12 weeks and injected with AAV8 - GFP or AAV8 - shDOCK5 or AAV8 - shDOCK5 + AAV8 - Cre via the tail vein . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 in the liver .

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

Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1 in the liver.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The expression of PEPCK and G6Pase mRNA and protein .

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

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The expression of PEPCK and G6Pase mRNA and protein.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . Total and phosphorylated InsR , IRS - 1Ser1101 and Akt .

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

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. Total and phosphorylated InsR, IRS-1Ser1101 and Akt.


MPHs from Raptor flox / flox mice were infected with Ad - GFP or Ad - shDOCK5 or Ad - shDOCK5 + Ad - Cre . The lysates were immunoblotted with indicated antibodies or β - actin . The levels of DOCK5 and Raptor protein , and total and phosphorylated mTOR and S6K1 .

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

MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1.


Rac1 GEF activation in MPHs from WT and DOCK5 - / - mice .

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

Rac1 GEF activation in MPHs from WT and DOCK5-/- mice.


SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes ( MPHs ) of DOCK5 - / - mice .

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

SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.


Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated - Hep1 - 6 cells .

O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated-Hep1-6 cells.


Coimmunoprecipitation ( Co - IP ) analysis of DOCK5 and Raptor expression in Hepa1 - 6 cells . After transfected with indicated plasmids , lysates from cells were immunoprecipitated ( IP ) with anti - DOCK5 antibody or normal IgG , and then immunoblotted ( IB ) with the indicated antibody .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O

Coimmunoprecipitation (Co-IP) analysis of DOCK5 and Raptor expression in Hepa1-6 cells. After transfected with indicated plasmids, lysates from cells were immunoprecipitated (IP) with anti-DOCK5 antibody or normal IgG, and then immunoblotted (IB) with the indicated antibody.


DOCK5 binding to Raptor fragments ( 445 - 887 aa ) during transient expression in Hepa1 - 6 cells .

B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

DOCK5 binding to Raptor fragments (445 - 887 aa) during transient expression in Hepa1-6 cells.


The DOCK5 and Raptor binding sites were identified using mutant DOCK5 ( DHR - 2V1559A ) and Raptor fragments ( 445 - 887 aa ) based on a Co - IP assay . VA , mutant DOCK5 DHR - 2 ; His , His - DHR - 2 .

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

The DOCK5 and Raptor binding sites were identified using mutant DOCK5 (DHR-2V1559A) and Raptor fragments (445 - 887 aa) based on a Co-IP assay. VA, mutant DOCK5 DHR-2; His, His-DHR-2.


B Immunofluorescence analysis of fibronectin ( green ) and collagen ( red ) fibers on de - cellularized ECM produced by human fibroblasts . Fibers orientation was quantified using ImageJ software . Percentages indicate oriented fibers accumulated in a range of ± 21 ° around the modal angle . Data is represented as mean ± s . d . ( n = 10 random fields from 2 independent determinations ) . . Scale bar , 50 µm . A representative image of picrosirius red staining from 10 analyzed is shown for each condition .

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

B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition.


D , E Quantification of proliferation of 1205Lu ( D ) and SKMEL5 ( E ) cells plated for 48 h on plastic , Coll - 1 or the indicated fibroblast - derived ECMs prior a 96 h treatment with vehicle , 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi . Cells were counted by Hoechst - labeled nuclei staining . Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments . ( D ) * * P = 0 . 015 , * * * P = 0 . 0009 , * * * * P < 0 . 0001 and ( E ) * P = 0 . 0204 , * * * P = 0 . 0006 , * * * * P < 0 . 0001 . Kruskal - Wallis followed by Dunn ' s multiple comparisons test .

O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O 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, E Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll-1 or the indicated fibroblast-derived ECMs prior a 96 h treatment with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst-labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P=0.015, ***P=0.0009, ****P<0.0001 and (E) *P=0.0204, ***P=0.0006, ****P<0.0001. Kruskal-Wallis followed by Dunn's multiple comparisons test.


F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic , Coll - 1 or the indicated fibroblast - derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0 . 01 µM MEKi . The percentage of cells in different phases of the cell cycle is indicated .

