C : CA9 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 CA9 + area . F ( 5 , 11 ) = 2 . 466 and P = 0 . 0961 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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 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

C: CA9 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 CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA.


D : Correlation between HIF - 1α levels and proliferation indexes in HT29 primary tumors . Left and middle / left : HIF - 1α and Ki67 expression in vehicle - treated controls ; consecutive sections are shown . Middle / right : HIF - 1α + vs Ki67 + proliferation index scatterplot . Each point represents median values for each tumor . Regression line ( red ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 27 ) = 34 . 45 and P = 10 - 4 ; slope ≠ 0 by F - test Right : Machine - learning quantification of the effect of monotherapies or doublet LDM / MTD regimens upon Ki67 + proliferative index . Median indexes per tumor are indicated . F ( 5 , 12 ) = 1 . 609 and P = 0 . 2312 ( not significant ) for overall treatment by Brown - Forsythe ANOVA .

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

D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors. Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown. Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.


A : Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size . Left : Histogram of pooled cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . A decreasing linear trend for median metastatic diameter was observed ( left to right , slope = - 143 . 2 , P = 0 . 043 by post - test for trend ) . Right : Dichotomized metastatic size at median diameter of vehicle - treated tumors . Liver nodule size was classified as small ( blue ) or large ( red ) . Individual nodule counts per group and size category ( small / large ) are plotted on the right ordinate . χ2 ( df = 5 ) = 6 . 80 ; P = 0 . 2361 ( not significant ) for overall effects on size ; χ2 ( df = 1 ) = 4 . 71 ; P = 0 . 0299 ( LDMCTX + LDMCPB vs vehicle ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle).


B : HIF - 1α levels in HT29 liver metastatic nodules . Left : vehicle - treated controls . Middle / left : Doublet LDM cyclophosphamide + capecitabine ( LDMCTX + LDMCPB ) . Middle / right : LDM cyclophosphamide + MTD capecitabine ( LDMCTX + MTDCPB ) . Right : Automatic quantification of the effect of monotherapies or doublet regimens on HIF - 1α + areas in individual metastatic nodules . F ( 2 , 13 ) = 4 . 796 and P = 0 . 028 for overall treatment by Brown - Forsythe ANOVA ; * , P = 0 . 038 LDMCTX + MTDCPB vs vehicle or P = 0 . 0123 LDMCTX + LDMCPB vs vehicle ; # , P = 0 . 0098 LDMCTX + LDMCPB 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 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 O O O O O O

B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test.


C : Correlation between metastatic and peri - metastatic HIF - 1α levels in the liver . Left : Nodule immunostaining and parenchymal HIF - 1α expression . Each point represents median values per nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 57 ) = 150 . 7 and P < 0 . 0001 ; slope ≠ 0 by F - test . Right : Correlation between intra - metastatic and peri - metastatic parenchymatous ring in the liver . Each point represents median values per nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 54 ) = 55 . 2 and P < 0 . 0001 ; slope ≠ 0 by F - test . .

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 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 O O O O O O O O O O O O O O O O O O O O O O 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: Correlation between metastatic and peri-metastatic HIF-1α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-1α expression. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,57) = 150.7 and P< 0.0001; slope ≠ 0 by F-test. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values per nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test..


D : Correlation between metastatic and peri - metastatic HIF - 2α levels in the liver . Left : Nodule immunostaining and parenchymal HIF - 2α expression . Right : Correlation between intra - metastatic and peri - metastatic parenchymatous ring in the liver . Each point represents median values for each nodule . Regression line ( blue ) and 95 % CI ( shaded blue area ) are indicated . F ( 1 , 54 ) = 55 . 2 and P < 0 . 0001 ; slope ≠ 0 by F - test . n , number of nodules .

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 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 O O

D: Correlation between metastatic and peri-metastatic HIF-2α levels in the liver. Left: Nodule immunostaining and parenchymal HIF-2α expression. Right: Correlation between intra-metastatic and peri-metastatic parenchymatous ring in the liver. Each point represents median values for each nodule. Regression line (blue) and 95% CI (shaded blue area) are indicated. F(1,54) = 55.2 and P< 0.0001; slope ≠ 0 by F-test. n, number of nodules.


A : EMT6 - CDDP lung metastatic nodule size . Left : Histogram of cross - sectional metastatic diameter ( Ø ) . Middle : Metastatic diameter ( Ø ) by chemotherapy regimen . F ( 5 , 99 ) = 0 . 9310 and P = 0 . 4644 ( not significant ) for overall treatment by Brown - Forsythe ANOVA . Right : Dichotomized metastatic size at median diameter of vehicle - treated tumors . Lung nodule size was classified as small ( blue ) or large ( red ) . Individual nodule counts per group and size category ( small / large ) are plotted on the right ordinate . χ2 ( df = 5 ) = 2 . 631 ; P = 0 . 7567 ( not significant ) for overall effects on size .

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A: EMT6-CDDP lung metastatic nodule size. Left: Histogram of cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. F(5,99) = 0.9310 and P= 0.4644 (not significant) for overall treatment by Brown-Forsythe ANOVA. Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Lung nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 2.631; P= 0.7567 (not significant) for overall effects on size.


B : Hypoxic fraction in lung metastatic nodules . Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 110 ) = 7 . 544 and P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicle ; # , P = 0 . 0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini , Krieger and Yekuteli post hoc test .

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B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test.


C : HIF - 1α levels in lung metastatic nodules . Automatic quantification of HIF - 1α levels measured as fractional positive areas per nodule ( rightmost panel ) . F ( 5 , 114 ) = 5 . 325 and P = 0 . 0004 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 vs vehicleby 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 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

C: HIF-1α levels in lung metastatic nodules. Automatic quantification of HIF-1α levels measured as fractional positive areas per nodule (rightmost panel). F(5,114) = 5.325 and P= 0.0004 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicleby Benjamini, Krieger and Yekuteli post hoc test.


D : Microvessel density in lung metastatic nodules . Automatic quantification of CD31 fractional areas per nodule ( rightmost panel ) . F ( 5 , 124 ) = 7 . 531 and P < 0 . 0001 for overall treatment by Brown - Forsythe ANOVA ; * , P < 0 . 01 ( LDM capecitabine , cyclophosphamide , or doublet capecitabine + cyclophosphamide vs vehicle ) , # , P = 0 . 0417 ( doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine ) by Benjamini , Krieger and Yekuteli post hoc test .

