Quantification of pADAM17 - positive cells and pERK1 / 2 MAPK - positive cells in LAC patients either KRAS wild - type ( WT , n = 13 ) or mutant ( MUT , n = 5 ) .

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

Quantification of pADAM17-positive cells and pERK1/2 MAPK-positive cells in LAC patients either KRAS wild-type (WT, n = 13) or mutant (MUT, n = 5).


Linear regression analyses of pADAM17 with pERK1 / 2 ( D ) and pp38 ( E ) MAPK staining in serial lung sections from LAC KRAS wild - type and mutant patients .

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

Linear regression analyses of pADAM17 with pERK1/2 (D) and pp38 (E) MAPK staining in serial lung sections from LAC KRAS wild-type and mutant patients.


Immunoblots of lysates from normal human bronchial epithelial cells ( HBEC ) and LAC cell lines A549 , NIH - H23 and NIH - H2228 with the indicated antibodies . Shown are lysates from 2 separate passages per cell line . Pro , proADAM17 ; Mat , mature ADAM17 . Densitometric semi - quantification of immunoblots showing the relative expression levels of pADAM17 ( relative to Actin ) , pro and mature forms of ADAM17 ( relative to Actin ) , and pERK1 / 2 and pp38 MAPK ( relative to their total counterparts ) protein levels .

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

Immunoblots of lysates from normal human bronchial epithelial cells (HBEC) and LAC cell lines A549, NIH-H23 and NIH-H2228 with the indicated antibodies. Shown are lysates from 2 separate passages per cell line. Pro, proADAM17; Mat, mature ADAM17. Densitometric semi-quantification of immunoblots showing the relative expression levels of pADAM17 (relative to Actin), pro and mature forms of ADAM17 (relative to Actin), and pERK1/2 and pp38 MAPK (relative to their total counterparts) protein levels.


ELISA of sIL - 6R protein levels in culture supernatants from matching cells in ( F ) cultured over 24 hours . Data are from 4 independent experiments .

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

ELISA of sIL-6R protein levels in culture supernatants from matching cells in (F) cultured over 24 hours. Data are from 4 independent experiments.


MTT viability assays of A549 cells transduced with nontargeted control sgRNA ( Ctl ) and ADAM17 sgRNA ( knockout , KO ) .

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

MTT viability assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).


ATP proliferation ( J ) assays of A549 cells transduced with nontargeted control sgRNA ( Ctl ) and ADAM17 sgRNA ( knockout , KO ) .

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

ATP proliferation (J) assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).


ELISA of sIL - 6R protein levels in culture supernatants from matching A549 cells ( I and J ) cultured over 24 hours . Data are from 3 independent experiments .

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

ELISA of sIL-6R protein levels in culture supernatants from matching A549 cells (I and J) cultured over 24 hours. Data are from 3 independent experiments.


Tumor volume ( mm3 ) of KO and Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 cells into NSG mice ( n = 5 per group ) .

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

Tumor volume (mm3) of KO and Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 cells into NSG mice (n = 5 per group).


Weight (grams) (M) of tumor xenografts


representative images (N) of tumor xenografts


ELISA of human sIL - 6R protein levels in serum of the indicated mice ( n = 5 per group ) . Levels of sIL - 6R are below detection in serum from KO xenograft mice . ND , not detected .

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

ELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). Levels of sIL-6R are below detection in serum from KO xenograft mice. ND, not detected.


Representative images of pADAM17 and pERK1 / 2 MAPK immunohistochemical staining on KO and Ctl A549 xenograft tumors . Scale bars , 100μm .

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

Representative images of pADAM17 and pERK1/2 MAPK immunohistochemical staining on KO and Ctl A549 xenograft tumors. Scale bars, 100μm.


Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies .

Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies.


Representative images of H & E - stained lung sections from KrasG12D mice treated with either vehicle ( control ) or the ADAM17 prodomain inhibitor , A17pro ( 1mg / kg , 3 times a week ) , for 6 weeks starting on the day after Cre inhalation . Scale bars , 3mm . Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups ( n = 6 per group )

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

Representative images of H&E-stained lung sections from KrasG12D mice treated with either vehicle (control) or the ADAM17 prodomain inhibitor, A17pro (1mg/kg, 3 times a week), for 6 weeks starting on the day after Cre inhalation. Scale bars, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups (n = 6 per group)


Representative images of lung sections from the indicated mice from ( A ) stained with antibodies against TTF - 1 ( C ) , PCNA ( E ) and pERK1 / 2 ( G ) . Scale bars , 100μm . Quantification of positive cells / high power field ( HPF ) in the lungs of mice from ( C ) , ( E ) and ( G ) stained for TTF - 1 ( D ) , PCNA ( F ) and pERK1 / 2 ( H ) . n = 6 per group .

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

Representative images of lung sections from the indicated mice from (A) stained with antibodies against TTF-1 (C), PCNA (E) and pERK1/2 (G). Scale bars, 100μm. Quantification of positive cells/high power field (HPF) in the lungs of mice from (C), (E) and (G) stained for TTF-1 (D), PCNA (F) and pERK1/2 (H). n = 6 per group.


ELISA of serum sIL - 6R protein levels in the indicated mice ( n = 6 per group )

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

ELISA of serum sIL-6R protein levels in the indicated mice (n = 6 per group)


Tumor volume ( mm3 ) of a KRAS mutant PDX treated with vehicle control ( Ctl ) or A17pro ( 1mg / kg ) every second day . Treatment started when tumors reached a volume of 100 - 150mm3 after injection of 2 × 106 cells in NSG mice . n = 5 per group .