O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic, Coll-1 or the indicated fibroblast-derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.


G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic , Coll - 1 or the indicated fibroblast - derived ECMs in the presence or not of BRAFi or BRAFi / MEKi for 96 h , using antibodies against P - ERK1 / 2 , ERK2 or cell cycle markers ( P - Rb , Rb , Cyclin D1 , p27KIP1 , Survivin , p53 ) . HSP60 , loading control .

O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O

G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic, Coll-1 or the indicated fibroblast-derived ECMs in the presence or not of BRAFi or BRAFi/MEKi for 96 h, using antibodies against P-ERK1/2, ERK2 or cell cycle markers (P-Rb, Rb, Cyclin D1, p27KIP1, Survivin, p53). HSP60, loading control.


B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays . Representative IHC images and quantification ( right bar histograms ) of DDR1 and DDR2 expression in normal skin , naevus , primary melanoma ( PM ) and lymph node melanoma metastases ( MM ) . Scale bar , 100 µm . Histological scoring of the samples was performed in blinded fashion . Samples were scored as low , medium or high for DDR1 or DDR2 expression ( naevus , n = 12 ; PM , n = 30 ; MM , n = 20 ) .

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

B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays. Representative IHC images and quantification (right bar histograms) of DDR1 and DDR2 expression in normal skin, naevus, primary melanoma (PM) and lymph node melanoma metastases (MM). Scale bar, 100 µm. Histological scoring of the samples was performed in blinded fashion. Samples were scored as low, medium or high for DDR1 or DDR2 expression (naevus, n = 12; PM, n = 30; MM, n = 20).


Immunoblotting of equal amounts of protein extracts from melanoma cell lines ( C ) using antibodies against DDR1 , DDR2 or markers of the melanoma cell differentiation AXL , MITF or SOX10 . ERK2 , loading control .

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Immunoblotting of equal amounts of protein extracts from melanoma cell lines (C) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.


Immunoblotting of equal amounts of protein extracts patient - derived short - term cell cultures ( D ) using antibodies against DDR1 , DDR2 or markers of the melanoma cell differentiation AXL , MITF or SOX10 . ERK2 , loading control .

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Immunoblotting of equal amounts of protein extracts patient-derived short-term cell cultures (D) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.


B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1 # 2 / siDDR2 # 2 prior being cultivated on FRC - or MAF - derived ECMs ( left and right panels , respectively ) and treated with vehicle , 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi , using antibodies against the indicated proteins . HSP60 , loading control .

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B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1#2/siDDR2#2 prior being cultivated on FRC- or MAF-derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control.


D Immunoblotting of protein extracts from 1205Lu , SKMEL5 and MM099 cells plated for 48 h on FRC - derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib ( 7 µM for 1205Lu , 10 µM for SKMEL5 and MM099 ) or DDR1 - IN - 1 ( 1 µM for 1205Lu , 5 µM for SKMEL5 , 3 µM for MM099 ) , using antibodies against the indicated proteins . HSP60 , loading control .

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D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.


A Representative images of 1205 cells cultivated on collagen - I ( Coll - I ) or FRC - derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0 . 01 µM MEKi for 96 h . Immunofluorescence for Phospho - DDR1 ( P ( Y792 ) - DDR1 ) ( red ; left panels ) and Phospho - DDR1 / 2 ( P ( Y796 ) - DDR1 / P ( Y740 ) - DDR2 ) ( red ; right panels ) , F - actin ( green ) and nuclei ( blue ) is shown . Enlarged images of P - DDR1 and P - DDR1 / 2 immunostaining are shown . White arrows indicate P - DDR1 and P - DDR1 / 2 cell membrane linear clustering . Scale bar , 25 µm ( enlarged images : scale bar , 10 µm ) .

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A Representative images of 1205 cells cultivated on collagen-I (Coll-I) or FRC-derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for Phospho-DDR1 (P(Y792)-DDR1) (red; left panels) and Phospho-DDR1/2 (P(Y796)-DDR1/P(Y740)-DDR2) (red; right panels), F-actin (green) and nuclei (blue) is shown. Enlarged images of P-DDR1 and P-DDR1/2 immunostaining are shown. White arrows indicate P-DDR1 and P-DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).