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D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.


B : HIFα - inducible colon cancer signature ( HIFi - CCS ) . Left and middle / left : List of HIFα targets and overall survival in TCGA ( left ) or aggregated microarray data ( middle / left ) from colon adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank test . Middle / right : HIFi - CCS and lymph node ( LN ) invasion status . Data are standardized as z - scores . t ( 194 ) = 4 . 399 and P < 0 . 0001 by Student t - test with Welch ' s correction . Right : HIFi - CCS is associated with accelerated distant metastasis . Cases are classified as low ( blue ) or high ( red ) HIFi - CCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: HIFα- inducible colon cancer signature (HIFi-CCS). Left and middle/left: List of HIFα targets and overall survival in TCGA (left) or aggregated microarray data (middle/left) from colon adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-CCS and lymph node (LN) invasion status. Data are standardized as z-scores. t(194) = 4.399 and P< 0.0001 by Student t-test with Welch's correction. Right: HIFi-CCS is associated with accelerated distant metastasis. Cases are classified as low (blue) or high (red) HIFi-CCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test.


C : HIFα - inducible breast cancer signature ( HIFi - BCS ) . Left and middle / left : List of HIFα targets and overall survival in METABRIC ( left ) or recurrence - free survival in microarray ( middle / left ) from breast adenocarcinomas . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS according to their relationship to the median . Kaplan - Meier analysis followed by log - rank test . Middle / right : HIFi - BCS and estrogen receptor ( ER ) status . Data are standardized as z - scores . t ( 112 ) = 3 . 789 and P = 0 . 0002 by Student t - test with Welch ' s correction . Right : HIFi - BCS is associated with worsened distant metastasis - free survival . Cases are classified as low ( blue ) or high ( red ) HIFi - BCS whenever they are below or above the distribution median . Kaplan - Meier analysis followed by log - rank 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: HIFα- inducible breast cancer signature (HIFi-BCS). Left and middle/left: List of HIFα targets and overall survival in METABRIC (left) or recurrence- free survival in microarray (middle/left) from breast adenocarcinomas. Cases are classified as low (blue) or high (red) HIFi-BCS according to their relationship to the median. Kaplan-Meier analysis followed by log-rank test. Middle/right: HIFi-BCS and estrogen receptor (ER) status. Data are standardized as z-scores. t(112) = 3.789 and P= 0.0002 by Student t-test with Welch's correction. Right: HIFi-BCS is associated with worsened distant metastasis-free survival. Cases are classified as low (blue) or high (red) HIFi-BCS whenever they are below or above the distribution median. Kaplan-Meier analysis followed by log-rank test.


D : Median RNAseq expression of individual HIFα targets as split by median HIFi - CCS ( left ) or HIFi - BCS ( right ) . Transcripts up / downregulated by ± 1 . 5 - fold are shown in blue or yellow , respectively . Error bars indicate 95 % CI of the median . Log2 scale ; n = 209 ( colon , TCGA ) or n = 609 ( breast , METABRIC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Median RNAseq expression of individual HIFα targets as split by median HIFi-CCS (left) or HIFi-BCS (right). Transcripts up/downregulated by ±1.5-fold are shown in blue or yellow, respectively. Error bars indicate 95% CI of the median. Log2 scale; n= 209 (colon, TCGA) or n= 609 (breast, METABRIC).


A - D Cells were infected at the indicated MOI . Viral replication ( Left ) and release ( Right ) were assessed by flow cytometry and RT - qPCR . A ) Caco2 / TC7 cells ( MOI 0 . 01 ) B ) Calu - 3 cells ( MOI 0 . 001 ) C ) Vero cells ( MOI 0 , 01 ) D ) primary human airway epithelial cells ( HAEC ) virus release ( Right ) and infectious virus release ( Left ) ( MOI 0 . 01 ) . Data are mean ± SD of at least 3 independent experiments . Statistical analysis : Mixed - effect analysis or Two ‐ way ANOVA compared to D614G reference , ns : non ‐ significant , * P < 0 . 05 , * * * * P < 0 . 0001 .

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A-D Cells were infected at the indicated MOI. Viral replication (Left) and release (Right) were assessed by flow cytometry and RT-qPCR. A) Caco2/TC7 cells (MOI 0.01) B) Calu-3 cells (MOI 0.001) C) Vero cells (MOI 0,01) D) primary human airway epithelial cells (HAEC) virus release (Right) and infectious virus release (Left) (MOI 0.01). Data are mean ± SD of at least 3 independent experiments. Statistical analysis: Mixed-effect analysis or Two‐way ANOVA compared to D614G reference, ns: non‐significant, *P < 0. 05, ****P < 0.0001.


( B ) Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for U2OS - ACE2 20h post infection at MOI 0 . 001 . Right Panel : Representative images of U2OS - ACE2 20h post infection , GFP - Split ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel .

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(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS-ACE2 20h post infection at MOI 0.001. Right Panel: Representative images of U2OS-ACE2 20h post infection, GFP-Split (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel.


( C ) Left Panel : Quantified fusion of Vero cells infected at MOI 0 . 01 . Right Panel : Representative images of Vero cells 48h post infection , GFP - Split ( Green ) and Hoechst ( Blue ) .

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(C) Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48h post infection, GFP-Split (Green) and Hoechst (Blue).


( B ) Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Vero GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( C ) Left Panel : Quantification of variant S protein mediated fusion in Vero - GFP split cells by video microscopy . Results are mean ± SD from three fields per condition from one representative experiment . Right Panel : Fusion quantification of at least 3 independent video microscopy experiments , 20h post transfection , normalized to D614G .

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(C) Left Panel: Quantification of variant S protein mediated fusion in Vero-GFP split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of at least 3 independent video microscopy experiments, 20h post transfection, normalized to D614G.


( B ) Left Panel : Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S . Right Panel : Representative images of fusion at 20h . Scale bar : 200 µm . Top and bottom are the same images with and without Hoechst channel .

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(B) Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.


( B ) Left Panel : EC50 values ( concentration of ACE2 needed for 50 % binding ) for Alpha and associated mutations . Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding . Right Panel : EC50 values for Beta and associated mutations . Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants .

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(B) Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Colour code corresponds to location on S protein functional domains and lower EC50 values signifies higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants.


( A ) Vero GFP - split cells were transfected with variant S proteins and imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Vero GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200 µm .

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(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.


( D ) Caco2 GFP - split cells were transfected with variant S proteins and imaged 18h post - transfection . Left Panel : Fusion was quantified by GFP area / number of nuclei and normalized to D614G for each of the transfected variant S proteins . Right Panel : Representative images of Caco2 GFP - split cells 18h post - transfection , GFP ( Green ) and Hoechst ( Blue ) . Top and bottom are the same images with and without Hoechst channel . Scale bars : 200

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

(D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm


( A ) Expression of all rate - limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA ( sh - ctrl ) , as measured by Western blot . β - actin was employed as a loading control throughout . ( B - I ) Expression of the pluripotency genes c - Myc , Oct - 4 , Nanog , Klf4 and Sox - 2 in R1 cells after knockdown of Hk2 ( B ) , Pfkl ( C ) , Hk1 ( D ) , Hk3 ( E ) , Pfkm ( F ) , Pkm2 ( G ) , Pklr ( H ) and Pfkp ( I ) glycolytic enzymes using two or three independent shRNAs , as measured by Western blot .

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(A) Expression of all rate-limiting glycolytic isoenzymes in R1 cells after knockdown of Stat3 using three independent shRNAs compared to an empty vector control shRNA (sh-ctrl), as measured by Western blot. β-actin was employed as a loading control throughout. (B-I) Expression of the pluripotency genes c-Myc, Oct-4, Nanog, Klf4 and Sox-2 in R1 cells after knockdown of Hk2 (B), Pfkl (C), Hk1 (D), Hk3 (E), Pfkm (F), Pkm2 (G), Pklr (H) and Pfkp (I) glycolytic enzymes using two or three independent shRNAs, as measured by Western blot. D


( A - C ) Expression of Pfkp mRNA levels ( top panels ) and Stat3 and Pfkp protein expression ( bottom panels ) after knockdown of Stat3 using three independent shRNAs in either R1 cells ( A ) or E14 cells ( B ) or after overexpression of Stat3 in R1 cells ( C ) , as measured by qPCR and Western blot , respectively . β - actin was employed as a loading control throughout .

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(A-C) Expression of Pfkp mRNA levels (top panels) and Stat3 and Pfkp protein expression (bottom panels) after knockdown of Stat3 using three independent shRNAs in either R1 cells (A) or E14 cells (B) or after overexpression of Stat3 in R1 cells (C), as measured by qPCR and Western blot, respectively. β-actin was employed as a loading control throughout.


ChIP assays against Stat3 p - Stat3 ( Y705 ) or isotype - matched immunoglobulin G ( IgG ) ( F ) , as measured by PCR , and images were representative of three independent experiments . Nanog promoter is shown as a positive control .

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ChIP assays against Stat3 p-Stat3 (Y705) or isotype-matched immunoglobulin G (IgG) (F), as measured by PCR, and images were representative of three independent experiments. Nanog promoter is shown as a positive control.


( G ) Transcriptional activity of the indicated pGL3 - based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3 , as measured by luciferase reporter assay ( top panel ) . Western blotting was used to verify Stat3 expression ( bottom panel ) .

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(G) Transcriptional activity of the indicated pGL3-based Pfkp promoter or mutant constructs expressed in HEK293T cells with or without overexpression of Stat3, as measured by luciferase reporter assay (top panel). Western blotting was used to verify Stat3 expression (bottom panel).


( B ) Representative images of alkaline phosphatase - stained colonies formed by R1 cells after overexpression of Pfkp . Images were representative of three independent experiments .

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(B) Representative images of alkaline phosphatase-stained colonies formed by R1 cells after overexpression of Pfkp. Images were representative of three independent experiments.


( K , L ) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation ( K ) or Foxa2 following definitive endoderm differentiation ( L ) . Green staining indicates positive cells with nuclei counterstained with DAPI . Images were representative of three independent experiments . ( M , N ) Quantitative analysis of Sox17 ( M ) and Foxa2 ( N ) in R1 cells from ( K ) and ( L ) , respectively as determined by flow cytometry . Representative profiles ( left panels ) and quantitation ( right panels ) as a percentage of total cells .

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(K, L) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation (K) or Foxa2 following definitive endoderm differentiation (L). Green staining indicates positive cells with nuclei counterstained with DAPI. Images were representative of three independent experiments. (M, N) Quantitative analysis of Sox17 (M) and Foxa2 (N) in R1 cells from (K) and (L), respectively as determined by flow cytometry. Representative profiles (left panels) and quantitation (right panels) as a percentage of total cells.


( A , B ) Protein interactome of Pfkp in R1 cells . SDS - PAGE gel comparing Pfkp and control ( IgG ) immunoprecipitates ( left ) with candidate Pfkp - interacting proteins identified by mass spectrometry ( right ) ( A ) . Validation of candidate Pfkp - interacting proteins using Western blotting ( B ) .

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(A, B) Protein interactome of Pfkp in R1 cells. SDS-PAGE gel comparing Pfkp and control (IgG) immunoprecipitates (left) with candidate Pfkp-interacting proteins identified by mass spectrometry (right) (A). Validation of candidate Pfkp-interacting proteins using Western blotting (B).


( E - F ) Co - IP assays mapping interactions between Pfkp and fragments of Lin41 ( E ) , interactions between Lin41 and fragments of Pfkp ( F ) in HEK293T cells .

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(E-F) Co-IP assays mapping interactions between Pfkp and fragments of Lin41 (E), interactions between Lin41 and fragments of Pfkp (F) in HEK293T cells.


( J , K ) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment ( J ) or with Lactacyctin treatment ( K ) , as determined by Western blot .

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(J, K) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment (J) or with Lactacyctin treatment (K), as determined by Western blot.


( F ) HEK293T cells were co - transfected with the indicated vectors to overexpress Pfkp in combination with HA - ubiquitin ( Ub ) and the individual wildtype Flag - Lin41 or serine substitution mutants . Lin41 ubiquitination levels were determined using Western blotting against HA ( top panels ) with input samples analyzed in parallel to verify transfection efficiency ( bottom panels ) .

