Flow cytometric analysis of total B cells and different B cell subsets in th splee of non - immunized Eβ - / - Prdm1ihCd2 / + ( red ) and Eβ - / - Prdm1 + / + ( gray ) chimeric mice . Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 ( Blimp1 ) in splenic FO B , GC B and naïve CD4 T cells from the chimeric mice of the indicated

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

Flow cytometric analysis of total B cells and different B cell subsets in th splee of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 (Blimp1) in splenic FO B, GC B and naïve CD4 T cells from the chimeric mice of the indicated genotypes


Presence of IgG antibodies detecting dsDNA , cardiolipin , SSA ( Ro - 52 ) and SSB ( La ) in the serum of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 , 4 and 12 months . The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units ( AU ) . The serum of 6 - month - old Faslgld / gld mice was used as positive

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Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive control


Representative images of anti - nuclear antibody ( ANA ) staining obtained with serum from 12 - month - old Prdm1ihCd2 / + or wild - type mice ( right ) , as detected by indirect immunofluorescence assay using HEp - 2 cells and an Alexa488 - conjugated anti - mouse IgG detection antibody . The serum of Faslgld / gld mice was used as positive control . The presence of ANA - IgG antibodies was analyzed for 22 Prdm1ihCd2 / + and 12 wild - type mice , as summarized in the pie - chart ( right )

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Representative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right)


Periodic acid - Schiff ( PAS ) staining of paraffin - embedded kidney sections of Prdm1ihCd2 / + and wild - type mice at 12 months of age . The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods . Representative pictures of glomeruli of Prdm1ihCd2 / + kidneys with different pathology scores are shown to the left , and dot plots with average scores for male and female mice ( Table EV2 ) are shown to the right . Arrows denote intracapillary hyaline " thrombi " , asterisks indicate mesangial sclerosis with obscured capillary loops , and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture . Statistical data ( A , C ) are shown as mean value with SEM and were analyzed by the Mann - Whitney test ( A ) or the Student " s t - test ( C ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Each dot

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

Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon


Hydrolysis of fluorogenic substrates ( Suc - LLVY - AMC ) for the chymotrypsin - like activity of human ( B ) or mouse ( C ) 20S constitutive proteasome ( CP ) or 20S immunoproteasome ( IP ) at various PRN1126 concentrations . Data are presented as the means ± s . d . from quadruplicate assays . The highest fluorescence value was set to 100 % . The experiments were repeated twice with similar results .

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Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.


Presentation of UTY246 - 254 on splenocytes after exposure to indicated concentrations of PRN1126 . Data are presented as the mean absorbance ± s . d . of three replicate cultures . The experiment has been performed twice , yielding similar results .

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Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 mice treated with the indicated concentrations of PRN1126 overnight . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM .

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Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM.


Splenocytes from C57BL / 6 mice ( C ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . ( C ) IL - 6 concentrations are presented as mean and individual data points from triplicate wells . The experiment has been performed twice , yielding similar results .

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Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results.


Splenocytes from human PBMCs ( D ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . Data are presented as single dots from three independent donors . The highest cytokine concentration was set to 100 % . * P < 0 . 05 . Unpaired student ´ s t - test .

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Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.


Colitis was induced by oral administration of 3 % DSS for 5 d . Mice were treated daily ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . Data points represent mean ± SEM of 15 mice pooled from three independent experiments .

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Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated three times a week ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . All data were statistically compared to the vehicle treated group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Two - way ANOVA . Shown are the means of the clinical scores ± SEM ( n = 6 per group ) .

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Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).


Hydrolysis of fluorogenic substrates ( Ac - PAL - AMC ) for LMP2 - activity of human ( upper panel ) or mouse ( lower panel ) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914 . Data are presented as the means of fluorescence ± s . d . from quadruplicate assays . The experiments were repeated three times with similar results .

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Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.


Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914 . Ficoll enriched lymphocytes from C57BL / 6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro . SDS - PAGE and immunoblotting against indicated proteins was performed . Shown are representative western blots out of three independent experiments with similar outcome .

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Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 wild type ( left side ) or LMP7 - / - ( right side ) mice treated with vehicle ( DMSO ) , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) overnight . Unstained splenocytes were used as negative control . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM . All data were statistically compared to the DMSO treated group . * * P < 0 . 01 , * * * P < 0 . 001 . One - way 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 O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.


Splenocytes from C57BL / 6 mice ( A ) or human PBMCs ( B ) were incubated ( continuous treatment ) with DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and stimulated with LPS overnight . The IL - 6 concentration in the supernatant was analyzed by ELISA . The highest IL - 6 concentration from each experiment was set to 100 % . ( A ) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates . ( B ) Data are presented as the mean ± SEM of 6 different healthy donors . All data were statistically compared to the DMSO treated group .

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

Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group.