E Kaplan - Meier survival curves of mice treated with vehicle , Imatinib , BRAFi or BRAFi plus Imatinib . Median time to progression was 18 , 20 , 36 and 48 days , respectively . Log rank ( Mantel - Cox ) for BRAFi vs BRAFi / Imatinib mesylate . * * * * P < 0 . 0001 and hazard ratio ( log rank ) : 0 . 2403 ( 95 % CI of ratio , 0 . 08123 to 0 . 7106 ) .

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E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106).


A Sections of 1205Lu xenografts from Fig . 6B were stained with picrosirius red and imaged under transmission light ( upper panels ) or polarized light ( middle panels ) ( scale bar , 500 µm ) or imaged by second harmonic generation ( SHG ) microscopy ( lower panels ) ( scale bar , 50 µm ) to examine collagen fiber network upon the mono or combined regimens .

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A Sections of 1205Lu xenografts from Fig. 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono or combined regimens.


A Immunoblot analysis of protein extracts from siCTRL - or siDDR1 / 2 - transfected SKMEL5 cells plated on FRC - or MAF - derived ECMs in the presence or not of 5 µM BRAFi , 2 µM BRAFi plus 0 . 01 µM MEKi for 96 h using antibodies against DDR1 , DDR2 , P - ERK1 / 2 , ERK2 , RelB , p100 / p52 and HSP60 as loading control were used .

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A Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected SKMEL5 cells plated on FRC- or MAF-derived ECMs in the presence or not of 5 µM BRAFi, 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P-ERK1/2, ERK2, RelB, p100/p52 and HSP60 as loading control were used.


F , G Immunoblot analysis of protein extracts obtained from SKMEL5 ( F ) or MM099 ( G ) cells plated on FRC - or MAF - derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1 - IN - 1 ( 5 µM for SKMEL5 and 3 µM for MM099 ) or 10 µM NIK inhibitor ( NIKi ) for 96 h . Antibodies against P - ERK1 / 2 , ERK2 , P - Rb , Rb , Survivin , Caspase 3 , cleaved Caspase 3 , RelB , p100 / p52 and HSP60 as loading control were used .

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F, G Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC- or MAF-derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1-IN-1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P-ERK1/2, ERK2, P-Rb, Rb, Survivin, Caspase 3, cleaved Caspase 3, RelB, p100/p52 and HSP60 as loading control were used.


Immunoblot of expression levels of MYC , TSC1 , TSC2 , and β - actin loading control in high MYC Burkitt ' s lymphoma ( BL ) cells compared to low MYC Hodgkin lymphoma ( HL ) cells .

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Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells.


Immunoblots showing expression levels of MYC , TSC1 , TSC2 or β - actin loading control in P493 - 6 cells treated with tetracycline for 72 hours ( + Tet ) or in untreated cells ( - Tet ) .

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Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).


Relative TSC1 and TSC2 mRNA expression levels determined by qRT - PCR for high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells treated for 24 h with tetracycline ( mean ± st . dev . , n = 3 technical replicates ) * p < 0 . 05 ; * * p < 0 . 01 ; statistical relevance was determined by unpaired t - test ( two - tailed ) .

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Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).


qRT - PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h - 72h ( + Tet ) . Immunoblots for 24h and 48h ( + Tet ) show S6K and phosphorylation ( P - ) of S6K as downstream mTORC1 target , and β - actin loading control . For 72 h ( + Tet ) the immunoblots show expression of MYC and phosphorylation ( P - ) of downstream mTORC1 targets S6K and S6 , and α - tubulin as loading control .

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qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.


Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1 - specific sh - RNAs compared to scrambled control sh - RNA in P493 - 6 cells . Other blots show the expression levels of TSC2 , S6K / P - S6K , S6 / P - S6 and α - tubulin for loading control .

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Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control.


Immunoblots of indicated proteins in P493 - 6 cells with high MYC ( - Tet , 72 hours ) or low MYC ( + Tet , 72 hours ) levels either treated with rapamycin or solvent .

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Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.