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(F) HEK293T cells were co-transfected with the indicated vectors to overexpress Pfkp in combination with HA-ubiquitin (Ub) and the individual wildtype Flag-Lin41 or serine substitution mutants. Lin41 ubiquitination levels were determined using Western blotting against HA (top panels) with input samples analyzed in parallel to verify transfection efficiency (bottom panels).


( G ) Control or Pfkp knockdown R1 cells were co - transfected with the individual wildtype Flag - Lin41 or serine substitution mutants and Lin41 ubiquitination levels determined as per ( F ) using Western blotting against Flag . ( H ) In vitro ubiquitination assays employing the products from in vitro kinase assays employing recombinant GST - Lin41 and Flag - Pfkp , with Lin41 ubiquitination measured by Western blotting against Lin41 . (

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(G) Control or Pfkp knockdown R1 cells were co-transfected with the individual wildtype Flag-Lin41 or serine substitution mutants and Lin41 ubiquitination levels determined as per (F) using Western blotting against Flag. (H) In vitro ubiquitination assays employing the products from in vitro kinase assays employing recombinant GST-Lin41 and Flag-Pfkp, with Lin41 ubiquitination measured by Western blotting against Lin41. (


( C - E ) Expression of selected expression of pluripotency and lineage specification markers in the cells from ( B ) determined by qPCR ( C ) with parallel Western blotting analysis of a panel of ectodermal ( D ) and endodermal ( E ) markers .

O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Expression of selected expression of pluripotency and lineage specification markers in the cells from (B) determined by qPCR (C) with parallel Western blotting analysis of a panel of ectodermal (D) and endodermal (E) markers.


( G - J ) RNA pull - down assays measuring interactions between Fgf5 ( G ) , Otx2 ( H ) , Sox1 ( I ) , and Pax2 ( J ) mRNAs and endogenous Lin41 in R1 cells . Recovery of each mRNA was compared in sense and anti - sense probes using qPCR ( top ) with Western blotting used to detect Lin41 ( bottom ) .

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(G-J) RNA pull-down assays measuring interactions between Fgf5 (G), Otx2 (H), Sox1 (I), and Pax2 (J) mRNAs and endogenous Lin41 in R1 cells. Recovery of each mRNA was compared in sense and anti-sense probes using qPCR (top) with Western blotting used to detect Lin41(bottom).


( K ) Relative stability of mRNAs for the ectodermal genes Fgf5 , Otx2 , Sox1 , and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6 , as determined in actinomycin D chase assays . ( L ) Relative stability of ectodermal gene mRNAs measured as per ( K ) in Day 6 Pfkp knockdown EBs transfected with or without Lin41 .

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(K) Relative stability of mRNAs for the ectodermal genes Fgf5, Otx2, Sox1, and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6, as determined in actinomycin D chase assays. (L) Relative stability of ectodermal gene mRNAs measured as per (K) in Day 6 Pfkp knockdown EBs transfected with or without Lin41.


( A ) The indicated yeast strains ( wild type , pex19Δ , pex3Δ , msp1Δ , pex19Δmsp1Δ , and pex3Δmsp1Δ ) expressing RFP - SKL ( peroxisomal marker ) and mitochondria - targeted green ﬂuorescent protein ( mGFP ) were grown to mid - log phase and imaged by ﬂuorescence microscopy . Representative images are shown , scale bar , 2 μm . DIC , differential interference contrast . Enhanced : the red signal intensity was set to ' best - fit ' in ZENPro microscopy software analysis .

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(A) The indicated yeast strains (wild type, pex19Δ, pex3Δ, msp1Δ, pex19Δmsp1Δ, and pex3Δmsp1Δ) expressing RFP-SKL (peroxisomal marker) and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and imaged by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. Enhanced: the red signal intensity was set to 'best-fit' in ZENPro microscopy software analysis.


( B ) Volcano plot showing the most enriched / decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 7 , biological replicates ; n = 2 , technical replicates ) . Effect sizes were calculated for Cohen ' s d A protein ' s relevance was determined using a p - value threshold of < 0 . 05 with an absolute effect size threshold of 1 or greater ( large effect ) . Gray dots represent all measured proteins and red dots highlight peroxisomal - associated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot showing the most enriched/decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry (n=7, biological replicates; n=2, technical replicates). Effect sizes were calculated for Cohen's d A protein's relevance was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins and red dots highlight peroxisomal-associated proteins.


( D ) The indicated yeast strains expressing PEX13 - RFP and mitochondria - targeted green ﬂuorescent protein ( mGFP ) were grown to mid - log phase and visualized by ﬂuorescence microscopy . Representative images are shown , scale bar , 2 μm . DIC , differential interference contrast .

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(D) The indicated yeast strains expressing PEX13-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast.


( F ) Total yeast cell lysate , post mitochondrial supernatant , and HistodenzTM - purified mitochondria from the indicated strain ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , and pex3Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pex13 ( α - V5 ) , cytochrome c ( α - cyt c ) , and HSP70 ( α - hsp70 ) . ( G ) Total yeast cell lysate , post mitochondrial supernatant , and HistodenzTM - purified mitochondria from the indicated strain ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , and pex3Δmsp1Δ ) expressing Pex11 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pex11 ( α - V5 ) and porin ( α - porin ) , cytochrome c ( α - cyt c ) , and HSP70 ( α - hsp70 ) .

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

(F) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex13 (α-V5), cytochrome c (α-cyt c), and HSP70 (α-hsp70). (G) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex11-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex11 (α-V5) and porin (α-porin), cytochrome c (α-cyt c), and HSP70 (α-hsp70).


( A ) Volcano plot ( log10 ) of the most enriched / decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 3 , biological replicates , n = 1 , technical replicate ) . 2010 ) . Effect sizes were calculated for Cohen ' s d A protein ' s relevance to the model was determined using a p - value threshold of < 0 . 05 with an absolute effect size threshold of 1 or greater ( large effect ) . Gray dots represent all measured proteins . Red dots highlight peroxisomal - associated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot (log10) of the most enriched/decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry (n=3, biological replicates, n=1, technical replicate). 2010). Effect sizes were calculated for Cohen's d A protein's relevance to the model was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins. Red dots highlight peroxisomal-associated proteins.


