SHG images of organization and quantification ( bars ) of collagen fractions in primary lung tumours and metastases at day 30 . Scale bars 10µm . Data information : All statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 for in vitro experiments and error bars represent the mean ± S . E . M .

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

SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars 10µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative H & E and quantification ( bars ) of picrosirius red staining of two independent regions of n = 5 H1299control and n = 3 H1299RASSF1A primary lung tumours . Scale bars 100µm , zoom 20µm . Data information : All statistical analyses were performed using Student ' s t - test ( 2 - tailed ) of n = 3 for in vitro experiments and error bars represent the mean ± S . E . M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Left : Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG ( green ) or DAPI ( blue ) . Scale bars 10µm . Right : Quantification of NANOG : DAPI nuclear co - localization ( upper graph ) and total NANOG fluorescence intensity ( bottom graph ) . Quantification of nuclear co - localization ( n = 300cells / experiment ) is represented by Pearson ' s coefficient . Data information : Statistical significance was determined by Student ' s t - test ( 2 - tailed ) of n = 3 experiments unless otherwise stated . Error bars represent the mean ± S . E . M .

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

Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence images show merge of NANOG ( green ) , DAPI ( blue ) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass . Scale bars 10µm . Right : Quantification of nuclear co - localization ( n = 200cells / experiment ) is represented by Pearson ' s coefficient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images show merge of NANOG (green), DAPI (blue) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass. Scale bars 10µm. Right: Quantification of nuclear co-localization (n=200cells/experiment) is represented by Pearson's coefficient.


Representative immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness . Scale bars 10µm . Bottom : Quantification of n = 200 cells / experiment . Data information : Statistical significance was determined by Student ' s t - test ( 2 - tailed ) of n = 3 experiments unless otherwise stated . Error bars represent the mean ± S . E . M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness. Scale bars 10µm. Bottom: Quantification of n=200 cells/experiment. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative images of immunofluorescence staining for the pluripotency marker NANOG and β - catenin in three - dimensional spheroids embedded in collagen matrix ( 2mg / ml ) . Scale bars 50µm .

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

Representative images of immunofluorescence staining for the pluripotency marker NANOG and β-catenin in three-dimensional spheroids embedded in collagen matrix (2mg/ml). Scale bars 50µm.


Representative images of H & E and immunohistochemical staining for NANOG and YAP1 in primary lung tumours , ( day 17 ) . Scale bars 100µm . Right : Graph bars represent quantification of NANOG and YAP1 staining based on strong ( 3 + , 2 + ) nuclear intensity ( % ) for at least 2 independent regions of n = 4 H1299control and n = 2 H1299RASSF1A primary tumours at day 17 . Data information : Statistical significance was determined by Student ' s t - test ( 2 - tailed ) of n = 3 experiments unless otherwise stated . Error bars represent the mean ± S . E . M .

Representative images of H&E and immunohistochemical staining for NANOG and YAP1 in primary lung tumours, (day 17). Scale bars 100µm. Right: Graph bars represent quantification of NANOG and YAP1 staining based on strong (3+, 2+) nuclear intensity (%) for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Relative mRNA expression levels of NANOG , OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells . Data information : Statistical significance was determined by Student ' s t - test ( 2 - tailed ) of n = 3 experiments unless otherwise stated . Error bars represent the mean ± S . E . M .

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

Relative mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three - dimensional collagen matrix ( 2mg / ml ) . Scale bars 10µm .

O O O O O O O O O O O O O O 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 immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three-dimensional collagen matrix (2mg/ml). Scale bars 10µm.


Western blots of H1299control and H1299RASSF1A lysates for NANOG , SOX2 and OCT4 in the presence of siNT or siYAP .

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

Western blots of H1299control and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.


Relative H1299control mRNA expression levels NANOG , OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP . Data information : Statistical significance was determined by Student ' s t - test ( 2 - tailed ) of n = 3 experiments unless otherwise stated . Error bars represent the mean ± S . E . M .

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

Relative H1299control mRNA expression levels NANOG, OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix ( 2mg / ml ) and stained for NANOG ( Green ) and YAP ( Red ) with or without 4μM DPCA . Scale bars 10µm .

O O O O O O O O O O O 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

Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4μM DPCA. Scale bars 10µm.


Western blots of H1299control lysates for NANOG , SOX2 and OCT4 in the presence of siNT , siP4HA2 or 4μM DPCA .

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

Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.


Western blot analyses of expression levels of TTF - 1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser .

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

Western blot analyses of expression levels of TTF-1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser.


Representative images of H & E and immunohistochemical staining for lung differentiation markers TTF - 1 and Mucin 5B for at least 2 independent regions of n = 4 H1299control and n = 2 H1299RASSF1A primary tumours at day 17 with quantification ( right ) of TTF - 1 based on nuclear intensity . Scale bars 100µm . Statistical significance was determined by Student ' s t - test ( 2 - tailed ) . Error bars represent the mean ± S . E .

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

Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF-1 and Mucin 5B for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF-1 based on nuclear intensity. Scale bars 100µm. Statistical significance was determined by Student's t-test (2-tailed). Error bars represent the mean ± S.E.M


Representative images of immunofluorescence staining of spheroids grown in collagen matrix ( 2mg / ml ) for differentiation markers TTF - 1 ( Green ) and Mucin 5B ( Red ) . Scale bars 50µm , Zoom 10µm .

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

Representative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm.


Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three - dimensional substrate . Scale bars 200µm .

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

Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.


SHAPE analysis of the complete sfRNAs of ZIKV ( D ) Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , and moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . The SHAPE reactivity were also annotated onto the corresponding secondary structures in ( B ) Nucleotides which SHAPE reactivity was not determined are labeled in gray in ( B )

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

SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B)


SHAPE analysis of the complete sfRNAs of WNV ( E ) . Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , and moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . The SHAPE reactivity were also annotated onto the corresponding secondary structures in ( C ) . Nucleotides which SHAPE reactivity was not determined are labeled in gray in ( C ) .

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

SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).


( A ) - ( D ) The scattering profiles ( A ) , pair distance distribution functions ( PDDFs ) ( B ) , dimensionless Kratky plots ( C ) and Porod - Debye plots ( D ) of the complete sfRNAs of DENV2 ( cyan line ) , ZIKV ( red line ) and WNV ( blue line ) . The fine features of P1 in the scattering profiles in ( A ) arise from helical interstrand pair - distance correlation . The PDDF profiles in ( B ) were calculated using GNOM ( qmax = 0 . 3 ) . The dimensionless Kratky plot and Porod - Debye plot for ZIKV xrRNA1 ( red dashed line ) were included in ( C ) and ( D ) for comparison , indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1 .