The differentiation of CD4 + T cells isolated from the spleens of C57BL / 6 mice that were exposed ( continuous treatment ) to DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and that were stimulated with plate bound antibodies to CD3 / CD28 in the presence of TGF - β and IL - 6 and scavenging antibodies neutralising IL - 4 and IFN - γ was measured in 3 days cultures . IL - 17 expression was detected by intracellular cytokine staining after a short restimulation with PMA / Ionomycin . IL - 17 expression in activated T cells cultured in the absence of Th17 - polarizing conditions is shown as a comparison ( no cocktail ) . Values reflect the percentage of CD4 + cells that are IL - 17A + . Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates . All data were statistically compared to the DMSO treated group .

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

The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group.


Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . ( A ) The body weight of individual mice was monitored daily and the percent weight loss ( y - axis ) was plotted versus time ( x - axis ) . All data were statistically compared to the vehicle treated group . *

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

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. (A) The body weight of individual mice was monitored daily and the percent weight loss (y-axis) was plotted versus time (x-axis). All data were statistically compared to the vehicle treated group. *


Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . On day 9 after initiation of DSS treatment , colon lengths were measured ( n = 15 ) . Naïve mice ( n = 5 ) were used as healthy controls . All data were statistically compared to the vehicle treated group .

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

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. On day 9 after initiation of DSS treatment, colon lengths were measured (n=15). Naïve mice (n=5) were used as healthy controls. All data were statistically compared to the vehicle treated group.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated intermittently with three times a week ( s . c . ) schedule with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the start of the experiment . Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments . All data were statistically compared to the vehicle treated group .

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

Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated intermittently with three times a week (s.c.) schedule with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the start of the experiment. Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments. All data were statistically compared to the vehicle treated group.


( C ) miR - 574 - 5p and miR - 574 - 3p were highly expressed in the explanted failing hearts from chronic HF patients ( n = 17 ) versus donor non - failing hearts ( n = 6 ) .

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(C) miR-574-5p and miR-574-3p were highly expressed in the explanted failing hearts from chronic HF patients (n=17) versus donor non-failing hearts (n=6).


( D ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice with isoproterenol ( ISO ) infusion ( 4 weeks ; n = 10 - 12 ) .

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(D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12).


( E ) miR - 574 - 5p and miR - 574 - 3p were induced in the hearts of mice under transverse aortic constriction ( TAC ) surgery ( 4 weeks ; n = 8 - 10 ) .

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(E) miR-574-5p and miR-574-3p were induced in the hearts of mice under transverse aortic constriction (TAC) surgery (4 weeks; n=8-10).


( A ) H & E of hearts from WT and miR - 574 - / - mice with or without isoproterenol ( ISO ) treatment for 4 weeks . Mice were between 8 - 10 weeks old females .

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(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females.


( B ) Ratio of HW / TL ( heart weight / tibia length ) in WT and miR - 574 - / - mice ( n = 8 / 7 / 8 / 6 for 4 groups of WT , Veh . ; WT , ISO ; KO , Veh . ; KO , ISO ) .

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(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).


( C ) WGA ( wheat germ agglutinin ) staining in WT and miR - 574 - / - mice . Cross - sectional area ( CSA ) of CMs was measured and quantified ( n ≥ 500 cells ) . Scale bar : 50 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group .

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(C) WGA (wheat germ agglutinin) staining in WT and miR-574-/- mice. Cross-sectional area (CSA) of CMs was measured and quantified (n≥500 cells). Scale bar: 50 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.


( D ) Picrosirius red staining in WT and miR - 574 - / - mice ( n = 6 per group ) . Scale bar : 1 mm .

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(D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm.


( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with ISO treatment ( n = 3 per group ) . ANF , atrial natriuretic factor ; BNP , B - type natriuretic peptide ; Myh6 / Myh7 , myosin heavy polypeptide 6 / 7 ; Col1a2 / Col3a1 , procollagen , type I , α2 ; type III , α1 .

(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1.


( F ) TUNEL assay for heart tissue sections from WT and miR - 574 - / - mice under ISO versus vehicle treatment ( n = 6 per group ) . Scale bar : 10 μm .

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(F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.


( A ) H & E staining of hearts from WT and miR - 574 - / - mice 4 weeks after transverse aortic constriction ( TAC ) surgery .

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(A) H&E staining of hearts from WT and miR-574-/- mice 4 weeks after transverse aortic constriction (TAC) surgery.


( B ) Ratio of HW / TL in WT and miR - 574 - / - mice ( n = 8 per group ) .

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(B) Ratio of HW/TL in WT and miR-574-/- mice (n=8 per group).


( C ) WGA staining of hearts from WT and miR - 574 - / - mice 4 weeks after TAC surgery ( n ≥ 500 cells ) . Scale bar : 20 μm . In the violin plot , black line shows median value for the group and peach dashed lines represent two quartile lines in each group .