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

Tumor volume (mm3) of a KRAS mutant PDX treated with vehicle control (Ctl) or A17pro (1mg/kg) every second day. Treatment started when tumors reached a volume of 100-150mm3 after injection of 2 × 106 cells in NSG mice. n = 5 per group.


representative images (L) of tumor PDXs


ELISA of human sIL - 6R protein levels in serum of the indicated mice ( n = 5 per group ) . *

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

ELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). *


Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro .

Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro.


Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor . Scale bar , 100μm .

Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor. Scale bar, 100μm.


Signal tracks showing cleavages generated by Med8 - MNase , Med17 - MNase , and PMED8 - MNase in YPD at three highly expressed SAGA - dependent ( A ) and three highly expressed TFIID - dependent ( B ) loci . All time points for a given factor were scaled to the same data range , and PMED8 - MNase tracks were scaled to the lower Mediator - MNase fusion range . Med14ChIP - seq and Med15ChIP - chip data are shown for comparison . TSSs are indicated by arrows .

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

Signal tracks showing cleavages generated by Med8-MNase, Med17-MNase, and PMED8-MNase in YPD at three highly expressed SAGA-dependent (A) and three highly expressed TFIID-dependent (B) loci. All time points for a given factor were scaled to the same data range, and PMED8-MNase tracks were scaled to the lower Mediator-MNase fusion range. Med14ChIP-seq and Med15ChIP-chip data are shown for comparison. TSSs are indicated by arrows.


( A ) Average plots of Mediator cleavages around the TSSs of SAGA - and TFIID - dependent genes . The distance of cleavage maximum to TSS is indicated by the dotted lines and corresponding numbers .

O O O O O O O O O O O O O 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) Average plots of Mediator cleavages around the TSSs of SAGA- and TFIID-dependent genes. The distance of cleavage maximum to TSS is indicated by the dotted lines and corresponding numbers.


( B ) Signal tracks of Med8 - MNase and free MNase cleavages at the previously characterized UASs of the CLB2 and RPS5 genes . TSSs are indicated by arrows .

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

(B) Signal tracks of Med8-MNase and free MNase cleavages at the previously characterized UASs of the CLB2 and RPS5 genes. TSSs are indicated by arrows.


( A ) Average plots of Med8 cleavages around the TSSs of SAGA - and TFIID - dependent genes in Kin28AS cells treated with DMSO ( - NA - PP1 ) or 6 μM NA - PP1 ( + NA - PP1 ) .

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

(A) Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes in Kin28AS cells treated with DMSO (-NA-PP1) or 6 μM NA-PP1 (+NA-PP1).


( B ) Signal tracks of Med8 - MNase cleavages at the upstream regions of several genes previously shown to have increased core promoter association of Mediator by ChIP - qPCR following NA - PP1 treatment of a Kin28AS strain ( Wong et al . , 2014 ) . All time points for a given treatment were concatenated to generate a combined track . TSSs are indicated by arrows .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Signal tracks of Med8-MNase cleavages at the upstream regions of several genes previously shown to have increased core promoter association of Mediator by ChIP-qPCR following NA-PP1 treatment of a Kin28AS strain (Wong et al., 2014). All time points for a given treatment were concatenated to generate a combined track. TSSs are indicated by arrows.


( A ) Average plots of Med8 cleavages around SAGA - and TFIID - dependent gene TSSs stratified into quintiles by the level of NET - seq signal in a 200 bp window downstream of the TSS .

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

(A) Average plots of Med8 cleavages around SAGA- and TFIID-dependent gene TSSs stratified into quintiles by the level of NET-seq signal in a 200 bp window downstream of the TSS.


( B ) Average plots of Med8 cleavage around the TSSs of the 87 most highly transcribed SAGA - and TFIID - dependent genes .

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

(B) Average plots of Med8 cleavage around the TSSs of the 87 most highly transcribed SAGA- and TFIID-dependent genes.


( C ) Boxplots of the transcription levels of the 87 most highly transcribed SAGA - and TFIID - dependent genes as determined by NET - seq . Significance was assessed by t - test .

O O O O O O O O O O O O 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) Boxplots of the transcription levels of the 87 most highly transcribed SAGA- and TFIID-dependent genes as determined by NET-seq. Significance was assessed by t-test.


( D ) Scatterplots of average NET - seq counts in a 200 bp window downstream of the TSS versus average Med8 cleavages in a 1 kb window upstream of the TSS for SAGA - and TFIID - dependent genes . Correspondence between the datasets was assessed by Spearman correlation .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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) Scatterplots of average NET-seq counts in a 200 bp window downstream of the TSS versus average Med8 cleavages in a 1 kb window upstream of the TSS for SAGA- and TFIID-dependent genes. Correspondence between the datasets was assessed by Spearman correlation.


( A ) Average plot of Med8 cleavages around 193 Gcn4ChIP - chip peak midpoints ( MacIsaac et al . , 2006 ) in WT and med15Δ .

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

(A) Average plot of Med8 cleavages around 193 Gcn4ChIP-chip peak midpoints (MacIsaac et al., 2006) in WT and med15Δ.


( B ) Average plots of Med8 cleavages around the TSSs of genes upregulated and downregulated ≥ 2 fold in SM ( Saint et al . , 2014 ) in WT and med15Δ . Control cleavages were subtracted from SM cleavages at each base position .