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

(A)-(D) The scattering profiles (A), pair distance distribution functions (PDDFs) (B), dimensionless Kratky plots (C) and Porod-Debye plots (D) of the complete sfRNAs of DENV2 (cyan line), ZIKV (red line) and WNV (blue line). The fine features of P1 in the scattering profiles in (A) arise from helical interstrand pair-distance correlation. The PDDF profiles in (B) were calculated using GNOM (qmax=0.3). The dimensionless Kratky plot and Porod-Debye plot for ZIKV xrRNA1 (red dashed line) were included in (C) and (D) for comparison, indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1.


( C , D ) The scattering profiles ( left ) , the PDDFs ( middle ) and the dimensionless Kratky plots ( right ) of individual xrRNA1s ( C ) and xrRNA2s ( D ) of DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) and MVEV ( green ) . The insets in ( C ) and ( D ) show the Guinier regions of the respective scattering profiles with a linear fit line . The back - calculated scattering profile of ZIKV - xrRNA1 crystal structure ( black , open circle ) can be nicely fitted onto its experimental SAXS data ( red line ) in ( C ) , while the back - calculated scattering profile of MVEV - xrRNA2 crystal structure ( black , open circle ) fits poorly to its experimental SAXS data ( green line ) in ( D ) . The asymmetric PDDFs of xrRNA1 ( C ) and xrRNA2 ( D ) , indicate slightly elongated molecules with asymmetric shapes . The dimensionless Kratky plots of xrRNA1 ( C ) and xrRNA2 ( D ) of DENV2 , ZIKV , WNV and MVEV suggest all individual xrRNA1s and xrRNA2s are well - folded and of reduced flexibility in solution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The scattering profiles (left), the PDDFs (middle) and the dimensionless Kratky plots (right) of individual xrRNA1s (C) and xrRNA2s (D) of DENV2 (cyan), ZIKV (red), WNV (blue) and MVEV (green). The insets in (C) and (D) show the Guinier regions of the respective scattering profiles with a linear fit line. The back-calculated scattering profile of ZIKV- xrRNA1 crystal structure (black, open circle) can be nicely fitted onto its experimental SAXS data (red line) in (C), while the back-calculated scattering profile of MVEV- xrRNA2 crystal structure (black, open circle) fits poorly to its experimental SAXS data (green line) in (D). The asymmetric PDDFs of xrRNA1 (C) and xrRNA2 (D), indicate slightly elongated molecules with asymmetric shapes. The dimensionless Kratky plots of xrRNA1 (C) and xrRNA2 (D) of DENV2, ZIKV, WNV and MVEV suggest all individual xrRNA1s and xrRNA2s are well-folded and of reduced flexibility in solution.


( A ) - ( C ) The scattering profiles ( A ) , the PDDFs ( B ) and the dimensionless Kratky plots ( C ) of xrRNA1 - 2s from DENV2 ( cyan ) , ZIKV ( red ) and WNV ( blue ) . The inset in ( A ) shows the guinier regions of the respective scattering profiles with linear fit lines . The dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed line ) was included in ( C ) for comparison , indicating that the structures of xrRNA1 - 2s are all well folded .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of xrRNA1-2s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of xrRNA1-2s are all well folded.


( A ) - ( C ) The scattering profiles ( A ) , the PDDFs ( B ) and the dimensionless Kratky plots ( C ) of DB12s from DENV2 ( cyan ) , ZIKV ( red ) and WNV ( blue ) . The inset in ( A ) shows the Guinier regions of the respective scattering profiles with linear fit lines . The dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed line ) was included in ( C ) for comparison , indicating that the structures of DB12s of ZIKV and WNV are well folded , but that of DB12 of DENV2 is partially folded . The Kratky plots for the two PK3 mutants of DB12s , the ZIKV - DB12 - PK3 ∆ ( green ) and WNV - DB12 - PK3 ∆ ( black ) , respectively , are also included for comparison , suggesting reduced compactness upon PK3 mutations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of DB12s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the Guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of DB12s of ZIKV and WNV are well folded, but that of DB12 of DENV2 is partially folded. The Kratky plots for the two PK3 mutants of DB12s, the ZIKV- DB12-PK3∆ (green) and WNV- DB12-PK3∆ (black), respectively, are also included for comparison, suggesting reduced compactness upon PK3 mutations.


( C ) E protein expression of ZIKV and the mutants showed by IFA .

(C) E protein expression of ZIKV and the mutants showed by IFA.


( D ) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT - PCR in three technical replicates . Two - way ANOVA and Dunnett ' s multiple comparison test was used for statistical analysis . The error bars represent the standard deviation . * * * p < 0 . 001 , * * * * 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

(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.


( E ) The luciferase activity of the ZIKV - Rep and the mutants were measured at different time points after transfection in three technical repelicates . The error bars represent the standard deviation .

O O O O O O O O O O O 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) The luciferase activity of the ZIKV-Rep and the mutants were measured at different time points after transfection in three technical repelicates. The error bars represent the standard deviation.


( A ) - ( C ) The scattering profiles ( A ) , the PDDFs ( B ) and the dimensionless Kratky plots ( C ) of 3 ' SLs from DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) and WNV - 3 ' SLM ( 438UAG441A → 438GUC441U ) mutant ( green ) . The inset in ( A ) shows the guinier regions of the respective scattering profiles with linear fit lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of 3'SLs from DENV2 (cyan), ZIKV (red), WNV (blue) and WNV-3'SLM (438UAG441A→438GUC441U) mutant (green). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines.


( A ) - ( C ) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 ( A ) , ZIKV ( B ) and WNV ( C ) , suggesting optimized minimal structural ensembles with 3 , 3 , 3 conformers , respectively . The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg .

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

(A)-(C) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C), suggesting optimized minimal structural ensembles with 3, 3 ,3 conformers, respectively. The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg.


( A ) Quantification of cell death in 293 cells24 h after transfection with luciferase , p55 / TNFR1 , DRP - 1 Δ73 or DAPk ΔCaM , and GFP . Graphs show the percentage of GFP - positive cells with altered cell morphology . ( B ) Photographs of GFP - positive cells cotransfected with luciferase ( 1 ) , p55 / TNFR1 ( 2 ) , DRP - 1 Δ73 ( 3 ) , or DAPk ΔCaM ( 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of cell death in 293 cells24 h after transfection with luciferase, p55/TNFR1, DRP-1 Δ73 or DAPk ΔCaM, and GFP. Graphs show the percentage of GFP-positive cells with altered cell morphology. (B) Photographs of GFP-positive cells cotransfected with luciferase (1), p55/TNFR1 (2), DRP-1 Δ73 (3), or DAPk ΔCaM (4).