( C ) Mitochondria from the indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) , expressing Pex13 - V5 ( under its endogenous promotor ) were either left untreated ( lane 1 , 2 , 3 ) or exposed to Na2CO3 and separated into the membrane pellet ( 4 , 5 , 6 ) or supernatant fraction ( 7 , 8 , 9 ) . After separation by SDS - PAGE the fractions were immunoblotted for Pex13 - V5 ( α - V5 ) and porin ( α - porin ) .

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(C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).


( B ) Digitonin - solubilized ( 1 or 4 g / g digitonin / protein ratio , as indicated ) mitochondrial membranes from the indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by BN - PAGE with a 3 - 18 % gradient and stained with Coomassie brilliant blue . BHM , bovine heart mitochondria ; V2 , complex V - dimer ; SL , supercomplex III2IV2 ; Ss , supercomplex III2IV1 ; V1 , complex V - monomer .

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(B) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and stained with Coomassie brilliant blue. BHM, bovine heart mitochondria; V2, complex V-dimer; SL,supercomplex III2IV2; Ss, supercomplex III2IV1; V1, complex V-monomer.


( F ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pex22 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE and immunoblotted for Pex22 - V5 ( α - Pex22 - V5 ) and porin ( α - porin ) . ( G ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pot1 - V5 ( α - Pot1 - V5 ) and porin ( α - porin ) . ( H ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1 Δ , and pex13Δmsp1Δpex19Δ ) expressing Mdh3 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE and immunoblotted for Mdh3 - V5 ( α - Mdh3 - V5 ) and porin ( α - porin ) .

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(F) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pex22-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Pex22-V5 (α-Pex22-V5) and porin (α-porin). (G) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ ) expressing Pot1-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pot1-V5 (α-Pot1-V5) and porin (α-porin). (H) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1 Δ, and pex13Δmsp1Δpex19Δ) expressing Mdh3-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Mdh3-V5 (α-Mdh3-V5) and porin (α-porin).


( I ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were either left untreated ( nt ) ( lane 1 , 4 , 7 ) or were treated with proteinase K in high fidelity buffer ( PK - HF ) ( cutting control , lane 2 , 5 , 8 ) or low fidelity buffer ( PK - LF ) ( protease protection assay , lane 3 , 6 , 9 ) and separated by SDS - PAGE and immunoblotted for Mia40 ( intermembrane space ) ( α - Mia40 ) , Pot1 - V5 ( α - V5 ) , porin ( outer membrane ) ( α - porin ) and Tom22 ( outer membrane but partially proteinase exposed ) ( α - Tom22 ) .

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

(I) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pot1-V5 (under its endogenous promotor) were either left untreated (nt) (lane 1,4,7) or were treated with proteinase K in high fidelity buffer (PK-HF) (cutting control, lane 2,5,8) or low fidelity buffer (PK-LF) (protease protection assay, lane 3,6,9) and separated by SDS-PAGE and immunoblotted for Mia40 (intermembrane space) (α-Mia40), Pot1-V5 (α-V5), porin (outer membrane) (α-porin) and Tom22 (outer membrane but partially proteinase exposed) (α-Tom22).


( A ) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients - cWT , cWT + ATAD1 , PEX3 - / - , and PEX3 - / - + ATAD1 - expressing PEX13 - GFP and stained with Mitotracker  Deep Red FM . Representative images are shown , scale bar , 10 μm .

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

(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm.


( B ) Electron microscopy of human fibroblast cell lines cWT , cWT + ATAD1 , PEX3 - / - , and PEX3 - / - + ATAD1 . Representative images are shown , scale bar 500 nm .

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

(B) Electron microscopy of human fibroblast cell lines cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1. Representative images are shown, scale bar 500 nm.


( A ) Bioenergetic profile of human fibroblast cell lines . OCR ( pmol / min / norm . unit ) for cWT , cWT + ATAD1 , PEX3 - / - , PEX3 - / - + ATAD1 , cWT - ATAD1 - / - , and PEX3 - / - atad1 - / - cells plotted against time ( XF96e Extracellular Flux Analyzer , Mito - Stress - Test ) . 1 μM Oligomycin A , 0 . 15 μM FCCP , and 1 μM rotenone + 1 μM antimycin A ( final concentrations ) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge . The graph is the compilation of two independent assays , error bars show mean + / - s . d . ( n = 4 , biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bioenergetic profile of human fibroblast cell lines. OCR (pmol/min/norm.unit) for cWT, cWT+ATAD1, PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1-/-, and PEX3-/- atad1-/- cells plotted against time (XF96e Extracellular Flux Analyzer, Mito-Stress-Test). 1 μM Oligomycin A, 0.15 μM FCCP, and 1 μM rotenone + 1 μM antimycin A (final concentrations) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge. The graph is the compilation of two independent assays, error bars show mean +/- s.d. (n=4, biological replicates).


( E ) Scatter plot representation of cardiolipin lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 11 detected cardiolipin species , log10 pareto - scaled is visualized . The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line . ( F ) Scatter plot representation of a subpopulation of the phosphoethanolamine ( PE ) lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 20 phosphoethanolamine ( PE ) species ( decreased in PEX3 - / - ) , log10 pareto - scaled , is visualized . The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line . ( G ) Scatter plot representation of ether - phospholipid lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 21 detected ether - phospholipid ( plasmalogen ) species , log10 pareto - scaled , is visualized . The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line .

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

(E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. (F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.


Time - series measurements of ( B ) E . coli abundance , ( C ) B . subtilis abundance ,

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

Time-series measurements of (B) E. coli abundance, (C) B. subtilis abundance,


( H ) Bar plot of measured extracellular pBB275 plasmid concentration at 0 hr and 6 hr in the co - culture composed of E . coli MG1655 - rfp and B . subtilis .

O O O O O O O O O O O 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) Bar plot of measured extracellular pBB275 plasmid concentration at 0 hr and 6 hr in the co-culture composed of E. coli MG1655-rfp and B. subtilis.


( B , C ) ( B ) Extracellular pBB275 plasmid concentration or ( C ) extracellular E . coli genomic DNA concentration at 0 or 6 hr in the co - cultures composed of a recA + or recA - E . coli plasmid donor and engineered B . subtilis .