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

(B) Average plots of Med8 cleavages around the TSSs of genes upregulated and downregulated ≥2 fold in SM (Saint et al., 2014) in WT and med15Δ. Control cleavages were subtracted from SM cleavages at each base position.


( C ) qRT - PCR analysis of SAGA - and TFIID - dependent genes in WT and med15Δ . Bars represent mean + SEM for two biological replicates performed in triplicate . * , p < 0 . 05 ; † , p < 0 . 01 ; ‡ , p < 0 . 005 by unpaired Student ' s t - test .

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

(C) qRT-PCR analysis of SAGA- and TFIID-dependent genes in WT and med15Δ. Bars represent mean + SEM for two biological replicates performed in triplicate. *, p < 0.05; †, p < 0.01; ‡, p < 0.005 by unpaired Student's t-test.


( A ) Average plot of Taf1 cleavages around the TSSs of SAGA - and TFIID - dependent genes . A nucleosome occupancy profile as determined by MNase - seq is also shown for comparison . Dotted lines represent Taf1 cleavage maxima in TFIID - dependent gene bodies . The NDR , taken to be the - 1 to + 1 nucleosome midpoint distance , is indicated by a black rectangle .

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

(A) Average plot of Taf1 cleavages around the TSSs of SAGA- and TFIID-dependent genes. A nucleosome occupancy profile as determined by MNase-seq is also shown for comparison. Dotted lines represent Taf1 cleavage maxima in TFIID-dependent gene bodies. The NDR, taken to be the -1 to +1 nucleosome midpoint distance, is indicated by a black rectangle.


( B ) Average plots of Taf1 cleavages around the TSSs of SAGA - and TFIID - dependent genes ± rapamycin to deplete Med14 - FRB .

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

(B) Average plots of Taf1 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Med14-FRB.


Average plots of Med8 cleavages around the TSSs of SAGA - and TFIID - dependent genes ± rapamycin to deplete Taf1 - FRB .

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

Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Taf1-FRB.


A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C . trachomatis L2 ( multiplicity of infection MOI = 1 ) in the presence or not of BP . In the indicated samples CP4d was added 2 h before infection . Cell lysates were run on SDS - PAGE , proteins were transferred to a membrane and BP incorporation was revealed with HRP - conjugated streptavidin . BP incorporation is enhanced in infected samples , and is inhibited by CP4d . The two main bands present in all samples correspond to naturally biotinylated host proteins ( Haneji & Koide , 1989 ) . After blotting the membrane was stained with Coomassie blue to control for equal loading . B - Same as in ( A ) , except that where indicated 250 μM doxycycline ( doxy , left ) or 7 μM cycloheximide ( CHX , right ) were added 24 h or 2 hpi , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. trachomatis L2 (multiplicity of infection MOI=1) in the presence or not of BP. In the indicated samples CP4d was added 2 h before infection. Cell lysates were run on SDS-PAGE, proteins were transferred to a membrane and BP incorporation was revealed with HRP-conjugated streptavidin. BP incorporation is enhanced in infected samples, and is inhibited by CP4d. The two main bands present in all samples correspond to naturally biotinylated host proteins (Haneji & Koide, 1989). After blotting the membrane was stained with Coomassie blue to control for equal loading. B - Same as in (A), except that where indicated 250 μM doxycycline (doxy, left) or 7 μM cycloheximide (CHX, right) were added 24 h or 2 hpi, respectively.


C - Whole cell lysates were prepared with TG2 + / + and TG2 - / - MEFs infected or not for 48 h with C . trachomatis L2 in the presence or not of BP , and analyzed as in ( A ) .

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

C - Whole cell lysates were prepared with TG2+/+ and TG2-/- MEFs infected or not for 48 h with C. trachomatis L2 in the presence or not of BP, and analyzed as in (A).


D - Western blot with anti - TG2 antibodies on total cell lysates infected or not with C . trachomatis L2 for the indicated time . The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments , with the results of the Student ' s ratio - paired t - test . NI : not infected .

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

D -Western blot with anti-TG2 antibodies on total cell lysates infected or not with C. trachomatis L2 for the indicated time. The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments, with the results of the Student's ratio-paired t-test. NI: not infected.


E - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . Where indicated , 40 μM CP4d was added 2 hpi . tgm2 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least four times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. Where indicated, 40 μM CP4d was added 2 hpi. tgm2 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least four times. P-values of Student's ratio-paired t-test <0.05 are shown.


F - Cells were treated for 18 h with the indicated concentration of human recombinant IL - 6 before measuring TG2 transcription relative to actin like in ( E ) . The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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

F - Cells were treated for 18 h with the indicated concentration of human recombinant IL-6 before measuring TG2 transcription relative to actin like in (E). The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


Cells were left uninfected or infected with C . trachomatis L2 in the presence of the indicated concentration of anti - IL - 6 receptor antibodies . Forty - eight h later TG2 transcription relative to actin was measured like in ( E ) . The data are presented as relative mRNA levels compared to uninfected / untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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

Cells were left uninfected or infected with C. trachomatis L2 in the presence of the indicated concentration of anti-IL-6 receptor antibodies. Forty-eight h later TG2 transcription relative to actin was measured like in (E). The data are presented as relative mRNA levels compared to uninfected/untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


A - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were disrupted and bacterial titers ( IFU = inclusion forming unit ) were determined by re - infecting fresh HeLa cells as described in the methods . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