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(C) WGA staining of hearts from WT and miR-574-/- mice 4 weeks after TAC surgery (n≥500 cells). Scale bar: 20 μm. In the violin plot, black line shows median value for the group and peach dashed lines represent two quartile lines in each group.


( D ) Picrosirius red staining of hearts from WT and miR - 574 - / - mice under TAC surgery ( n = 8 per group ) . Scale bar : 1 mm .

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(D) Picrosirius red staining of hearts from WT and miR-574-/- mice under TAC surgery (n=8 per group). Scale bar: 1 mm.


( E ) RT - qPCR of fetal cardiac genes in WT and miR - 574 - / - mice with TAC surgery ( n = 4 per group ) .

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(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with TAC surgery (n=4 per group).


( F ) Echocardiography measurement of cardiac functions of hearts from WT and miR - 574 - / - mice 4 weeks post TAC ( n = 8 per group ) . FS , fractional shortening .

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(F) Echocardiography measurement of cardiac functions of hearts from WT and miR-574-/- mice 4 weeks post TAC (n=8 per group). FS, fractional shortening.


The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations . HW / TL ratio is measured ( n = 4 - 10 per group ) . Ctrl - miR : negative control miRNA mimics .

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The schematic of therapeutic treatment of the TAC model using miRNA mimics followed by phenotypic characterizations. HW/TL ratio is measured (n=4-10 per group). Ctrl-miR: negative control miRNA mimics.


( B ) H & E and WGA staining of murine hearts in the therapeutic models . Scale bar : 1 mm for H & E and 50 μm for WGA . Cardiomyocyte surface area was quantified from WGA staining in the right panel . > 120 CMs are quantified in each group . The black line shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(B) H&E and WGA staining of murine hearts in the therapeutic models. Scale bar: 1 mm for H&E and 50 μm for WGA. Cardiomyocyte surface area was quantified from WGA staining in the right panel. >120 CMs are quantified in each group. The black line shows medium value for the group and the dotted lines represent two quartile lines in each group.


( C ) Picrosirius red staining of murine hearts in the therapeutic models . Scale bar : 1 mm . The fibrotic area was quantified in the right panel ( n = 4 - 10 per group ) .

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(C) Picrosirius red staining of murine hearts in the therapeutic models. Scale bar: 1 mm. The fibrotic area was quantified in the right panel (n=4-10 per group).


( D ) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end - point of 6 weeks post - surgery ( n = 4 - 10 per group ) .

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(D) Echocardiography analysis of the cardiac function of mice in the therapeutic models at the end-point of 6 weeks post-surgery (n=4-10 per group).


( A ) Volcano curve analysis of dysregulated genes in miR - 574 - / - versus WT heart . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure . The dots above the dashed line show Padj < 0 . 05 .

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(A) Volcano curve analysis of dysregulated genes in miR-574-/- versus WT heart. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure. The dots above the dashed line show Padj<0.05.


( B ) Heatmap of significantly upregulated genes in miR - 574 - / - mice at baseline analyzed by RNA - Seq . P60 male mice , n = 3 per group , Padj < 0 . 05 . The Padj is the P - values adjusted for multiple testing with correction using the Benjamini - Hochberg procedure .

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(B) Heatmap of significantly upregulated genes in miR-574-/- mice at baseline analyzed by RNA-Seq. P60 male mice, n=3 per group, Padj<0.05. The Padj is the P-values adjusted for multiple testing with correction using the Benjamini-Hochberg procedure.


( E ) Expression of Fam210a mRNA in the heart of miR - 574 - / - and WT mice from 3 biological replicates .

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(E) Expression of Fam210a mRNA in the heart of miR-574-/- and WT mice from 3 biological replicates.


( F ) Expression of FAM210A protein in the heart of miR - 574 - / - and WT mice . Protein intensity was quantified in the right panel ( n = 6 per group ) .

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(F) Expression of FAM210A protein in the heart of miR-574-/- and WT mice. Protein intensity was quantified in the right panel (n=6 per group).


( H ) Dual luciferase reporter assays with co - transfection of miR - 574 - 5p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates .

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(H) Dual luciferase reporter assays with co-transfection of miR-574-5p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.


( I ) Dual luciferase reporter assays with co - transfection of miR - 574 - 3p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates .

(I) Dual luciferase reporter assays with co-transfection of miR-574-3p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.


( B ) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database . TPM : transcripts per million of reads . The central band shows the median value and the box indicates the first and third quartiles . The sample size of human samples used for quantification in GTEx Portal database ( from testis to pancreas ) : n = 361 / 429 / 432 / 555 / 803 / 240 / 432 / 226 / 406 / 578 / 241 / 202 / 85 / 328 / 755 .

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(B) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database. TPM: transcripts per million of reads. The central band shows the median value and the box indicates the first and third quartiles. The sample size of human samples used for quantification in GTEx Portal database (from testis to pancreas): n=361/429/432/555/803/240/432/226/406/578/241/202/85/328/755.