( C ) Photographs showing the overlay of GFP and phase - contrast images of 293 cells24 h after transfection with GFP and luciferase ( 1 ) or with GFP - DAPk ( 2 and 3 ) ; both a blebbing GFP - DAPk - expressing cell ( 2 , arrows indicate blebs ) and spread nontransfected cells are shown ( 3 ) .

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

(C) Photographs showing the overlay of GFP and phase-contrast images of 293 cells24 h after transfection with GFP and luciferase (1) or with GFP- DAPk (2 and 3); both a blebbing GFP-DAPk-expressing cell (2, arrows indicate blebs) and spread nontransfected cells are shown (3).


( D ) Hoechst staining ( 4 - 6 ) of GFP - positive 293 cells ( 1 - 3 ) 60 h after cotransfection with GFP and DAPk ΔCaM ( 1 and 4 ) , DRP - 1 Δ73 ( 2 and 5 ) , or p55 / TNFR1 ( 3 and 6 ) . Arrows point to condensed nuclei resulting from DRP - 1 Δ73 or DAPk ΔCaM transfections ( 4 and 5 ) .

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

(D) Hoechst staining (4-6) of GFP-positive 293 cells (1-3) 60 h after cotransfection with GFP and DAPk ΔCaM (1 and 4), DRP-1 Δ73 (2 and 5), or p55/TNFR1 (3 and 6). Arrows point to condensed nuclei resulting from DRP-1 Δ73 or DAPk ΔCaM transfections (4 and 5).


( A ) Scoring loss of mitochondrial membrane potential ( ΔΨm ) . 293 cells expressing luciferase ( black ) , DRP - 1 Δ73 ( pink ) , DAPk ΔCaM ( purple ) , and Bax ( green ) were assayed for loss of mitochondrial membrane potential 24 h after transfection using flow cytometric analysis of TMRM fluorescence . The fraction of TMRM - negative cells calculated from three experiments is shown in the graph below . The small increase in TMRM fluorescence in the kinase - transfected cells is not statistically significant .

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(A) Scoring loss of mitochondrial membrane potential (ΔΨm). 293 cells expressing luciferase (black), DRP-1 Δ73 (pink), DAPk ΔCaM (purple), and Bax (green) were assayed for loss of mitochondrial membrane potential 24 h after transfection using flow cytometric analysis of TMRM fluorescence. The fraction of TMRM-negative cells calculated from three experiments is shown in the graph below. The small increase in TMRM fluorescence in the kinase-transfected cells is not statistically significant.


( B ) Scoring the release of cytochrome C from mitochondria . 293 cells cotransfected with luciferase ( 1 and 2 ) , Bax ( 3 and 6 ) , DRP - 1 Δ73 ( 7 and 8 ) , or DAPk ΔCaM ( 9 and 10 ) and GFP constructs were incubated for 24 h and immunostained using anti - cytochrome C antibodies . Cells were visualized for cytochrome C ( left ) or GFP ( right ) fluorescence . Bax - transfected spread ( 3 and 4 ) or blebbed ( 5 and 6 ) cells are shown . In fields that also contain nontransfected cells , arrows point to GFP - positive cytochrome C - stained cells .

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(B) Scoring the release of cytochrome C from mitochondria. 293 cells cotransfected with luciferase (1 and 2), Bax (3 and 6), DRP-1 Δ73 (7 and 8), or DAPk ΔCaM (9 and 10) and GFP constructs were incubated for 24 h and immunostained using anti-cytochrome C antibodies. Cells were visualized for cytochrome C (left) or GFP (right) fluorescence. Bax-transfected spread (3 and 4) or blebbed (5 and 6) cells are shown. In fields that also contain nontransfected cells, arrows point to GFP-positive cytochrome C-stained cells.


( C ) Assessing the effects of Bcl - 2 and Bcl - XL . 293 cells were cotransfected with luciferase , DRP - 1 Δ73 , DAPk ΔCaM , or Bax , and either Bcl - 2 or Bcl - XL together with GFP . GFP - positive cells were visualized by fluorescent microscopy and scored for the appearance of cell death morphology 24 h after transfection . Total cell death refers to the sum of all morphological changes observed .

(C) Assessing the effects of Bcl-2 and Bcl-XL. 293 cells were cotransfected with luciferase, DRP-1 Δ73, DAPk ΔCaM, or Bax, and either Bcl-2 or Bcl-XL together with GFP. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of cell death morphology 24 h after transfection. Total cell death refers to the sum of all morphological changes observed.


( A ) Transmission electron micrographs of 293 cells transfected with luciferase ( 1 ) p55 / TNFR1 ( 2a - 2c ) , DRP - 1 Δ73 ( 3a and 3b ; part of fig . 3b is magnified on its right ) , and DAPk ΔCaM ( 4 ) . The different stages of autophagic development are depicted as follows : immature autophagic vesicles ( double arrow ) , mature autophagic vesicles and autolysosomes ( black arrow ) , and residual bodies ( dashed arrow ) . Empty vacuoles ( white arrow ) are also shown . Arrowheads show condensed and fragmented chromatin . m , mitochondria ; g , Golgi apparatus . Bar , 1 μm .

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(A) Transmission electron micrographs of 293 cells transfected with luciferase (1) p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b; part of fig. 3b is magnified on its right), and DAPk ΔCaM (4). The different stages of autophagic development are depicted as follows: immature autophagic vesicles (double arrow), mature autophagic vesicles and autolysosomes (black arrow), and residual bodies (dashed arrow). Empty vacuoles (white arrow) are also shown. Arrowheads show condensed and fragmented chromatin. m, mitochondria; g, Golgi apparatus. Bar, 1 μm.


( B ) Transmission electron micrographs of MCF - 7 cells transfected with luciferase ( 1 ) , p55 / TNFR1 ( 2a - 2c ) , DRP - 1 Δ73 ( 3a and 3b ) , and DAPk ΔCaM ( 4 ) . Immature autophagic vesicles ( double arrow ) , autophagic vesicles ( black arrow ) , and residual bodies ( dashed arrow ) are shown . Insets in B , 2b and 2c , correspond to empty vacuoles , whereas those in B , 3a and 4 , correspond to autophagic vesicles . Arrowheads show condensed and fragmented chromatin . m , mitochondria ; dm , darkened mitochondria ; n , nucleus ; g , Golgi apparatus ; v , vacuoles ; ly , lysosomes ; er , endoplasmic reticulum .