B - HeLa cells were transfected with control siRNA or two siRNAs against TG2 . Two days later , the efficiency of the silencing was assessed by western blot using anti - TG2 antibodies and anti - actin antibodies as loading control ( bottom ) . Duplicate wells were infected with L2incDGFP and progeny was analyzed as in ( A ) ( top ) . The mean ± SD of four independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were transfected with control siRNA or two siRNAs against TG2. Two days later, the efficiency of the silencing was assessed by western blot using anti-TG2 antibodies and anti-actin antibodies as loading control (bottom). Duplicate wells were infected with L2incDGFP and progeny was analyzed as in (A) (top). The mean ± SD of four independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


C - Same as in ( A ) except that C . trachomatis serovar L2 ( left ) or D ( right ) were grown in primary cells isolated from fallopian tubes . For serovar D , IFU were determined 48 hpi . The mean ± SD of four to five independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Same as in (A) except that C. trachomatis serovar L2 (left) or D (right) were grown in primary cells isolated from fallopian tubes. For serovar D, IFU were determined 48 hpi. The mean ± SD of four to five independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


D - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( left ) and the mean fluorescence of the infected population ( right ) ± SD are shown for three independent experiments . P - values of Student ' s paired t - test are indicated when < 0 . 05 . A representative field for each condition is shown , scale bar = 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm.


E - Primary epithelial cells isolated from fallopian tubes were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with C . trachomatis serovar L2 ( left ) or D ( right ) . Twenty - four hours later the cells were fixed , bacteria were stained using FITC - labeled anti - Chlamydia - LPS antibodies , and the mean size of the inclusions manually determined using ImageJ , on twenty inclusions per experiment . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


F - Mice were infected intravaginally with 105 IFU of C . muridarum . Twenty - five days later the mice were sacrificed and the upper genital tract , from the uterine horn to the oviduct , was collected . The right part was used for bacterial burden assessment ( top left ) . The left part was rinsed with PBS and observed with a binocular magnifier ( right ) to determine the hydrosalpinx score ( bottom left ) . Each dot represents one mouse , the mean ± SD is shown . P - values of Mann - Whitney test are indicated when < 0 . 05 .

F - Mice were infected intravaginally with 105 IFU of C. muridarum. Twenty-five days later the mice were sacrificed and the upper genital tract, from the uterine horn to the oviduct, was collected. The right part was used for bacterial burden assessment (top left). The left part was rinsed with PBS and observed with a binocular magnifier (right) to determine the hydrosalpinx score (bottom left). Each dot represents one mouse, the mean ± SD is shown. P-values of Mann-Whitney test are indicated when <0.05.


A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria ( MOI = 0 . 2 ) . Cells were disrupted 30 h later and the bacterial titer determined by re - infecting fresh wild type cells . The mean ± SD of three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria (MOI = 0.2). Cells were disrupted 30 h later and the bacterial titer determined by re-infecting fresh wild type cells. The mean ± SD of three independent experiments are shown.


B - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 48 h . Where indicated 40 μM CP4d was added 2 hpi . GLUT - 1 and GLUT - 3 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated four times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 48 h. Where indicated 40 μM CP4d was added 2 hpi. GLUT-1 and GLUT-3 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated four times. P-values of Student's ratio-paired t-test are indicated when <0.05.


C - Relationship between TGM2 and GLUT - 1 ( top ) and GLUT - 3 ( bottom ) expression across 265 HGSOCs . Expression comparisons were performed using Spearman ' s rank correlation test .

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

C -Relationship between TGM2 and GLUT-1 (top) and GLUT-3 (bottom) expression across 265 HGSOCs. Expression comparisons were performed using Spearman's rank correlation test.


D - HeLa cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . HIF - 1α transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative

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

D -HeLa cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. HIF-1α transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative


E - HeLa cells were transfected with control siRNA or two siRNAs against HIF - 1α . Two days later , cells were infected with C . trachomatis L2 ( MOI = 1 ) for 48 h . The indicated transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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

E - HeLa cells were transfected with control siRNA or two siRNAs against HIF-1α. Two days later, cells were infected with C. trachomatis L2 (MOI=1) for 48 h. The indicated transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


F - TG2 - / - MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C . trachomatis L2 ( MOI = 1 ) . Mouse GLUT - 1 transcripts were measured by real - time RT - PCR and normalized to mouse actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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

F - TG2-/- MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C. trachomatis L2 (MOI=1). Mouse GLUT-1 transcripts were measured by real-time RT-PCR and normalized to mouse actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


A - HeLa cells were infected with C . trachomatis ( MOI = 1 ) and 40 µM CP4d was added or not 2 hpi . After 24 h 0 . 5 mM BP was added and cells were lysed at 48 hpi . Lysates were precipitated with streptavidin - coated beads . After separation with SDS - PAGE , proteins were transferred to a membrane and blotted with anti - GFPT antibody followed with HRP - conjugated secondary antibody .

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

A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.


B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP . Samples were incubated for 3 h at 37 ° C before separation by SDS - PAGE . Proteins were transferred to a membrane and BP was revealed using HRP - conjugated streptavidin . rhGFPT1 is 77 . 5 kDa .

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

B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.


D - In vitro assay was performed as described in B using wild type rhGFPT1 ( WT ) , rhGFPT1 Q58N , rhGFPT1 Q328N or rhGFPT1 Q555N as substrates . The reaction was performed at 37 ° C for 30 min . After probing with HRP - Streptavidin the membrane was washed and probed with anti - GFPT antibodies followed with HRP - conjugated secondary antibodies . The ratio of modified protein ( streptavidin signal ) to the total GFPT is shown , normalized to its value with WT rhGFPT1 . The mean ± SD of five independent experiments is shown , the p - value of the Student ' s ratio - paired t - test is indicated when < 0 . 05 .