( C ) Cellular localization of FAM210A in primary mouse ACMs detected by IF . Scale bar : 30 μm .

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(C) Cellular localization of FAM210A in primary mouse ACMs detected by IF. Scale bar: 30 μm.


( D ) Cellular localization of recombinant FAM210A in human AC16 CM cells . FAM210A - 3xFlag expression plasmid was transfected into AC16 cells for 48 hrs . Scale bar : 40 μm .

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(D) Cellular localization of recombinant FAM210A in human AC16 CM cells. FAM210A-3xFlag expression plasmid was transfected into AC16 cells for 48 hrs. Scale bar: 40 μm.


( E ) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts . VDAC1 and tubulin are used as fraction controls . Cyto : cytoplasm ; Mito : mitochondria . A representative image is shown from three biological replicates .

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(E) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts. VDAC1 and tubulin are used as fraction controls. Cyto: cytoplasm; Mito: mitochondria. A representative image is shown from three biological replicates.


( F ) Sub - organelle localization of FAM210A in isolated mouse cardiac mitochondria . TOM20 and ATP5B are used as the outer membrane ( OM ) and mitoplast ( MP ) markers , respectively .

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(F) Sub-organelle localization of FAM210A in isolated mouse cardiac mitochondria. TOM20 and ATP5B are used as the outer membrane (OM) and mitoplast (MP) markers, respectively.


( B ) Top list of FAM210A - interacting proteins in IP - MS ( immunoprecipitation - mass spectrometry ) analysis . PSM : peptide spectrum match .

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(B) Top list of FAM210A-interacting proteins in IP-MS (immunoprecipitation-mass spectrometry) analysis. PSM: peptide spectrum match.


C ) Confocal imaging of co - localization of FAM210A - 3xFLAG and endogenous EF - Tu by immunostaining . Scale bar : 40 μm .

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C) Confocal imaging of co-localization of FAM210A-3xFLAG and endogenous EF-Tu by immunostaining. Scale bar: 40 μm.


( D ) Left panel : IP - IB validates FAM210A - interacting proteins using pre - immune IgG and FAM210A antibodies . Right panel : IP - IB validates FAM210A - interacting proteins in HEK293T cell lysates subjected to IP using pre - immune IgG and anti - FLAG antibodies . A representative image is shown in three biological replicates .

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(D) Left panel: IP-IB validates FAM210A-interacting proteins using pre-immune IgG and FAM210A antibodies. Right panel: IP-IB validates FAM210A-interacting proteins in HEK293T cell lysates subjected to IP using pre-immune IgG and anti-FLAG antibodies. A representative image is shown in three biological replicates.


( E ) Mapping of interacting domains in FAM210A for binding EF - Tu .

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(E) Mapping of interacting domains in FAM210A for binding EF-Tu.


( F , G ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( F ) siRNA knockdown of FAM210A . ( G ) FAM210A overexpression .

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(F,G) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (F) siRNA knockdown of FAM210A. (G) FAM210A overexpression.


( A ) miR - 574 - 5p and miR - 574 - 3p overexpression reduced FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) . ( B ) miR - 574 - 5p and miR - 574 - 3p inhibition increased FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) .

(A) miR-574-5p and miR-574-3p overexpression reduced FAM210A mRNA expression in AC16 cells (n=3 in each group). (B) miR-574-5p and miR-574-3p inhibition increased FAM210A mRNA expression in AC16 cells (n=3 in each group).


( C , D ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( C ) miR - 574 - 5p or miR - 574 - 3p overexpression . ( D ) Transfection of anti - miR - 574 - 5p or anti - miR - 574 - 3p inhibitor .

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(C,D) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (C) miR-574-5p or miR-574-3p overexpression. (D) Transfection of anti-miR-574-5p or anti-miR-574-3p inhibitor.


( E , F ) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment ( 10 μM for 24 hr ) with overexpression of miR - 574 - 5p or miR - 574 - 3p . > 120 cells / group were quantified in ( E ) and n = 4 biological replicates in ( F ) . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(E,F) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment (10 μM for 24 hr) with overexpression of miR-574-5p or miR-574-3p. >120 cells/group were quantified in (E) and n=4 biological replicates in (F). The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.


( G , H ) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR - 574 - / - mice under ISO treatment . Scale bar : 10 μm . > 110 cells / group were quantified in bottom panel of ( G ) and n = 5 in ( H ) .

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(G,H) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR-574-/- mice under ISO treatment. Scale bar: 10 μm. >110 cells/group were quantified in bottom panel of (G) and n=5 in (H).


( I ) transfection of miR - 574 - 5p / 3p in the absence or presence of FAM210A overexpression . All the Western blot images were provided as representative data from 3 - 4 biologically replicated experiments .

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(I) transfection of miR-574-5p/3p in the absence or presence of FAM210A overexpression. All the Western blot images were provided as representative data from 3-4 biologically replicated experiments.