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(B) Transmission electron micrographs of MCF-7 cells transfected with luciferase (1), p55/TNFR1 (2a-2c), DRP-1 Δ73 (3a and 3b), and DAPk ΔCaM (4). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and residual bodies (dashed arrow) are shown. Insets in B, 2b and 2c, correspond to empty vacuoles, whereas those in B, 3a and 4, correspond to autophagic vesicles. Arrowheads show condensed and fragmented chromatin. m, mitochondria; dm, darkened mitochondria; n, nucleus; g, Golgi apparatus; v, vacuoles; ly, lysosomes; er, endoplasmic reticulum.


( A ) Quantification of autophagy in 293 and MCF - 7 cells . Graphs show the percentage of MDC - positive cells resulting from luciferase or DRP - 1 Δ73 transfections ( mean ± SD calculated from triplicates of 100 cells each ; note that total cell number comprises transfected and nontransfected cells ) . These experiments were repeated three times with reproducible results .

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(A) Quantification of autophagy in 293 and MCF-7 cells. Graphs show the percentage of MDC-positive cells resulting from luciferase or DRP-1 Δ73 transfections (mean ± SD calculated from triplicates of 100 cells each; note that total cell number comprises transfected and nontransfected cells). These experiments were repeated three times with reproducible results.


( B ) Photographs of MDC - stained MCF - 7 cells from the same experiment shown in A . Arrows point to MDC - positive cells . Transfections : luciferase ( 1a and 1b ) and DRP - 1 Δ73 ( 2a and 2b ) at low ( a ) and high ( b ) magnifications .

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(B) Photographs of MDC-stained MCF-7 cells from the same experiment shown in A. Arrows point to MDC-positive cells. Transfections: luciferase (1a and 1b) and DRP-1 Δ73 (2a and 2b) at low (a) and high (b) magnifications.


( A ) Quantification of cell death - associated morphologies in 293 cells . Graphs show the percentage of cells harboring all spectrum of morphological changes occurring as a result of luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM transfections in the presence or absence of the caspase inhibitors BD - fmk or z - VAD - fmk ( 100 μM ; mean ± SD calculated from triplicates of 100 cells each ) . This experiment was repeated three times with reproducible results .

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(A) Quantification of cell death-associated morphologies in 293 cells. Graphs show the percentage of cells harboring all spectrum of morphological changes occurring as a result of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM transfections in the presence or absence of the caspase inhibitors BD-fmk or z-VAD-fmk (100 μM; mean ± SD calculated from triplicates of 100 cells each). This experiment was repeated three times with reproducible results.


( B ) Quantification of cell death - associated morphologies in MCF - 7 cells . Experimental conditions were performed as in A in the presence or absence of BD - fmk ( 100 μM ) .

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(B) Quantification of cell death-associated morphologies in MCF-7 cells. Experimental conditions were performed as in A in the presence or absence of BD-fmk (100 μM).


( C ) Graphs show the percentage of cells harboring the complete spectrum of death - associated morphological changes occurring as a result of cotransfections of luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM with the caspase inhibitor crmA or control luciferase vector ( mean ± SD calculated from triplicates of 100 cells each ) . Immunoblot shows the expression levels of transfected HA - tagged DAPk and DRP - 1 Δ73 , confirming that cotransfection with crmA did not alter expression of the death - inducing proteins .

(C) Graphs show the percentage of cells harboring the complete spectrum of death-associated morphological changes occurring as a result of cotransfections of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM with the caspase inhibitor crmA or control luciferase vector (mean ± SD calculated from triplicates of 100 cells each). Immunoblot shows the expression levels of transfected HA-tagged DAPk and DRP-1 Δ73, confirming that cotransfection with crmA did not alter expression of the death-inducing proteins.


( D ) Transmission electron micrographs of 293 cells transfected with p55 / TNFR1 ( 1 ) or DRP - 1 Δ73 ( 2a and 2b ; parts of these figures are magnified at the right ) and treated with the caspase inhibitor BD - fmk ( 100 μM ) . Immature autophagic vesicles ( double arrow ) , autophagic vesicles ( black arrow ) , and autolysosomes ( white arrow ) are shown . m , mitochondria ; g , Golgi apparatus .

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(D) Transmission electron micrographs of 293 cells transfected with p55/TNFR1 (1) or DRP-1 Δ73 (2a and 2b; parts of these figures are magnified at the right) and treated with the caspase inhibitor BD-fmk (100 μM). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and autolysosomes (white arrow) are shown. m, mitochondria; g, Golgi apparatus.


( E ) Western blots of cell lysates prepared from HeLa cells transfected with luciferase , p55 / TNFR1 , DRP - 1 Δ73 , or DAPk ΔCaM and GFP and incubated for 24 h . Blots were reacted with anti - PARP antibodies , anti - caspase - 8 antibodies , anti - caspase - 3 antibodies , anti - HA antibodies for DRP - 1 Δ73 and DAPk ΔCaM detection , and anti - β - tubulin antibodies to quantitate protein loading .

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(E) Western blots of cell lysates prepared from HeLa cells transfected with luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM and GFP and incubated for 24 h. Blots were reacted with anti-PARP antibodies, anti-caspase-8 antibodies, anti-caspase-3 antibodies, anti-HA antibodies for DRP-1 Δ73 and DAPk ΔCaM detection, and anti-β-tubulin antibodies to quantitate protein loading.


( A ) 293 cells were cotransfected with p55 / TNFR1 in combination with DAPk death domain fused to GFP or DRP - 1 K42A and pEGFP - N1 vector . A control luciferase vector was also used . GFP - positive cells were visualized by fluorescent microscopy and scored for the appearance of membrane blebbing 24 h after transfection .

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(A) 293 cells were cotransfected with p55/TNFR1 in combination with DAPk death domain fused to GFP or DRP-1 K42A and pEGFP-N1 vector. A control luciferase vector was also used. GFP-positive cells were visualized by fluorescent microscopy and scored for the appearance of membrane blebbing 24 h after transfection.


( B ) Photographs of the various categories of p55 / TNFR1 - transfected 293 cells : spread cells ( 1 ) , blebbed cells ( 2 ) , condensed cells ( 3 ) , cells displaying a fragmented cytoplasm ( 4 ) , and cells carrying fragmented nuclei within a noncondensed cytoplasm ( 5 ) . ( C ) Quantification of each death morphology observed 24 h after transfection with GFP , p55 / TNFR1 , and either luciferase or DRP - 1 K42A .

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(B) Photographs of the various categories of p55/TNFR1-transfected 293 cells: spread cells (1), blebbed cells (2), condensed cells (3), cells displaying a fragmented cytoplasm (4), and cells carrying fragmented nuclei within a noncondensed cytoplasm (5). (C) Quantification of each death morphology observed 24 h after transfection with GFP, p55/TNFR1, and either luciferase or DRP-1 K42A.