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

D - In vitro assay was performed as described in B using wild type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N or rhGFPT1 Q555N as substrates. The reaction was performed at 37 °C for 30 min. After probing with HRP-Streptavidin the membrane was washed and probed with anti-GFPT antibodies followed with HRP-conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown, normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, the p-value of the Student's ratio-paired t-test is indicated when <0.05.


E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 ° C for 45 min with fructose - 6 - P and glutamine . The production of glucosamine - 6 - P was measured using HPAEC - PAD . Results of three independent experiments are shown , with mean ± SD , and p - value of the Student ' s paired t - test is indicated ( * P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).


B - Endocervical epithelial cells were pre - treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin ( or an equivalent volume of DMSO ) and 0 . 5 µM BP . Six hours later whole cell lysates were analyzed by western blot . The membrane was first blotted with HRP - conjugated streptavidin to detect TG2 activity , then extensively washed and probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . Last the membrane was probed with anti - actin as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.


C - The same experimental procedure as described in ( B ) was applied to HeLa cells treated for 48 h prior to ionomycin treatment ( 8 µM ) with siRNA control or directed against TG2 .

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

C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.


A - HeLa cells were infected or not ( NI ) with C . trachomatis ( MOI = 1 ) , then lysed 24 or 48 hpi . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . After extensive washes the membrane was blotted again with anti - GFPT and anti - actin antibodies before revelation with HRP - conjugated secondary antibodies .

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

A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.


B - HeLa cells were infected or not with C . trachomatis ( MOI = 1 ) . Twenty - four hours later , 8 µM ionomycin ( or DMSO alone ) and 0 . 5 mM BP were added . After 6 h of treatment , cells were lysed and proteins revealed as in ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).


C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - GFPT and anti - actin antibody before revelation with HRP - conjugated secondary antibodies .

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

C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.


D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2 . Two days later the cells were infected for the indicated times ( MOI = 0 . 3 ) before fixation , rupture of the cells and measurement of bacterial diameter . The mean diameter ± SD and p - values of Student ' s paired t - test on 4 independent experiments are shown .

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

D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.


E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not ( siCTRL ) were infected with C . trachomatis ( MOI = 0 . 15 ) . Thirty hours later cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( top ) and the mean fluorescence of the infected population ( middle ) ) ± SD are shown for at least four independent experiments . Duplicate wells were lysed and used to re - infect fresh HeLa cells to determine the bacterial titer ( bottom ) . P - values of Student ' s ratio - paired t - test are indicated .

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

E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.


C - E KRAS and TP53 alterations in the cancer genome atlas ( TCGA ) pan - cancer MPM dataset ( n = 86 patients ) . Shown are clinical and molecular data plot with alteration frequencies ( C ) and patients reclassified as KRAS - or TP53 - altered ( asterisks ) , copy number variation data summary ( D ) , and segments of the KRAS and TP53 loci ( E ) .

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

C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients ( A ) and data summaries of mononucleotide change signatures ( B ) , of indices of genomic instability and mutation burden ( C ) , of clinical

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

Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients (A) and data summaries of mononucleotide change signatures (B), of indices of genomic instability and mutation burden (C), of clinical features


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients overall survival ( F ) .

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

Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients overall survival (F).


H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from ( A - G ) .

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

H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from (A-G).


Pleural fluid cell pellets and supernatants from 10 patients ( called CRCINA # ) with pleural effusion and pleural tumor samples from 17 patients ( called TR # ) with MPM were subjected to digital droplet polymerase chain reaction ( ddPCR ) for the detection of mutant ( MUT ) copies of KRAS codon 12 / 13 ( KRASG12 / 13 ) and KRAS codon 61 ( KRASQ61 ) , as well as copies of TP53 and TERT . Diagnoses were lung adenocarcinoma ( LUAD ; n = 4 ) and MPM ( n = 23 ) . The assays were designed for detection of down to 1 : 20000 copies using EKVX ( KRASWTTP53G610T ) , A549 ( KRASG12STP53WT ) , NCI - H460 ( KRASQ61HTP53WT ) , NCI - H3122 ( KRASWTTP53E285V ) , and NCI - H3255 ( KRASWTTP53G560 - 1A ) human LUAD cells as controls . Shown are individual patient ( KRAS plot ) and individual sample ( TP53 plot ) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot ( A ) Any number of KRAS - mutant droplets detected in any sample ( KRAS plot in A ) and any patient that failed to achieve normal TP53 ploidy by any sample ( TP53 plot in A ) was deemed altered .

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

Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.


F Representative May - Gruenwald - Giemsa - stained pleural fluid cytocentrifugal specimen from a KRASG12D ; Trp53f / f mouse showing macrophages ( MΦ , black arrow ) , lymphocytes ( LΦ , purple arrow ) , and neutrophils ( NΦ , green arrow ) and summary of cellular and biochemical features of effusions of KRASG12D ; Trp53f / f mice ( n = 10 ) .

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

F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).


G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [ n is given in table in ( E ) ] .

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

G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].