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

(B, C) (B) Extracellular pBB275 plasmid concentration or (C) extracellular E. coli genomic DNA concentration at 0 or 6 hr in the co-cultures composed of a recA+ or recA- E. coli plasmid donor and engineered B. subtilis.


( E ) Extracellular pBB275 or pUC19 plasmid concentration in the E . coli MG1655 monoculture or or co - culture with engineered B . subtilis .

O O O 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) Extracellular pBB275 or pUC19 plasmid concentration in the E. coli MG1655 monoculture or or co-culture with engineered B. subtilis.


( F ) HGT efficiencies of pBB275 plasmid ( with or without a f1 ori ) at 6 hr in the co - culture composed of E . coli MG1655 and engineered B . subtilis .

O O O O O O O O O O O O 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) HGT efficiencies of pBB275 plasmid (with or without a f1 ori) at 6 hr in the co-culture composed of E. coli MG1655 and engineered B. subtilis.


( B ) E . coli and B . subtilis abundances in the co - culture in the presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG at 6 hr .

O O O O O O O O O O O O O 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) E. coli and B. subtilis abundances in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG at 6 hr.


Time - series measurements of extracellular plasmid or E . coli gDNA concentrations in the co - culture in the presence of ( D ) 0 . 02 mM , ( E ) 0 . 2 mM or ( F ) 2 mM IPTG .

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

Time-series measurements of extracellular plasmid or E. coli gDNA concentrations in the co-culture in the presence of (D) 0.02 mM, (E) 0.2 mM or (F) 2 mM IPTG.


( H ) Abundance of transformants at 6 hr in the co - culture in the presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG .

O O O O O O O O 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) Abundance of transformants at 6 hr in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG.


( C ) Scatter plot of the initial abundance of heat - killed E . coli MG1655 - rfp and extracellular pBB275 plasmid concentration at 0h or 6 hr in the co - culture .

(C) Scatter plot of the initial abundance of heat-killed E. coli MG1655-rfp and extracellular pBB275 plasmid concentration at 0h or 6 hr in the co-culture.


Time - series measurements of ( E ) the abundance of E . coli MG1655 - rfp , ( F ) the abundance of engineered B . subtilis Unpaired t - test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different . Stars ( * ) indicate p - value of 0 . 0237 and 0 . 0283 for 4 hr and 8 hr , 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

Time-series measurements of (E) the abundance of E. coli MG1655-rfp, (F) the abundance of engineered B. subtilis Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different. Stars (*) indicate p-value of 0.0237 and 0.0283 for 4 hr and 8 hr, respectively.


Time - series measurements of ( G ) extracellular pBB275 plasmid concentration , Unpaired t - test was used to determine if the abundances in the presence and absence of

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

Time-series measurements of (G) extracellular pBB275 plasmid concentration, Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol


( C ) Histogram of the number of E . coli plasmid replication origins ColE1 , p15A , CloDF13 or pSC101 identified in Bacillus or non - Bacillus DNA sequences with a sequence length larger than 106 bp ( indicating potential genomic integration ) . ColE1 was found in 6 Bacillus genomes and 43 non - Bacillus genomes . CloDF13 was found in 23 non - Bacillus genomes . p15A and pSC101 were not detected in this NCBI RefSeq dataset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histogram of the number of E. coli plasmid replication origins ColE1, p15A, CloDF13 or pSC101 identified in Bacillus or non-Bacillus DNA sequences with a sequence length larger than 106 bp (indicating potential genomic integration). ColE1 was found in 6 Bacillus genomes and 43 non-Bacillus genomes. CloDF13 was found in 23 non-Bacillus genomes. p15A and pSC101 were not detected in this NCBI RefSeq dataset.


D Ratio of toxicity and viability of HCT116 , U87 and BxPC3 cells grown in spheroids ( 3D ) and treated as indicated for 24h ( BxPC3 ) or 48h ( HCT116 , U87 ) ( NEN 1 . 2 µM ; Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) . Data information : Data are presented as mean ( SD ) ( N = 3 ) and were analyzed by two - way ANOVA with Tukey post - hoc test Significance is indicated for multiple comparisons as indicated . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ratio of toxicity and viability of HCT116, U87 and BxPC3 cells grown in spheroids (3D) and treated as indicated for 24h (BxPC3) or 48h (HCT116, U87) (NEN 1.2 µM; Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunoblot analysis of CHOP , ATF4 , phosphorylated ( p - ) and t - ) eIF2alpha ( eIF2a ) of HCT116 cells treated as indicated for 16h ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp ) , Amitriptyline ( Ami ) , each 30 µM ) .

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

B Immunoblot analysis of CHOP, ATF4, phosphorylated (p-) and t-) eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM).


F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h ( NEN 1 . 2 µM , Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM , ISRIB 1 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

F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).


A Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ( ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) , determined by qPCR . Data information : In ( A ) ( first ( TFE3 ) and third ( CD68 ) graph ( N = 3 ) ; second ( TFE3 ) and fourth ( CD68 ) graph ( N = 5 , besides NEN + Clomi ( N = 4 ) and NEN + Desi ( N = 4 ) ) data are presented as mean ( SD ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . In the second and the fourth graph , significance is indicated for the comparison of combinatorial treatments ( NEN + TCAs ) to controls ( Ctrl ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ((NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM), determined by qPCR. Data information: In (A) (first (TFE3) and third (CD68) graph (N=3); second (TFE3) and fourth (CD68) graph (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) data are presented as mean (SD) and were analyzed by a one-way ANOVA with Tukey post-hoc test. In the second and the fourth graph, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls (Ctrl). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunofluorescence ( IF ) of HCT116 cells treated with DMSO ( Control ( Ctrl ) ) , NEN , Domperidone ( Domp ) or NEN + Domp as indicated ( NEN , 1 . 2 µM ; Domp , 30 µM ) for 16 h and stained with anti - Lamp1 ( top panel ) , Alexa - 555 LC3 ( middle panel ) co - stained with Alexa - 647 p62 ( bottom panel ) . Nuclei , Blue ( dapi ) . Scale bar 20 µm . Mean ( SEM ) fluorescence of Lamp1 ( Ctrl , NEN , Domp ( N = 20 cells ) ; NEN + Domp ( N = 24 cells ) ) , LC3 - II ( Ctrl , NEN + Domp ( N = 16 cells ) ) ; NEN , Domp ( N = 16 cells ) ) or P62 ( Ctrl ( N = 38 cells ) ; NEN ( N = 27 cells ) ; Domp ( N = 28 cells ) ; NEN + Domp ( N = 21 cells ) ) was quantified and analyzed by one - way ANOVA with Tukey post - hoc test ( for LC3 - II ) or Kruskal - Wallis with Dunn ´ s post - hoc test ( for Lamp1 and p62 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O 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 Immunofluorescence (IF) of HCT116 cells treated with DMSO (Control (Ctrl)), NEN, Domperidone (Domp) or NEN+Domp as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h and stained with anti-Lamp1 (top panel), Alexa-555 LC3 (middle panel) co-stained with Alexa-647 p62 (bottom panel). Nuclei, Blue (dapi). Scale bar 20 µm. Mean (SEM) fluorescence of Lamp1 (Ctrl, NEN, Domp (N=20 cells); NEN+Domp (N=24 cells)), LC3-II (Ctrl, NEN+Domp (N=16 cells)); NEN, Domp (N=16 cells)) or P62 (Ctrl (N=38 cells); NEN (N=27 cells); Domp (N=28 cells); NEN+Domp (N=21 cells)) was quantified and analyzed by one-way ANOVA with Tukey post-hoc test (for LC3-II) or Kruskal-Wallis with Dunn´s post-hoc test (for Lamp1 and p62). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control ( siCtrl ) or combined TFE3 - and MITF - targeting ( siTFE3 / siMITF ) siRNAs and treated as indicated ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , ISRIB 1 µM ) , determined by qPCR . Data information : data ( N = 3 ) are presented as mean ( SD ) and were analyzed by two - way ANOVA with Tukey post - hoc

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 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

C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control (siCtrl) or combined TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h ( NEN ( low ) 0 . 6 µM or NEN ( High ) 1 . 2 µM , Domperidone ( Domp ) 30 µM ) Cells were transfected either with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 48h prior to treatment .

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

F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.


H Ratio of toxicity and viability of HCT116 spheroids ( 3D ) treated as indicated ( NEN 1 . 2 µM , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM ) . Cells were transfected with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 24h prior to spheroid formation . Data information : data ( N = 3 ) are presented as mean ( SD ) and were analyzed by two - way ANOVA with Tukey post - hoc

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

H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM , Clomipramine ( Clomi ) 20 µM ) . Data information : , data are presented as mean ( SD ) ( N = 3 ) and were analyzed by Kruskal - Wallis with Dunn ´ s post - hoc test ( A , left graph ) , one - way ANOVA with Tukey post - hoc test ( A , right graph ) In ( A ) , significance is indicated for the comparison of the different treatments to controls . * * p < 0 , 01 ; * * * * p < 0 , 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001.


D Caspase activity in HCT116 cells treated as indicated for 24h ( NEN 0 . 6 µM , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 20 µM , Clomipramine ( Clomi ) 10 µM , A77126 ( A77 ) 50 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by two - way ANOVA with Tukey post - hoc

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

D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test


A Immunofluorescence ( IF ) images of HCT116 cells treated as indicated for 12h ( Imipramine and Domperidone , each 30 µM ) and stained for cholesterol content using filipin III . B Immunofluorescence ( IF ) images of HCT116 cells treated with Domperidone and co - stained for cholesterol and Lamp1 .

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

A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol content using filipin III. B Immunofluorescence (IF) images of HCT116 cells treated with Domperidone and co-stained for cholesterol and Lamp1.


E Relative mRNA expression levels of the specified genes in HCT116 cells , treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , Cyclodextrin ( CyD ) 0 . 75 % ) . F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h ( NEN 1 . 2 µM , Domperidone ( Domp ) 30 µM , Cyclodextrin ( CD ) 0 . 75 % ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

E Relative mRNA expression levels of the specified genes in HCT116 cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%). F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between single and combined drug treatments as indicated ( NEN 1 . 2 µM or 2 . 5 µM , Amitriptyline ( Ami , A ) , Imipramine ( Imi , I ) , Desipramine ( Desi , D ) , each 30 µM , Clomipramine ( Clomi , C ) 20 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between drug treatments as indicated to the control condition ( NEN ( N ) 1 . 2 µM or 2 . 5 µM , Amitriptyline ( A ) , Imipramine ( I ) 20 µM , Paclitaxel ( Pacli ) , 2 , 20 or 200nM ) . As references for sensitization effects to standard chemotherapy , the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue . Bars for controls ( Ctrl ) , Paclitaxel ( Pacli ) , NEN ( N ) alone and combined NEN + Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A), Imipramine (I) 20 µM, Paclitaxel (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A . Immunoblot of cell extracts following depletion of WDR26 , RanBP9 , ARMC8 , and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells . Endogenous levels of the indicated proteins were monitored by western blotting ( n = 3 ) .

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

A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3).


Western blotting of samples after ectopic overexpression of HBP1 ( B ) alone , or together with WDR26 or GID4 in HEK - 293T cells . HBP1 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

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

Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


Western blotting of samples after ectopic overexpression of ZMYND19 ( C ) alone , or together with WDR26 or GID4 in HEK - 293T cells . ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


C . , D . Western blot analysis of in vitro ubiquitinated HBP1 , which was performed by mixing purified HBP1 with ubiquitin E1 , UBCH5a , and ubiquitin in the presence of the indicated hGID sub - complexes ( n = 3 ) .

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

C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).


E . Immunoblots of in vitro ubiquitinated ZMYND19 , which was performed by mixing purified ZMYND19 with ubiquitin E1 , UBE2H , ubiquitin and the 6 - subunit hGID complex ( ARCM8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) in the presence or absence of GID4 and a 10 - fold excess of the PGLV GID4 - specific peptide ( n = 2 )

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

E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)


D . Western blotting of samples following ectopic overexpression of HBP1 either alone , or with full - length ( FL ) or WD40 - truncated WDR26 ( ΔWD40 ) in HEK - 293T cells . HBP1 levels were monitored in cells treated with MG132 or DMSO for 12 - 14 h ( n = 3 ) .

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 B-CONTROLLED_VAR 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

D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).


E . Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 ( WT ) or the WDR26 ( ΔWD40 ) mutant ( n = 2 ) .