Elevated TSC1 and MYC expression in BL ( cohort 1 ) . Example of immune staining of TSC1 , MYC , the B - cell marker CD20 , and DAPI nuclear DNA - staining in germinal centers of control lymph nodes ( upper rows ) and samples from BL patients ( lower rows ) . Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples ( see materials and methods ) ( the horizontal line shows the median , whiskers show maximum and minimum data points and the box represents the first to the third quartiles , n = 56 fields for tumor samples and n = 21 fields for control germinal centers ; scale bar = 100 μm ) .

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Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).


Quantification of immunoblot analysis of TSC expression in BL ( cohort 2 ) ( n = 13 ) compared to healthy tonsils ( n = 3 ) or reactive lymph nodes ( n = 3 ) ( mean ± st . dev . ) shown in Fig EV2B .

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Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n=13) compared to healthy tonsils (n=3) or reactive lymph nodes (n=3) (mean ± st. dev.) shown in Fig EV2B.


Left graph shows the multiplication rate of P493 - 6 ( - Tet ) cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA determined by viable cell counting 3 days after seeding of equal number of viable cells ( determined by Trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) . Right graph shows percentage of apoptotic P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or a TSC1 - specific sh - RNA determined by FACS analysis of AnnexinV / 7AAD stained cells ( mean ± st . dev . , n = 3 biological replicates ) .

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Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).


Rapamycin treatment recovers survival of TSC1 knockdown in P493 - 6 cells . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 14 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 30pM rapamycin where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

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Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown is synthetic lethal with MYC deregulation . U2OS - MYC - ER cells expressing either scrambled control sh - RNA or TSC1 - specific sh - RNA were treated with hydroxytamoxifen to induce MYC and rapamycin ( 100 nM ) where indicated . Percentage of apoptotic cells was determined with Annexin / 7AAD staining 4 days after MYC induction ( mean ± st . dev . , n = 3 biological replicates ) .

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TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).


Survival rate of different BL cell lines upon TSC1 knockdown . Graphs show numbers of viable cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA 3 days after seeding of equal number of viable cells ( determined by trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) .

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Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).


Immunoblots of control - or TSC1 - shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity ( 10nM ) or titrate the activity to control levels ( 30 pM ) , and survival rate of these cells over 7 days ( mean ± st . dev . , n = 3 biological replicates ) ; ( Bl2 cells were selected for stable knockdown with Puromycin , DG75 cells without selection ) .

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Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).


Ramos cells expressing either a TSC1 - specific or a control shRNA were inoculated into NOD / SCID mice and tumor volume was measured regularly . The immunoblot shows the level of knockdown of TSC1 ( sh - TSC1b ) compared to control ( sh - contr ) , and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation . Tumor growth curves show mean ± SEM ( n = 8 / group ) . Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation . The top panels show tumors derived from Ramos cells stably expressing control sh - RNA ( sh - contr ) ; the lower panels show tumors derived from Ramos cells stably expressing TSC1 - specific sh - RNA ( sh - TSC1b ) .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD 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 B-ORGANISM B-CELL_LINE B-DISEASE O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O

Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or TSC - specific sh - RNA , treated with 20 nM rapamycin for 12 h , or tetracycline to repress MYC for 48 h where

B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O

TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated


TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated ( B ) ( mean ± st . dev . , n = 6 biological replicates ) .

B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).


Ratio of mitochondrial to genomic DNA determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD 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

Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


expression of cytochrome C ( CYCS ) or ATP5G1 mRNAs ( D ) determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD 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

expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).


TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner . FACS analysis of DCF - DA stained P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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

TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).


TSC1 knockdown results in increased phosphorylation of SAPK / JNK . Immunoblot of SAPK / JNK Thr183 / Tyr185 - phosphorylation in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA . α - tubulin expression serves as loading control .

B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O

TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.


Antioxidant treatment rescues cells from death caused by TSC1 knockdown . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 3 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 10 μM of the antioxidant BHA where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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

Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).


Accumulation of EU - labeled ( Click - It , Invitrogen ) TSC1 - mRNA over 3 h in high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells ( mean ± st . dev . , n = 3 technical replicates ) .

O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O

Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).