Wild - type ( Wt ) , KRASG12D , and Trp53f / f mice were intercrossed , and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad - Cre and were sacrificed when moribund . In parallel , C57BL / 6 mice received ten consecutive weekly intraperitoneal injections of 1 g / Kg urethane and were sacrificed after six months . Data summary ( heatmap ) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma ( MPM ) and lung adenocarcinoma ( LUAD ) . n = 10 mice / group were analyzed for each marker . Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining . Note the ubiquitous strong expression of Wilms ' tumor 1 ( WT1 ) , patchy moderate expression of vimentin ( VIM ) , ubiquitous moderate expression of mesothelin ( MSLN ) , ubiquitous strong expression of calretinin ( CALB2 ) , podoplanin ( PDPN ) , and osteopontin ( SPP1 ) , patchy moderate expression of cytokeratin 5 / 6 ( CK5 / 6 ) , and the absence of expression of surfactant protein C ( SFTPC ) in murine KRAS - driven mesotheliomas . Note also the ubiquitous strong expression of WT1 , the patchy moderate expression of VIM , the ubiquitous low - level expression of MSLN , the ubiquitous strong expression of CALB2 and SPP1 , the ubiquitous low - level expression of PDPN , the variable moderate expression of CK5 / 6 , and the ubiquitous moderate expression of SFTPC in murine KRASG12D - driven and urethane - induced LUAD .

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

Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.


C Data summaries from in vitro MTT reduction ( top ; 2 x 104 cells / well ; n = 6 independent experiments ) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL / 6 mouse ( bottom ; n = 5 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group).


E , F RT - PCR ( E ) and qPCR ( F ) of KPM cells and parental tumors show Trp53f / f allele deletion ( Δ ) and Bap1 and Cdkn2a overexpression compared with PMC .

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

E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.


A Kaplan - Meier survival plot with survival table .

A Kaplan-Meier survival plot with survival table.


F , G C57BL / 6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1 % DMSO or 15 mg / Kg deltarasin in 1 % DMSO at day 9 post - tumor cells . Shown are data summaries of MPE volume ( n = 8 and 7 DMSO and deltarasin - treated mice / group , respectively ) and pleural fluid nucleated cells at day 19 post - KPM1 cells ( F ) , as well as representative images of pleural effusions ( dashed lines ) and tumors ( t in ( G ) ) .

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

F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).


B Volcano plot showing some top KPM versus PMC differentially expressed genes .

B Volcano plot showing some top KPM versus PMC differentially expressed genes.


C KPM and PMC expression of classic mesothelioma markers ( top ) and top KPM versus PMC overexpressed genes ( bottom ) .

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

C KPM and PMC expression of classic mesothelioma markers (top) and top KPM versus PMC overexpressed genes (bottom).


( A ) Flow - cytometry for CD1d expression ( top ) and CD1d mean fluorescence intensity ( MFI ; bottom ) on CD45 - cells , ILCs ( Lin - CD45 + CD127 + ) , DCs ( CD11c + ) and B cells ( B220 + ) of the depicted tissues from WT mice ( n = 4 ) .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4).


( B ) Flow - cytometry profiles showing gating strategy ( dot - plots ) , CD1d expression ( histograms ) and CD1d MFI ( bars ) in T - bet + ( empty profile ) , GATA - 3 + ( dark grey ) , RORγt + ( light grey ) and T - bet + RORγt + ( dotted line ) ILCs in mLN , spleen , PP and SI - LP ( n = 5 ) . Numbers indicate percentage of cells in the depicted gates .

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

(B) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates.


( C ) Quantitative RT - PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s ( from mLN of CD1d - deficient mice and mLN and spleen of WT mice ) , B cells and DCs ( n = 3 ) . Results are normalized to those of GAPDH ; RE = relative expression . * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(C) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test).


( D ) Flow - cytometry profiles showing NKp46 and RORγt expression in Lin - CD45 + cells ; CD1d and CCR6 expression in NCR - ILC3s and CD1d and CD4 expression in NCR - CCR6 + ILC3s from mLN and SI - LP ( n = 4 ) . Numbers indicate percentage of cells in the depicted gates .

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

(D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+cells; CD1d and CCR6 expression in NCR-ILC3s and CD1d and CD4 expression in NCR-CCR6+ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates.


( E ) CD1d expression ( histograms ) and CD1d MFI in NCR + ( empty profile ) , NCR - CCR6 - ( dark grey ) and NCR - CCR6 + ( light grey ) ILC3s from mLN and SI - LP ( n = 4 ) .

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

(E) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).


( A ) Cells were cultured in the presence or absence of BODIPY - αGalCer at 4oC or 37oC as indicated . Flow cytometry profiles and percentages of BODIPY - αGalCer + DC or ILC3 from the indicated tissues incubated at 4oC ( white bars ) or 37oC ( grey bars ) are shown ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).


( B ) WT mice were intravenously injected with BODIPY - αGalCer or PBS ( control ) ; lipid uptake was analysed by flow - cytometry in the depicted populations from the spleen 16 h after lipid injection ( n = 3 ) . Numbers indicate percentage of cells in the depicted gates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.


( C ) IL - 2 secretion by DN32 . D3 cells co - cultured with ILC3 sort - purified from mLN or spleen as indicated , pulsed ( + ) or not ( - ) with αGalCer and with or without antiCD1d - blocking antibody .

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

(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) and adoptively transferred into WT recipients . ( E ) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort - purified from spleen , mLN or SI - LP of mice injected with αGalCer - loaded ILC3s . Gene expression was measured by qPCR , normalized to GAPDH and presented as expression relative to iNKT cells sort - purified from mice injected with control ILC3s ( n = 3 - 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5).