( J ) Increased protein expression of FAM210A and ND1 in miR - 574 KO hearts compared to WT hearts upon TAC surgery . N = 5 hearts ( ~ 110 - 150 CM cells ) were measured per group . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(J) Increased protein expression of FAM210A and ND1 in miR-574 KO hearts compared to WT hearts upon TAC surgery. N=5 hearts (~110-150 CM cells) were measured per group. The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.


( A - C ) qRT - PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages ( A ) , THP1 cells ( B ) and human peripheral blood mononuclear cells ( PBMC ) ( C ) that were left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib ( 0 . 1 , 1 , 10 μM ) . The data are presented as fold induction of Ifnb1 / Gapdh transcripts . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by one - way ANOVA followed by Dunnett ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 .

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(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.


( D ) MTS detection of the viability of mouse peritoneal macrophages ( PM ) , THP1 and PBMC cells left untreated ( DMSO ) or treated with Palbociclib ( 1 μM , 10 μM ) for 4 h . The data shown are mean ± SEM from n = 3 biological replicates . Statistical significance was determined by one - way ANOVA . ns , not significant .

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(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.


( E - F ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( E ) and THP1 cells ( F ) stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( G - H ) qRT - PCR measurement of Il1b ( G ) and Il6 ( H ) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant . ( I - J ) qRT - PCR measurement of Il1b ( I ) and Il6 ( J ) transcripts in THP1 cells left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant .

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(G-H) qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant. (I-J) qRT-PCR measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.


( K - L ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( K ) and THP1 ( L ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( M - N ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( M ) and THP1 ( N ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( A ) A real - time OR - ID assay showing the association - dissociation curves of surface - immobilized Palbociclib with recombinant STING protein . The data shown are representative of n = 3 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 B-MEASURED_VAR O O O O O O O O O O O O O O

(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with Flag - STING ( C ) The data shown are representative of n = 3 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 O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) mouse peritoneal macrophages ( D ) The data shown are representative of n = 3 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 O O O O O O O O O O O O O O

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) THP1 cells ( E ) . The data shown are representative of n = 3 biological replicates .

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates.


( F ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - c - diAMP ( 2 μM ) and recombinant SUNO - tagged STING protein ( 10 μg / mL ) in the presence of DMSO and increasing concentrations of Palbociclib ( 0 , 1 , 10 μM ) . The data shown are representative of n = 3 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 O O O O O O O O O O O O O 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) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - STING in the presence of DMSO and indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

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 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

(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( H ) . Scale bar , 25 μm .

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 O O O O O O

Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm.


STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( I ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * * , P < 0 . 0001 .

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

STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001.


( J ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STEEP and HA - STING in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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 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

(J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( K ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - TBK1 in the presence of DMSO or indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

(K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


( C ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with corresponding Flag - STING mutants . The data shown are representative of n = 3 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 O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING mutants. The data shown are representative of n=3 biological replicates.


( D ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING or Flag - STING Y167A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( E ) . Scale bars , 25 μm .

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 O O O O O

Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm.


STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( F ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - TBK1 and Flag - STING or Flag - STING Y167A . The data shown are representative of n = 3 biological replicates .

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(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-TBK1 and Flag-STING or Flag-STING Y167A. The data shown are representative of n=3 biological replicates.


( H ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING C91A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( B ) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; ns, not significant.


The representative image of colon tissue harvested from mice of indicated groups is shown in ( C ) .

The representative image of colon tissue harvested from mice of indicated groups is shown in (C).


The length of colon is analyzed in ( D ) . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

The length of colon is analyzed in (D). Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( E - F ) Representative H & E staining of the histological changes in colon tissues harvested from mice of indicated groups ( E ) . Scale bar , 200 μm . The quantification of histological score was shown in ( F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative H&E staining of the histological changes in colon tissues harvested from mice of indicated groups (E). Scale bar, 200 μm. The quantification of histological score was shown in (F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Il6 ( H ) and Il1b ( I ) in mouse colon tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 ; ns , not significant .

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 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

(G-I) qRT-PCR measurement of transcripts of Ifnb1 (G), Il6 (H) and Il1b (I) in mouse colon tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not significant.