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

E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).


C . Transiently expressed FLAG - GID4 and HSS - tagged ARMC8 isoforms ( α or β ) in HEK293T cells . The presence of GID4 in isoform - specific ARMC8 immunoprecipitates was visualized by immunoblotting ( left panels ) . The right panel shows a western blot of transiently expressed and immunoprecipitated HSS - WDR26 from HEK - 293T cells and the presence of endogenous ARMC8 isoforms ( α or β ) was probed by immunoblotting ( n = 2 ) .

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 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 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

C. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2).


D . Baculoviral co - expression in Sf9 cells of the 5 - subunit hGID complex ( 5mer ; Strep - RanBP9 , His - WDR26 , FLAG - MAEA , His - RMND5a and His - TWA1 ) along with His - GID4 in the presence of ARMC8α or ARMC8β . Strep - or His - pulldowns revealed the presence of GID4 in ARMC8α , but not in ARMC8β , complexes ( 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 B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

D. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3).


E . Immunoblot analysis of in vitro ubiquitinated GID4 - hGID complexes containing either ARMC8α or ARMC8β ( 6mer ; ARMC8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) . Where indicated , the reaction was carried out in the presence of 20 - fold molar excess of the PGLV GID4 - binding peptide ( n = 3 ) .

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

E. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3).


F . Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5 - subunit complex in the presence of the different ARMC8 isoforms ( α or β ) ( n = 2 ) .

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

F. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2).


B , heatmap of log10 scaled PBMC community frequencies for all samples ( n = 42 patients × 2 timepoints = 84 samples ) . Clinical variables are depicted for all samples across the top of the heatmap ; convalescent post - symptom onset anti - CHIKV antibody titer and viral titer ( which was measured during the acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) and communities ( Y axis ) . Four major clusters of communities and two major clusters of samples ( largely separating acute and convalescent samples ) are highlighted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of log10 scaled PBMC community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and communities (Y axis). Four major clusters of communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


C , fold changes in log10 frequencies for PBMC communities between acute and convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) . Error bars represent the 95 % confidence interval . The acute phase frequency for each community is depicted with the purple heatmap . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR P < 0 . 001 .

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

C, fold changes in log10 frequencies for PBMC communities between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


E , differences in CHIKV E2 protein expression between acute - phase and convalescent - phase samples per MetaHybridLouvain sub - community . Sub - communities are ordered by largest to smallest difference and filtered to sub - communities where the median of the channel means per sample was higher in the acute - phase samples at a significance threshold of FDR < 0 . 05 . Sub - communities are named by their parent canonical community name plus an arbitrary

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, differences in CHIKV E2 protein expression between acute-phase and convalescent-phase samples per MetaHybridLouvain sub-community. Sub-communities are ordered by largest to smallest difference and filtered to sub-communities where the median of the channel means per sample was higher in the acute-phase samples at a significance threshold of FDR < 0.05. Sub-communities are named by their parent canonical community name plus an arbitrary number


F , heatmap of log10 scaled PBMC sub - community frequencies for all samples ( n = 42 patients × 2 timepoints = 84 samples ) . Clinical variables are depicted for all samples across the top of the heatmap ; convalescent post - symptom onset anti - CHIKV antibody titer and viral titer ( which was measured during the acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) and sub - communities ( Y axis ) . Four major clusters of sub - communities and two major clusters of samples ( largely separating acute and convalescent samples ) are highlighted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of log10 scaled PBMC sub-community frequencies for all samples (n = 42 patients × 2 timepoints = 84 samples). Clinical variables are depicted for all samples across the top of the heatmap; convalescent post-symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and sub-communities (Y axis). Four major clusters of sub-communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


, fold changes in log10 frequencies for PBMC sub - communities contrasted between acute and convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) . Error bars represent the 95 % confidence interval . The acute phase frequency for each sub - community is depicted with the purple heatmap . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR P < 0 . 001 .

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

, fold changes in log10 frequencies for PBMC sub-communities contrasted between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each sub-community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


A , relative expression of CD14 + and CD16 + in CD14 + CD16 + sub - communities , depicted as boxplots of the mean expression levels for all samples ( n = 42 patients × 2 timepoints = 84 samples ) , indicates that sub - community 1 has a CD14 + CD16 + + ( aka " non - classical " ) phenotype , while sub - community 2 has a CD14 + + CD16 + ( aka " intermediate " ) phenotype . Differences shown here are significant at FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) ; for a view of all differences significant at this

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

A, relative expression of CD14+ and CD16+ in CD14+CD16+ sub-communities, depicted as boxplots of the mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples), indicates that sub-community 1 has a CD14+CD16++ (aka "non-classical") phenotype, while sub-community 2 has a CD14++CD16+ (aka "intermediate") phenotype. Differences shown here are significant at FDR < 0.05 (moderated paired t-test, mixed effects model); for a view of all differences significant at this threshold


B , heatmap of relative expression of markers that most differentiate the sub - communities of CD14 + monocytes by difference in mean expression levels for all samples ( n = 42 patients × 2 timepoints = 84 samples ) . Markers are filtered to those different at a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) and fold change > 1 . 3 ; for a view of all differences significant at FDR < 0 . 05 The mean intensity of each channel across all sub - communities is shown by the adjacent purple heatmap .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of relative expression of markers that most differentiate the sub-communities of CD14+ monocytes by difference in mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples). Markers are filtered to those different at a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model) and fold change > 1.3; for a view of all differences significant at FDR < 0.05 The mean intensity of each channel across all sub-communities is shown by the adjacent purple heatmap.


( n = 42 pairs of samples , 1 pair per patient ) . Wilcoxon signed - rank test was used to determine statistical significance , with a Benjamini - Hochberg adjustment for FDR ( 40 comparisons ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A , CXC chemokines . B , CC chemokines . C , interferons . D , TNF superfamily cytokines . E , interleukins . F , colony - stimulating factors .

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

(n = 42 pairs of samples, 1 pair per patient). Wilcoxon signed-rank test was used to determine statistical significance, with a Benjamini-Hochberg adjustment for FDR (40 comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. A, CXC chemokines. B, CC chemokines. C, interferons. D, TNF superfamily cytokines. E, interleukins. F, colony-stimulating factors.