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) and adoptively transferred into WT recipients . ( F ) Intracellular cytokine staining ( left ) and percentage of cytokine + cells ( right ) in spleniciNKT cells from ILC3 - recipient mice ( n = 3 ) . Numbers indicate percentage of cells in the depicted gates . * * p < 0 . 01 , two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.


( A - B ) Flow - cytometry plots showing gating strategy ( A ) and percentage of ILC populations ( from Lin - CD45 + CD127 + cells , B ) in mLN from WT ( n = 4 ) and CD1d - deficient ( n = 3 ) mice .

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

(A-B) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.


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


( C ) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody - mediated CD1d crosslink ( n = 3 ) . Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s . * p < 0 . 05 two - tailed unpaired t - test .

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

(C) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody-mediated CD1d crosslink (n=3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *p<0.05 two-tailed unpaired t-test.


( D ) Fold change in IL22 mRNA expression in ILC3s after antibody - mediated CD1d crosslink and / or IL - 23 stimulation ( n = 4 ) . Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s . * * p < 0 . 01 two - tailed unpaired t - test .

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

(D) Fold change in IL22 mRNA expression in ILC3s after antibody-mediated CD1d crosslink and/or IL-23 stimulation (n=4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **p<0.01 two-tailed unpaired t-test.


( E ) IL - 22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and / or IL - 23 ( n = 3 ) . * p < 0 . 05 two - tailed unpaired t -

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

(E) IL-22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL-23 (n=3). *p<0.05 two-tailed unpaired t-test


( F - G ) Intracellular cytokine staining ( F ) and percentage of IL - 22 + ILC3s ( G ) after stimulation with αCD1d , IL - 23 and / or IL - 1β . * p < 0 . 05 , * * p < 0 . 01 two - tailed unpaired t - test ( n = 4 - 8 )

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

(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)


( H ) WT mice were i . v . injected with αGalCer or PBS ( control ) and activation of splenicILC3s was assessed by flow - cytometry as CD69 up - regulation at 6 h or 16 h after injection . Grey filled profile = ILC3 from αGalCer - injected mice ; Empty profile = ILC3 from PBS - injected mice ; Dotted line = CD45 - cells . Right graph , percentage of CD69 + ILC3s 6 h after injection of αGalCer ( grey ) or PBS ( white ) . ( n = 3 ) * p < 0 . 05 two - tailed unpaired t - test .

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

(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.


( I - J ) Fold change in mRNA expression for the indicated cytokines in ILC3s sort - purified from spleen ( I ) or SI - LP ( J ) 6 h after intravenous ( I ) or oral ( J ) αGalcer administration . Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort - purified from PBS - injected mice ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 two - tailed unpaired t - test .

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

(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.


( A ) Neural precursors derived from human iPS cells ( iNPCs ) were mock or ZIKV - infected and co - stained for Centrin ( green ) , Sox2 ( blue ) and flavivirus envelope ( red ) . All cells were fixed 16 hours post infection ( hpi ) . ( B ) Mock and ZIKV - infected U87 and H4 cells in S phase co - stained for Centrin ( green ) , flavivirus envelope ( red ) and DNA ( blue ) . ( C ) Quantification of the percentage of mock or ZIKV - infected iNPC , U87 or H4 cells in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 100nm .

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

(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi). (B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue). (C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 100nm.


( A ) Mock or ZIKV - infected U87 cells in S phase co - stained for Centrin ( red ) and the centrosomal microcephaly - associated protein , CEP63 ( green ) . ( B ) The fluorescence intensity ± s . d . of CEP63 at the centrosome ( 0 . 5μm around the Centrin signal ) and around the centrosome ( 1μm surrounding the Centrin signal minus the centrosomal CEP63 ) were quantified in mock and ZIKV infected U87 cells . For fluorescence quantifications 8 cells were analyzed per experiment ( n = 3 ) . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green). (B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test). Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( D ) Immunoblot analysis of lysate from mock or ZIKV - infected U87 cells probed for CEP63 and ZIKV NS3 . Actin served as a loading control . Asterisk denotes specific band for CEP63 .

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

(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.


( E ) S - phase mock and ZIKV infected control and Cep63gt / gt MEFs co - stained for Centrin ( green ) and flavivirus envelope ( red ) . ( F ) Quantification of the percentage of mock or ZIKV - infected Het and Cep63gt / gt MEFs in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red). (F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( B ) Mock or ZIKV - infected U87 cell lysates were immunoprecipitated for CEP63 or c - Myc , which served as a negative control for this and other immunoprecipitations ( Control ) . Precipitated proteins were immunoblotted for CEP63 and NS3 . Asterisk denotes specific band .

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

(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.


( C ) We expressed Myc - tagged Brazilian Zika NS3 in 293T / 17 cells and immunoprecipitated c - Myc . Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63 . The centriole distal end component , CP110 served as a negative control . Asterisk denotes specific band for CEP63 .

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

(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.


( G ) HeLa cells transfected with Control or Myc - tagged Brazilian ZIKV NS3 ( Br NS3 ) co - stained for Myc ( red ) and Centrin ( green ) . Data information : Scale bars indicate 5μm for all images .

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

(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green). Data information: Scale bars indicate 5μm for all images.


( H ) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . For all quantifications at least 100 cells were counted per experiment ( biological replicates = 3 ) . p < 0 . 005 ( paired Student ' s t test ) for the comparison of NS3 to mock transfected cells was denoted with asterisk .

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

(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.


( A ) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1 . Precipitated proteins were immunoblotted for CEP63 and TBK1 .