( B - F ) Representative H & E staining of the histological changes in heart , stomach , tongue and thigh muscle tissues harvested from mice of indicated groups ( B ) . Scale bar , 200 μm . The quantification of histological score was shown in ( C - F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * , P < 0 . 05 ; * * , P < 0 . 01 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative H&E staining of the histological changes in heart, stomach, tongue and thigh muscle tissues harvested from mice of indicated groups (B). Scale bar, 200 μm. The quantification of histological score was shown in (C-F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. *, P<0.05; **, P<0.01; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Cxcl10 ( H ) and Il6 ( I ) in mouse stomach tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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 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

(G-I) qRT-PCR measurement of transcripts of Ifnb1(G), Cxcl10 (H) and Il6 (I) in mouse stomach tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( A ) Heatmap showing viability measurements for the 192 samples ( rows ) subject to each of 17 microenvironmental stimuli ( columns ) . The data are shown on a robust z - score scale , i . e . , the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row . Limits were applied to scaling factor for optimal visualisation . Red indicates increased viability , blue indicates decreased viability . Samples are annotated for genetic features , sex and pre - treatment status .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap showing viability measurements for the 192 samples (rows) subject to each of 17 microenvironmental stimuli (columns). The data are shown on a robust z-score scale, i.e., the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row. Limits were applied to scaling factor for optimal visualisation. Red indicates increased viability, blue indicates decreased viability. Samples are annotated for genetic features, sex and pre-treatment status.


Logarithm of the relative viability after treatment with Resiquimod ( A , n = 169 ) , IL4 ( B , n = 174 ) stratified by trisomy 12 , and in A , also by IGHV status . P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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

Logarithm of the relative viability after treatment with Resiquimod (A, n=169), IL4 (B, n=174) stratified by trisomy 12, and in A, also by IGHV status. P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Logarithm of the relative viability after treatment with TGFβ ( C , n = 174 ) stratified by trisomy 12 , P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , 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

Logarithm of the relative viability after treatment with TGFβ (C, n=174) stratified by trisomy 12, P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A ) Graphical representation of the four drug - stimulus interaction categories . Categories are defined according to the nature of the interaction ( synergistic or antagonistic ) , and whether the viability is increased or decreased by the stimulus ( positive or negative ) . x - axis shows treatment type , y - axis shows viability with each treatment . Red and blue points and lines depict a representative treatment response pattern for given interaction type . Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug - stimulus ( i . e . additive effects ) , black horizontal lines represent the measured viability after combinatorial treatment . The difference between the black and blue lines indicates the impact of the interaction on expected viability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graphical representation of the four drug-stimulus interaction categories. Categories are defined according to the nature of the interaction (synergistic or antagonistic), and whether the viability is increased or decreased by the stimulus (positive or negative). x-axis shows treatment type, y-axis shows viability with each treatment. Red and blue points and lines depict a representative treatment response pattern for given interaction type. Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug-stimulus (i.e. additive effects), black horizontal lines represent the measured viability after combinatorial treatment. The difference between the black and blue lines indicates the impact of the interaction on expected viability.


( D - I ) Examples of drug - stimulus interactions , for each category . Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus . Each line represents one patient sample linked across treatments . Black horizontal lines in each single treatment indicate the expected viability based on linear modelling . Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus ( blue ) , and the expected viability accounting for the additive effect and the interaction ( black ) . ( D + E ) Ibrutinib , a clinically used BTK inhibitor , is blocked by IL4 and IFNγ . ( F ) The JAK inhibitor pyridone - 6 inhibits the pro - survival effect of sCD40L + IL4 stimulation . ( G ) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro - survival effect not observed in either single treatment . ( H ) TLR agonists , including CpG ODN ( shown ) increase sensitivity to BTK inhibition by ibrutinib , despite increasing viability as single treatments . ( I ) Soluble anti - IgM sensitises CLL samples to HSP90 inhibition by luminespib .

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

(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus. Each line represents one patient sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus (blue), and the expected viability accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK inhibitor, is blocked by IL4 and IFNγ. (F) The JAK inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L + IL4 stimulation. (G) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro-survival effect not observed in either single treatment. (H) TLR agonists, including CpG ODN (shown) increase sensitivity to BTK inhibition by ibrutinib, despite increasing viability as single treatments. (I) Soluble anti-IgM sensitises CLL samples to HSP90 inhibition by luminespib.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( C ) fludarabine + CpG ODN and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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

Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (C) fludarabine + CpG ODN and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( D ) ibrutinib + IL4 single and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (D) ibrutinib + IL4 single and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A Mean ( A ) pSTAT6 staining intensity in CLL - infiltrated ( n = 100 for pSTAT and tumour - free lymph node biopsies ( n = 98 for pSTAT6 after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

(A Mean (A) pSTAT6 staining intensity in CLL-infiltrated (n=100 for pSTAT and tumour-free lymph node biopsies (n=98 for pSTAT6 after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


B ) Mean ( B ) pIRAK4 staining intensity in CLL - infiltrated ( n = 100 for pIRAK4 ) and tumour - free lymph node biopsies n = 100 for pIRAK4 ) after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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B) Mean (B) pIRAK4 staining intensity in CLL-infiltrated (n=100 for pIRAK4) and tumour-free lymph node biopsies n=100 for pIRAK4) after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Example images of immunohistochemistry sections : ( C + D ) pSTAT6 levels in ( C ) CLL - infiltrated and ( D ) tumour - free samples . Scale bar defines 20µm in all images .

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Example images of immunohistochemistry sections: (C + D) pSTAT6 levels in (C) CLL-infiltrated and (D) tumour-free samples. Scale bar defines 20µm in all images.


Example images of immunohistochemistry sections : ( E + F ) pIRAK4 levels in ( E ) CLL - infiltrated and ( F ) tumour - free samples . Scale bar defines 20µm in all images .

Example images of immunohistochemistry sections: (E + F) pIRAK4 levels in (E) CLL-infiltrated and (F) tumour-free samples. Scale bar defines 20µm in all images.


A , B . MDCK - SIAT1 cells co - incubated with Luc - WSN / 33 and MH - S CM fractions . Replication was quantified by luminescence . ( A ) Replication curves display average luminescence reads from 5 wells per condition ( measured at 7 . 5 min intervals - displayed here at 30 min intervals ) from 1 experiment representative of 7 independent experiments . ( B ) Mean calculated area under the luminescence curves ( AUC ) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments .

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A, B. MDCK-SIAT1 cells co-incubated with Luc-WSN/33 and MH-S CM fractions. Replication was quantified by luminescence. (A) Replication curves display average luminescence reads from 5 wells per condition (measured at 7.5 min intervals - displayed here at 30 min intervals) from 1 experiment representative of 7 independent experiments. (B) Mean calculated area under the luminescence curves (AUC) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments.


C . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with flow cytometry - quantified MH - S AM - EVs at specified EV : cell ratios .

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C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios.


D . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with CM fractions of J2 retrovirus - immortalized primary mouse AMs .

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D. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with CM fractions of J2 retrovirus-immortalized primary mouse AMs.


E . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with primary rat AM - derived EVs .

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E. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with primary rat AM-derived EVs.


F . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with human THP - 1 macrophage - derived EVs .

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F. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with human THP-1 macrophage-derived EVs.


A , B . Luc - WSN / 33 replication co - incubated with MH - S EVs was assessed in ( A ) human A549 and ( B ) mouse MLE - 12 AECs . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments .

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A, B. Luc-WSN/33 replication co-incubated with MH-S EVs was assessed in (A) human A549 and (B) mouse MLE-12 AECs. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.


C . MLE - 12 AECs incubated with MH - S EVs infected with PR / 8 influenza . Replication was quantified by RT - qPCR at 12 h post infection . Data represent mean M gene transcripts normalized to β actin from 3 independent experiments .

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C. MLE-12 AECs incubated with MH-S EVs infected with PR/8 influenza. Replication was quantified by RT-qPCR at 12 h post infection. Data represent mean M gene transcripts normalized to β actin from 3 independent experiments.


E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT - qPCR . Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments ( 4 - 5 mice per group ) .

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E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT-qPCR. Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments (4-5 mice per group).


A . EVs isolated from MH - S AMs and A549 AECs were co - incubated in parallel with Luc - WSN / 33 ( MOI = 0 . 5 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments .

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A. EVs isolated from MH-S AMs and A549 AECs were co-incubated in parallel with Luc-WSN/33 (MOI = 0.5) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.


B . EVs isolated from MH - S cells treated with increasing concentrations of CSE were co - incubated with Luc - WSN / 33 in MLE - 12 cells . Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM - EVs from 3 independent experiments per condition .

B. EVs isolated from MH-S cells treated with increasing concentrations of CSE were co-incubated with Luc-WSN/33 in MLE-12 cells. Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM-EVs from 3 independent experiments per condition.


A . MLE - 12 AECs co - incubated with the listed WT strains ( MOI = 0 . 4 ) plus AM - EVs ( grey bars , EV : cell = 1 ) . Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain ( white bars ) , each normalized to β actin from 2 - 4 independent experiments per strain .

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A. MLE-12 AECs co-incubated with the listed WT strains (MOI = 0.4) plus AM-EVs (grey bars, EV:cell = 1). Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain (white bars), each normalized to β actin from 2-4 independent experiments per strain.


C . MHS EVs were co - incubated with the listed Luc - expressing strains plus AM - EVs ( grey bars , EV : cell = 2 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain ( white bars ) from 3 independent experiments per strain .

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C. MHS EVs were co-incubated with the listed Luc-expressing strains plus AM-EVs (grey bars, EV:cell = 2) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain (white bars) from 3 independent experiments per strain.


A . MDCK - SIAT1 cells were pretreated with MH - S EVs ( EV : cell = 2 , ' EV pretreat ' ) or control for 8 h , washed , and subsequently incubated with Luc - WSN / 33 ( MOI = 1 ) . Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments .

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A. MDCK-SIAT1 cells were pretreated with MH-S EVs (EV:cell = 2, 'EV pretreat') or control for 8 h, washed, and subsequently incubated with Luc-WSN/33 (MOI = 1). Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments.


MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( B ) RNA isolation ( B ) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition , each normalized to β actin from 3 independent experiments .

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MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (B) RNA isolation (B) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition, each normalized to β actin from 3 independent experiments.


C . MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( C ) immunofluorescence microscopy . ( C ) Cells in both conditions were fixed and stained with anti - NP or isotype control Ab . Images are representative of two independent experiments . Scale bars = 50

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C. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (C) immunofluorescence microscopy. (C) Cells in both conditions were fixed and stained with anti-NP or isotype control Ab. Images are representative of two independent experiments. Scale bars = 50 μm


D . A549 cells were incubated with WSN / 33 or HK / 14 + AM - EVs for 40 min , washed , fixed , and permeabilized . Cells were subsequently stained with Anti - NP ( green ) , Anti - Rab - 7 ( purple ) Abs , and DAPI ( blue ) . Images shown are representative of 8 independent experiments . Scale bars = 5

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D. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μm


E , F . Co - localization analysis of ( E ) NP : DAPI and ( F ) NP : Rab7 for each condition was performed on Z - stack confocal images images of WSN / 33 ( black ) or HK / 14 ( red ) infected A549s . Data represent the mean NP : DAPI co - localization ratios and the mean NP : Rab7 Mander ' s co - localization coefficient for individual cells from these 8 experiments .

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E, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.


A - D . Replication assessed by luminescence following 10 nM Baf - A1 addition at the indicated times following synchronized uptake of bound Luc - expressing virions . ( A , B ) Shown are examples from 1 experiment representative of a total of 3 - 11 independent experiments per strain . Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0 . 1 % DMSO ( dashed line ) . ( C , D ) The kinetics of endosomal escape by Luc - WSN / 33 and HK / 14 strains are depicted by the loss of inhibition by high dose Baf - A1 between ( C ) 0 - 20 min and ( D ) 60 - 80 min . Data represent mean slope ( change in % inhibition / time interval ) from 5 wells per plate from 3 - 11 independent experiments .

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A-D. Replication assessed by luminescence following 10 nM Baf-A1 addition at the indicated times following synchronized uptake of bound Luc-expressing virions. (A, B) Shown are examples from 1 experiment representative of a total of 3-11 independent experiments per strain. Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0.1% DMSO (dashed line). (C, D) The kinetics of endosomal escape by Luc-WSN/33 and HK/14 strains are depicted by the loss of inhibition by high dose Baf-A1 between (C) 0-20 min and (D) 60-80 min. Data represent mean slope (change in % inhibition/time interval) from 5 wells per plate from 3-11 independent experiments.


Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Baf - A1 on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments .

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Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Baf-A1 on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.


F . Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Chl on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments .

F. Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Chl on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.


A . Data are mean 525em ratio from 488 / 445 nm excitation intensities of internalized FDx particles vs . pH of clamping buffer fit to a standard curve from two independent experiments .

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A. Data are mean 525em ratio from 488/445 nm excitation intensities of internalized FDx particles vs. pH of clamping buffer fit to a standard curve from two independent experiments.


B . Representative images of PBS - and DR - EV - treated MDCK - SIAT1 cells . Scale bars for full size images = 50 μm . Scale bars for cropped images = 10 μm . FDx particles are shown green and DR - EV particles are shown purple . White arrows are FDx endosomes containing DR - EVs .

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B. Representative images of PBS- and DR-EV-treated MDCK-SIAT1 cells. Scale bars for full size images = 50 μm. Scale bars for cropped images = 10 μm. FDx particles are shown green and DR-EV particles are shown purple. White arrows are FDx endosomes containing DR-EVs.


C . Measured pH of FDx endosomes in PBS - ( left bar ) and DR - EV - treated ( right bar ) MDCK - SIAT1 cells . The pH of FDx endosomes containing DR - EVs in DR - EV - treated cells ( middle bar ) ( corresponding to white arrows in Fig 7B ) . Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments .

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C. Measured pH of FDx endosomes in PBS- (left bar) and DR-EV-treated (right bar) MDCK-SIAT1 cells. The pH of FDx endosomes containing DR-EVs in DR-EV-treated cells (middle bar) (corresponding to white arrows in Fig 7B). Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments.


( A ) Dually labeled D . discoideum amoebae producing MCS components fused to GFP , and either calnexin ( CnxA ) - mCherry ( pAW012 ) , P4C - mCherry ( pWS032 ) , or AmtA - mCherry were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) , fixed with 4 % PFA , and imaged by confocal fluorescence microscopy . Merged images are shown . Scale bars : 3 μm ( insets : 1 μm ) .

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(A) Dually labeled D. discoideum amoebae producing MCS components fused to GFP, and either calnexin (CnxA)-mCherry (pAW012), P4C-mCherry (pWS032), or AmtA-mCherry were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed with 4 % PFA, and imaged by confocal fluorescence microscopy. Merged images are shown. Scale bars: 3 μm (insets: 1 μm).