( A and B ) . Quantification of autophagy in MCF - 7 cells . MCF - 7 cells were transfected with luciferase or FLAG - DRP - 1 K42A followed by steroid withdrawal ( serum starvation plus 10 − 6 M tamoxifen treatment ) ( A ) or by serum and amino acid starvation ( B ) ( mean ± SD calculated from triplicates of 100 cells each ) . These experiments were repeated three times with reproducible results . Proteins extracted from the transfected cells were subjected to Western blot analysis with anti - FLAG and anti - β - tubulin antibodies .

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(A and B). Quantification of autophagy in MCF-7 cells. MCF-7 cells were transfected with luciferase or FLAG-DRP-1 K42A followed by steroid withdrawal (serum starvation plus 10−6 M tamoxifen treatment) (A) or by serum and amino acid starvation (B) (mean ± SD calculated from triplicates of 100 cells each). These experiments were repeated three times with reproducible results. Proteins extracted from the transfected cells were subjected to Western blot analysis with anti-FLAG and anti-β-tubulin antibodies.


( C ) Photographs of the MDC - stained steroid withdrawn MCF - 7 cells taken from the same experiment shown in A . Transfections : luciferase ( 1a and 1b ) and DRP - 1 K42A ( 2a and 2b ) at low ( a ) and high ( b ) magnifications .

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(C) Photographs of the MDC-stained steroid withdrawn MCF-7 cells taken from the same experiment shown in A. Transfections: luciferase (1a and 1b) and DRP-1 K42A (2a and 2b) at low (a) and high (b) magnifications.


( A ) Photographs of MDC - stained MCF - 7 cells . ( Top ) Steroid withdrawal . Cells were either grown in complete medium ( 1 ) , steroid depleted ( 2 ) , or steroid depleted in the presence of the broad caspase inhibitor BD - fmk ( 100 μM ) ( 3 ) for 3 d . ( Bottom ) amino acid starvation . Cells were either grown in complete medium ( 1 - 3 ) or under amino acid starvation condition ( 4 - 6 ) without ( 1 and 4 ) or with ( 2 and 5 ) the presence of the broad caspase inhibitor BD - fmk ( 100 μM ) or the general inhibitor of autophagy 3 - methyladenine ( 10 mM ) ( 3 and 6 ) for 3 d .

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(A) Photographs of MDC-stained MCF-7 cells. (Top) Steroid withdrawal. Cells were either grown in complete medium (1), steroid depleted (2), or steroid depleted in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (Bottom) amino acid starvation. Cells were either grown in complete medium (1-3) or under amino acid starvation condition (4-6) without (1 and 4) or with (2 and 5) the presence of the broad caspase inhibitor BD-fmk (100 μM) or the general inhibitor of autophagy 3-methyladenine (10 mM) (3 and 6) for 3 d.


( B ) PARP cleavage in MCF - 7 - starved cells . Proteins extracted from 4 - d steroid - depleted ( left ) or 12 - h TNF - α - treated ( 100 ng / ml ) cells ( right ) were subjected to Western blot analysis with anti - PARP antibodies . The proteins were prepared from the same experiment shown in A .

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(B) PARP cleavage in MCF-7-starved cells. Proteins extracted from 4-d steroid-depleted (left) or 12-h TNF-α-treated (100 ng/ml) cells (right) were subjected to Western blot analysis with anti-PARP antibodies. The proteins were prepared from the same experiment shown in A.


( C ) Graph showing the cumulative phase microscopy scores of dead ( shrunk and detached ) MCF - 7 cells in tamoxifen - treated ( filled ) or nontreated ( unfilled ) cultures in the presence ( ▴ ) or absence ( • ) of the indicated caspase inhibitors ( 25 μM ) .

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(C) Graph showing the cumulative phase microscopy scores of dead (shrunk and detached) MCF-7 cells in tamoxifen-treated (filled) or nontreated (unfilled) cultures in the presence (▴) or absence (•) of the indicated caspase inhibitors (25 μM).


( A ) Quantitation of autophagy in HeLa cells . Cells were either grown in complete medium ( 1 ) , treated with IFN - γ ( 1 , 000 U / ml ) ( 2 ) , or treated with IFN - γ in the presence of the broad caspase inhibitor BD - fmk ( 100 μM ) ( 3 ) for 3 d . ( B ) Photographs of the MDC - stained HeLa cells corresponding to the experiment in A . Treatments : complete medium ( 1 ) , IFN - γ ( 1 , 000 U / ml ) ( 2 ) , and IFN - γ ( 1 , 000 U / ml ) + BD - fmk ( 3 ) .

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(A) Quantitation of autophagy in HeLa cells. Cells were either grown in complete medium (1), treated with IFN-γ (1,000 U/ml) (2), or treated with IFN-γ in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (B) Photographs of the MDC-stained HeLa cells corresponding to the experiment in A. Treatments: complete medium (1), IFN-γ (1,000 U/ml) (2), and IFN-γ (1,000 U/ml) + BD-fmk (3).


( C ) PARP cleavage in HeLa IFN - γ - treated cells . Proteins extracted from IFN - γ - treated ( left ) or TNF - α - treated ( 100 ng / ml ) cells ( right ) were subjected to Western blot analysis with anti - PARP antibodies . The proteins were prepared from the same experiment shown in A .

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(C) PARP cleavage in HeLa IFN-γ-treated cells. Proteins extracted from IFN-γ-treated (left) or TNF-α-treated (100 ng/ml) cells (right) were subjected to Western blot analysis with anti-PARP antibodies. The proteins were prepared from the same experiment shown in A.


( D ) Transmission electron micrographs of nontreated ( top ) and IFN - γ - treated ( bottom ) HeLa cells . Arrows point to autophagic vesicles .

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(D) Transmission electron micrographs of nontreated (top) and IFN-γ-treated (bottom) HeLa cells. Arrows point to autophagic vesicles.


( E ) DAPk antisense - transfected and control DHFR - transfected polyclonal populations of HeLa cells were incubated with hygromycin B ( 200 μg / ml ) in the presence or absence of IFN - γ ( 1 , 000 U / ml ) and BD - fmk ( 50 μM ) . Detection of autophagic vesicles was performed 2 d later using the MDC dye . Graphs represent mean ± SD of MDC - positive cells calculated from triplicates of 100 cells each .

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(E) DAPk antisense-transfected and control DHFR-transfected polyclonal populations of HeLa cells were incubated with hygromycin B (200 μg/ml) in the presence or absence of IFN-γ (1,000 U/ml) and BD-fmk (50 μM). Detection of autophagic vesicles was performed 2 d later using the MDC dye. Graphs represent mean ± SD of MDC-positive cells calculated from triplicates of 100 cells each.


DRP - 1 is localized to the lumen of autophagic vesicles . Immunogold staining of 293 cells expressing HA - DRP - 1 reveals a specific staining of DRP - 1 in the lumen of double membrane autophagosomes containing cytoplasmic material ( A and B ) in autophagosomes containing organelles and vacuoles ( C ) and inside the body of autolysosomes containing digested matter ( D ) . The gold particles do not overlap with the phagocytosed organelles in C or the lysosomes in D .

DRP-1 is localized to the lumen of autophagic vesicles. Immunogold staining of 293 cells expressing HA-DRP-1 reveals a specific staining of DRP-1 in the lumen of double membrane autophagosomes containing cytoplasmic material (A and B) in autophagosomes containing organelles and vacuoles (C) and inside the body of autolysosomes containing digested matter (D). The gold particles do not overlap with the phagocytosed organelles in C or the lysosomes in D.


( C Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P8 ( C ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

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(C Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


D ) Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P34 ( D ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


ASPC - 1 , MiaPaCa - 2 , Colo - 357 , Panc - 1 , Panc - 89 and PancTu - 1 cells were grown in the absence or presence of the indicated concentrations of omeprazole , 5 - fluorouracile , pantoprazole or gemcitabine , respectively , for 4 days to determine the IC50 values . The IC50 ies and the sigmoidicities are gradually differing between the cell lines . In lower concentrations a slight growth - stimulatory effect ( hormesis ) was observed .

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ASPC-1, MiaPaCa-2, Colo-357, Panc-1, Panc-89 and PancTu-1 cells were grown in the absence or presence of the indicated concentrations of omeprazole, 5-fluorouracile, pantoprazole or gemcitabine, respectively, for 4 days to determine the IC50 values. The IC50 ies and the sigmoidicities are gradually differing between the cell lines. In lower concentrations a slight growth-stimulatory effect (hormesis) was observed.


The cell lines ASPC - 1 , MiaPaCa - 2 , Colo357 , Panc - 1 , Panc89 and PancTu1 were untreated or treated with 5 - FU alone or in combination with the indicated concentrations of OMP for 4 days . At lower doses of 5 - FU alone ( black lines , 0 µg / ml OMP ) a growth - stimulatory effect ( hormesis ) was observed in the cell lines ASPC - 1 , Panc - 1 and PancTu - 1 . The data points show the means of 8 measurements . The curves are fitted in these cell lines using the Brain - Cousens model ( see sub - section 4 . 11 ) . In MiaPaCa - 2 and Panc - 89 cells no hormesis occured , these curves were fitted using a three parameter logistic model . In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations ( data points shown ) . The red , green and blue lines indicate the dose - effect curves of 5 - FU when various concentrations of OMP were added ( 10 , 20 and 40 µg / ml , respectively ) . In ASPC - 1 and Panc - 1 cells the hormesis of 5 - FU was reversed and in PancTu - 1 cell it was mitigated by OMP depending on the concentration of 5 - FU . In MiaPaCa - 2 cells we found an additive interaction of 5 - FU with OMP . In Panc - 89 cells the interaction was antagonistic .

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

The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.


Acridine Orange was added to living untreated ASPC - 1 and MiaPaCa - 2 cells and cells treated with 5 - FU , OMP or the combination of both for 30 minutes or 24 hours . Microscopical life images were taken at 525 nm ( green ) and 650 nm ( red ) to detect changes in the lysosomal pH value ( three images per plate , three plates per group ) . The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann - Whitney - U - test . Significant differences compared to control are marked by * . In ASPC - 1 cells , after 30 min of treatment , intralysosomal acidity increased upon treatment with OMP ( p : 0 . 0051 ) and 5 - FU + OMP ( p < 0 . 0001 ) . After 24 hours , the acidity is increased upon all treatment regimens ( 5 - FU - p : 0 . 0002 ; OMP - p < 0 . 00001 ; OMP + 5 - FU - p : 0 . 037 ) . In MiaPaCa - 2 cells the acidity is elevated after 30 min upon 5 - FU ( p : 0 . 005 ) and decreased after treatment with OMP ( p : 0 . 037 ) , 5 - FU ( p : 0 . 00026 ) and 5 - FU + OMP ( p : 0 . 011 ) after 24 hours .

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

Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.


( A ) ASPC - 1 cell without treatment ( 800 fold ) . ( B ) ASPC - 1 cell undergoing apoptosis upon 160 µg / ml OMP after 24 hours ( 800 fold ) . Vacuolisation of the cytoplasma and condensation of the nucleus are visible . ( C ) Phagophores and autophagosomes in a segment of an ASPC - 1 cell treated with omeprazole 80 µg / ml for 24 hours ( 2800fold enlargement ) . The phagophores are characterised by a cup - like shape ( white arrows ) . Autophagosomes are closed particles , the number of which is increased in treated cells ( black arrows ) . ( D ) Early phagophores and autophagosomes are also found in MiaPaCa - 2 cells treated with OMP 80 µg / ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex . In contrast to ASPC - 1 cells , early signs of apoptosis such as vacuolization , are also present .

O O O O 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 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 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

(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present.


( E ) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa - 2 and ASPC - 1 cells untreated or treated with 5 - FU , OMP or the combination of both for 24 hours with standard errors . Significant differences compared to control are marked by * . In ASPC - 1 cells there were significant differences compared to the control in the OMP group ( p : 0 . 03 ) and the 5 - FU + OMP group ( p : 0 . 03 ) . In MiaPaCa - 2 cells the 5 - FU + OMP group differed significantly from control ( 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

(E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).


The ASPC - 1 cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . The most obvious changes is that the PC / Cho ratio is significantly lower in the OMP group compared to the control group . Upon 5 - FU , the Cho / Acetate ratio decrease is the only significant change . In the 5 - FU + OMP group , there are several significant changes , i . e . the FACH2 / CH = CH ratio is significantly higher . Moreover , the Cho / Acetate ratio changed upon 5 - FU + OMP as in the 5 - FU group , but also the citrate / GSH ratio . The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline , Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ASPC-1 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. The most obvious changes is that the PC/Cho ratio is significantly lower in the OMP group compared to the control group. Upon 5-FU, the Cho/Acetate ratio decrease is the only significant change. In the 5-FU+OMP group, there are several significant changes, i.e.the FACH2/CH = CH ratio is significantly higher. Moreover, the Cho/Acetate ratio changed upon 5-FU+OMP as in the 5-FU group, but also the citrate/GSH ratio. The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline, Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


The cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . Upon OMP , the PC / Cho ratios are significantly lower compared to control . Furthermore the acetate / FACH2 ratio is significantly decreased in the OMP group . The latter also showed a higher CH = CH level , the ratio to FACH2 is , however , decreased . Upon 5 - FU and 5 - FU + OMP , similar changes could be observed . Furthermore , in contrast to ASPC - 1 cells , the Ala / Gln / AMP pathway is also involved . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline . Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


( B ) Western blot analysis of LAMP - 1 , a late endosome marker , which was nearly identically distributed over the first two fractions compared to Cathepsin - D , in the controle group , but ocurred only in the first fraction in the OMP treated group . β - COP as an indicator of the Golgi - complex , is found strongly in the lower fraction in the control group , but very weakly in the OMP treated group . Cathepsin - D , an early endosome marker , which can be found in the upper and middle fraction of the control group , but regarding the OMP group , it was only found in the first one .

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

(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.


The experiment was repeated three times with nearly identical results . The LC3 - I fractions can be clearly distinguished above the LC3 - II signals . Bafilomycin A1 elevated slightly the LC3 - II signal intensity compared to control in both cell lines . There was a marked and dose - dependent increase of the signal strength of both fractions when OMP was used . 5 - FU alone did not relevantly influence the LC3 - level . The corresponding β - Actin level is shown below the LC3 - WB to confirm the correctness of this semiquantitative evaluation .

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

The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.


Quantification of mRNAs in ASPC - 1 cells untreated or treated with OMP 80 µg / ml , 5 - FU 5 µg / ml or the combination of both was performed at different time points throughout 24 hours , and the means of three replicates are shown for every time point . The stars indicate significant differences after 24 hours compared to control . The pro - apoptotic bad - mRNA was upregulated after 5 - FU - and 5 - FU + OMP - treatment . The antiapoptotic bcl - 2 mRNA was downregulated by 5 - FU and bcl - XL by 5 - FU + OMP . OMP led to downregulation of bad and survivin and to upregulation of the mdr - 1mRNA .

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

Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1mRNA.


mRNA quantification in MiaPaCa - 2 cells untreated or treated with OMP 80 µg / ml , 5 - FU 5 µg / ml or the combination of both was performed at different time points throughout 24 hours , and the means of three replicates are shown for every time point . While the mdr - 1 mRNA is not signicantly changed , the vATPase mRNA is upregulated in the 5 - FU + OMP group after 24 hours compared to control .

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

mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.


The Bad , Puma and Atg12 proteins were detected in MiaPaCa - 2 and ASPC - 1 cells . The Bad expression is slightly increased by 5 - FU and suppressed by OMP and 5 - FU + OMP in ASPC - 1 , but not in MiaPaCa - 2 cells . Puma is induced by OMP in both cell lines indicating involvement of the BH3 - only pathway . Atg12 is strongly enhanced by OMP in both cell lines . Abbreviations : K - control , F - 5 - FU , O - OMP , FO - 5 - FU + OMP .

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

The Bad , Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP.


A , B Western blot analysis of ΔKu80 , PKAc1 - 3Ty ( A ) parasite lysates probed with αTy antibodies . Profilin ( αPRF ) served as loading control .

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

A, B Western blot analysis of ΔKu80, PKAc1-3Ty (A) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.


A , B Western blot analysis of ΔKu80 , PKAr - 3Ty ( B ) parasite lysates probed with αTy antibodies . Profilin ( αPRF ) served as loading control .

A, B Western blot analysis of ΔKu80, PKAr-3Ty (B) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.


C , D Peripheral localization of PKAc1 - 3Ty ( C ) shown by IFA using anti - Ty antibodies . Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC , 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

C, D Peripheral localization of PKAc1-3Ty (C) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.


C , D Peripheral localization of PKAr - 3Ty ( D ) shown by IFA using anti - Ty antibodies . Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC , respectively .

C, D Peripheral localization of PKAr-3Ty (D) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.


E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti - Ty after aerolysin treatment , indicating that PKAr - 3Ty localises at the IMC .

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

E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti-Ty after aerolysin treatment, indicating that PKAr-3Ty localises at the IMC.


F The N - terminus putative acylation site of PKAr is essential for its IMC targeting .

F The N-terminus putative acylation site of PKAr is essential for its IMC targeting.


G A second copy of DDmyc - PKAc1 is stabilised after 1 hour in presence of Shld - 1 .

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G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.


H A second copy of DDmyc - PKAc1 stabilised with Shld - 1 is targeted to the IMC in presence of a second copy of PKArWT - Ty . When of a second copy of PKArG2A - Ty is provided , stabilised DDmyc - PKAc1 does not localise at the cell periphery . Data information : Scale bars = 2 μm .

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H A second copy of DDmyc-PKAc1 stabilised with Shld-1 is targeted to the IMC in presence of a second copy of PKArWT-Ty. When of a second copy of PKArG2A-Ty is provided, stabilised DDmyc-PKAc1 does not localise at the cell periphery. Data information: Scale bars = 2 μm.


A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells . Destabilisation of PKAr - TyDD leads to the absence of plaque formation .

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A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells. Destabilisation of PKAr-TyDD leads to the absence of plaque formation.


B A drop in PKAr - TyDD expression is detected 5 hours after Shld - 1 removal and a significant parasite growth defect is observed as early as 12 hours later . Profilin ( αPRF ) served as loading control .

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B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.


C IFA analysis showed that the down regulation of PKAr - TyDD leads to the abnormal segregation of the apicoplast - associated thioredoxin family protein ( ATrx1 ) 24 hours after Shld - 1withdrawal .

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C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.


D Replication assay of PKAr - TyDD parasites grown for 24 hours + / - Shld - 1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation . ( 100 parasites were counted in three independent replicates ) .

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D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates).


E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to the complete block of cell growth indicated by plaque assay ( E )

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E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by plaque assay (E)


E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to the complete block of cell growth indicated by western blot analysis ( F ) .

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E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by western blot analysis (F).


G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld - 1 ( 100 parasites were counted in three independent replicates ) . Data information : In C and G , data are presented as mean ± SEM . Scale bars = 2 μm ( C and G ) , 3 mm ( A and E ) .

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G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E).


G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld - 1 ( 100 parasites were counted in three independent replicates ) .

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G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).


A A drop in PKAc1 - Ty - iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours .

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A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours.


B Conditional down - regulation of PKAc1 - Ty - iKD leads to the absence of plaque formation . Scale bars = 1 mm ( B )

B Conditional down-regulation of PKAc1-Ty-iKD leads to the absence of plaque formation.Scale bars = 1 mm (B)


C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress ( data is from three independent biological replicates ) . Scale bars = 20 μm ( C )

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C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Scale bars = 20 μm (C)


C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress ( data is from three independent biological replicates ) . Data information : In D , data are presented as mean ± SD .

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C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Data information: In D, data are presented as mean ± SD.


E Prematurely egressed PKAc1 - Ty - iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non - treated parasites invade and initiate a new lytic cycle . Scale bars = 2 . 5 mm ( E )

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E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle. Scale bars = 2.5 mm (E)


B Addition of Shld - 1 leads to the rapid stabilisation of DDmyc - PKArWT - Ty and DDmyc - PKArG321E - Ty .

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B Addition of Shld-1 leads to the rapid stabilisation of DDmyc-PKArWT-Ty and DDmyc-PKArG321E-Ty.


C PKAc1 is co - immunoprecipitated with DDmyc - PKArWT - Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [ 35S ] - labeled methionine / cysteine metabolic labelling . Conversely , PKAc1 is co - immunoprecipitated with DDmyc - PKArG321E - Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP . The autoradiogram shown is representative of two independent biological replicates . Data information : In C and E - H , data are presented as mean ± SEM .

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C PKAc1 is co-immunoprecipitated with DDmyc-PKArWT-Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [35S]-labeled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co-immunoprecipitated with DDmyc-PKArG321E-Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.Data information: In C and E-H, data are presented as mean ± SEM.


D Conditional overexpression of a dominant - negative DDmyc - PKArG321E - Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over - expression of DDmyc - PKArWT - Ty is well tolerated by the parasite . Scale bars = 3 mm ( D )

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D Conditional overexpression of a dominant-negative DDmyc-PKArG321E-Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over-expression of DDmyc-PKArWT-Ty is well tolerated by the parasite.Scale bars = 3 mm (D)


E Overexpression of the dominant negative DDmyc - PKArG321E - Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA . Images are representative of ~ 80 % of the parasite population . Blue = DAPI ; red = GAP45 ; green = GRA3 . Data information : In C and E - H , data are presented as mean ± SEM . Scale bars = 10 μm ( E )

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E Overexpression of the dominant negative DDmyc-PKArG321E-Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ~80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 10 μm (E)


F In heavily infected cells , addition of Shld - 1 at 32 hours post - inoculation for 4 hours leads to premature egress of DDmyc - PKArG321E - Ty parasites but not of the DDmyc - PKAr - Ty control line as assessed by IFA . Data information : In C and E - H , data are presented as mean ± SEM . Scale bars = 5 µm ( F )

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F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 5 µm (F)


G Quantification of extracellular DDmyc - PKArG321E - Ty parasites released from heavily infected cells after addition of Shld - 1 at 32 hours post - inoculation for 4 hours ( 100 parasites were counted in three independent replicates ) . Data information : In C and E - H , data are presented as mean ± SEM .

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G Quantification of extracellular DDmyc-PKArG321E-Ty parasites released from heavily infected cells after addition of Shld-1 at 32 hours post-inoculation for 4 hours (100 parasites were counted in three independent replicates).Data information: In C and E-H, data are presented as mean ± SEM.


H A non - selective PKA inhibitor , KT5270 , significantly enhances the premature egress of DDmyc - PKArWT - Ty parasites in presence of Shld - 1 ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) . Data information : In C and E - H , data are presented as mean ± SEM .

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H A non-selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc-PKArWT-Ty parasites in presence of Shld-1 (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).Data information: In C and E-H, data are presented as mean ± SEM.


I Prematurely egressed DDmyc - PKArG321E - Ty parasites after 4 hours of Shld - 1 treatment invade and exit host cells , fully lysing the monolayer of HFF cells while the non - treated parasites invade and initiate a new lytic cycle . Scale bars = 2 . 5 mm ( I ) .

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I Prematurely egressed DDmyc-PKArG321E-Ty parasites after 4 hours of Shld-1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.Scale bars = 2.5 mm (I).


B Upon Shld - 1 addition , a putative cGMP - specific PDE , PDE2 was more phosphorylated at Serine 1317 in DDmyc - PKArG321E - Ty parasites compared with DDmyc - PKArWT - Ty parasites suggesting a crosstalk between cAMP and cGMP signaling .

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B Upon Shld-1 addition, a putative cGMP-specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc-PKArG321E-Ty parasites compared with DDmyc-PKArWT-Ty parasites suggesting a crosstalk between cAMP and cGMP signaling.


C Chemical inhibition of the cGMP - dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO , a PDE inhibitor , or by stabilisation of DDmyc - PKArG321E - Ty in presence of Shld - 1 . A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0 . 2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only ( data is from three independent biological replicates ) . Data information : In C and D , data are presented as mean ± SD .

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C Chemical inhibition of the cGMP-dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc-PKArG321E-Ty in presence of Shld-1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data is from three independent biological replicates).Data information: In C and D, data are presented as mean ± SD.


D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer . Anti - GRA1 antibodies were used as a loading control and anti - catalase antibodies served as a lysis control ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) Data information : In C and D , data are presented as mean ± SD .

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D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer. Anti-GRA1 antibodies were used as a loading control and anti-catalase antibodies served as a lysis control (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test) Data information: In C and D, data are presented as mean ± SD.


A The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected ( data is from three independent biological replicates ) .

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A The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected (data is from three independent biological replicates).


B Transferring supernatants of Shld - 1 treated cultures from cells infected with DDmyc - PKArG321E - Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours ( data is from three independent biological replicates ; statistical analysis was done by two - tailed t - test ) .

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B Transferring supernatants of Shld-1 treated cultures from cells infected with DDmyc-PKArG321E-Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).


C Acidification of the culture medium induces parasite egress in less than 10 minutes . The effect is inhibited in presence of compound 1 ( data is from two independent biological replicates ) .

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C Acidification of the culture medium induces parasite egress in less than 10 minutes. The effect is inhibited in presence of compound 1 (data is from two independent biological replicates).


D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc - PKArG321E - Ty stabilisation . This block is circumvented by addition of the Ca2 + ionophore A23187 or the PDE inhibitor BIPPO ( data is from three independent biological replicates ) . Data information : Data are presented as mean ± SD .

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D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc-PKArG321E-Ty stabilisation. This block is circumvented by addition of the Ca2+ ionophore A23187 or the PDE inhibitor BIPPO (data is from three independent biological replicates). Data information: Data are presented as mean ± SD.


A - D Localization of PDE1 - 3Ty ( A ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm .

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A-D Localization of PDE1-3Ty (A) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of PDE2 - 3Ty ( B ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm .

A-D Localization of PDE2-3Ty (B) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of GCα - 3Ty ( C ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm .

A-D Localization of GCα-3Ty (C) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of PKG - 3Ty shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , and myc - GAP70 were used as markers of the plasma membrane , IMC , residual body , and apical pole , respectively . Data information : Scale bars = 2 μm .

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A-D Localization of PKG-3Ty shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.