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

(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.


( B ) Mock and ZIKV - infected U87 cells were co - stained for γ - tubulin ( red ) and p - TBK1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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

(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green). Data information: Scale bars indicate 5μm for all images.


( C ) Lysate from mock or ZIKV - infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3 , TBK1 , and p - TBK1 . Actin served as a loading control .

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

(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.


( D ) RT - ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV - infected H4 cells . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented .

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

(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.


( E ) Control and Cep63gt / gt MEFs were co - stained with antibodies to γ - tubulin ( red ) and p - Tbk1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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

(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green). Data information: Scale bars indicate 5μm for all images.


( F ) Immunoblot analysis of control and Cep63gt / gt MEFs probed with antibodies to Tbk1 and p - Tbk1 . Actin served as a loading control .

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

(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.


( G ) HeLa cell lysate was immunoprecipitated with antibodies to CEP63 , DTX4 or c - Myc ( negative control ) . Precipitated proteins were blotted with antibodies to CEP63 and DTX4 .

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

(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.


( H ) Mock and ZIKV - infected U87 cells co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

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

(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( I ) Total cell lysate from mock and ZIKV - transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4 . Actin served as a loading control .

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

(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.


( J ) SC and CEP63 siRNA treated HeLa cells were co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

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

(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( K ) TBK1 or c - Myc ( negative control ) were immunoprecipitated from Control or Cep63 mutant MEFs . Precipitating proteins were probed with antibodies to TBK1 and K48 - linked ubiquitin .

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

(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.


( L ) 16hpi Mock and ZIKV - infected U87 cell lysate was immunoprecipitated for TBK1 or c - Myc . The immunoprecipitations were probed using antibodies to TBK1 and K - 48 - linked ubiquitin .

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

(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.


A , B : serum levels of S1P ( A ) and apoM ( B ) in HLT ( n = 47 ) and COV ( n = 111 ) patients . The box - plots represent the interquartile range with median value ( central line ) ; the whiskers represent the measured range of HLT and COV . Each measurement was run in triplicate , and performed at least twice . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P and apoM ( C ) Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between S1P and RBC number ( D ) , HGB concentration ( E ) , HCT value ( F ) . Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


A , B : correlations between S1P and HDL - C ( A ) , and albumin ( B ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each S1P measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


C , D : correlations between apoM and HDL - C ( C ) and albumin ( D ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each apoM measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure .

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

C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


( A - D ) The serum concentrations of S1P ( A ) , apoM ( B ) , albumin ( C ) and HDL - C ( D ) in noICU ( n = 89 ) and ICU ( n = 22 ) patients are shown . The box - plots represent the interquartile range with median ( central line ) ; the whiskers represent the measured range of noICU and ICU patients . Each S1P and apoM measurement was run in triplicate , and performed at least twice in independent assays . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Pearson correlation between S1P ( A - C ) PSI days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


Pearson correlation between apoM ( D - F ) and PSI , days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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

Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


( A , B ) Cumulative risk for ICU admission ( A ) and cumulative survival ( B ) in COV patients ( n = 111 ) , grouped for cut - off value of S1P serum level of 0 . 60 μM . Statistical analysis was performed by Cox regression . Exact p values are reported .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.


b . Multiplexed Error Robust Fluorescent in situ hybridization ( MERFISH ) and DAPI stained nuclei in the mouse hippocampus . Each gene is represented by a different color . For the entire hippocampus ( left ) , only the mRNA spots are shown with a scale bar of 500 microns . On the zoomed - in section ( right ) , each gene is represented by a different color dot , and the DAPI intensity is displayed in white . The scale bar is 20 microns .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Multiplexed Error Robust Fluorescent in situ hybridization (MERFISH) and DAPI stained nuclei in the mouse hippocampus. Each gene is represented by a different color. For the entire hippocampus (left), only the mRNA spots are shown with a scale bar of 500 microns. On the zoomed-in section (right), each gene is represented by a different color dot, and the DAPI intensity is displayed in white. The scale bar is 20 microns.


a . Normalized expression of Tox in CA1sp1 , Leng8 in CA3sp3 , Hecw1 in DG3 , and Thra in Astro1 show variable expression throughout the hippocampus . Scale bar is 500 microns . spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell ( sub ) type neighborhoods .

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

a. Normalized expression of Tox in CA1sp1, Leng8 in CA3sp3, Hecw1 in DG3, and Thra in Astro1 show variable expression throughout the hippocampus. Scale bar is 500 microns. spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell (sub)type neighborhoods.


A : HIF - 1α levels in HT29 primary tumors . Left : vehicle - treated controls Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Inset , high - magnification image of the region marked with a green asterisk ( * ) . Right : Quantification of the effect of monotherapies or doublet LDM / MTD regimens on HIF - 1α + area . F ( 5 , 12 ) = 8 . 791 and P = 0 . 001 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 046 vs vehicle ; # , P < 0 . 0001 vs LDMCTX + MTDCPB by Benjamini , Krieger and Yekuteli post hoc test .

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

A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test.


B : HIF - 2α levels in HT29 primary tumors . Left : Example of immunostaining in vehicle - treated controls . Inset , high - magnification image of the region marked with a green asterisk ( * ) . Right : Quantification of the effect of monotherapies or doublet LDM / MTD regimens on HIF - 2α + area . F ( 5 , 18 ) = 1 . 215 and P = 0 . 3424 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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

B: HIF-2α levels in HT29 primary tumors. Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA.