C Quantification of the data from repeats of the experiment as shown in B . The upper two graphs show the percent of cells with high CHOP : : GFP or high XBP : : Turq for each transfection . The lower panel provides quantitation of the median fluorescence intensity for each reporter . Mean ± SD , n = 4 , biological replicates .

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C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.


B On the left are immunoblots of AMPylated proteins detected with pan - AMP , BiP and eIF2α ( a loading control ) reactive antibodies in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) for the indicated time . In the panel on the right are immunoblots of samples immunoprecipitated with a BiP - specific antiserum from the lysates of the samples shown on the left .

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B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.


C Immunoblots as in B in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) and / or thapsigargin ( Tg ) for the indicated periods prior to harvest . D Quantification of ratio of the BiP - AMP to total BiP signal ( expressed in arbitrary units ) of repeats of the experiment shown in C . Mean ± SD of the BiP - AMP signal normalized to total BiP signal from the same samples are depicted , ( P values by two tailed t - test , n = 6 , biological replicates ) .

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C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).


A Representative immunoblot of AMPylated proteins in FICD ∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes ( replicated three times ) . Note that the hyperactive E234G mutant bearing the K124E - H131A mutations that compromise its ability to interact with BiP , is nonetheless consistently auto - AMPylated .

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A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.


C Bar diagram of the amount of alkaline phosphatase [ normalised to cytoplasmic luciferase ( a transfection marker ) ] secreted from FICD ∆ CHO cells co - expressing the indicated FICD enzymes . The K124E - H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [ Mean ± SD , P values by ANOVA with Šídák ' s multiple comparisons test , n = 8 - 16 ( biological replicates ) ] .

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C Bar diagram of the amount of alkaline phosphatase [normalised to cytoplasmic luciferase (a transfection marker)] secreted from FICD∆ CHO cells co-expressing the indicated FICD enzymes. The K124E-H131A mutations that interfere with engagement of BiP as a substrate reverse the secretion defect imposed by the hyperactive FICD enzymes [Mean ± SD, P values by ANOVA with Šídák's multiple comparisons test, n = 8-16 (biological replicates)].


WT and SNX10 KO Caco - 2 cells were treated with indicated doses of OMVs for 24h . Protein expression of E - cadherin and SNX10 was determined by immunoblots ( A ) and quantified by ImageJ software ( B ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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WT and SNX10 KO Caco-2 cells were treated with indicated doses of OMVs for 24h. Protein expression of E-cadherin and SNX10 was determined by immunoblots (A) and quantified by ImageJ software (B) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


G , H Confocal images of E - cadherin staining in Caco - 2 cells treated with OMVs ( 100 μg / mL ) for the indicated time were captured ( G ) , and the fluorescence intensity of E - cadherin staining was quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Scale bar : 20 μm . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

G, H Confocal images of E-cadherin staining in Caco-2 cells treated with OMVs (100 μg/mL) for the indicated time were captured (G), and the fluorescence intensity of E-cadherin staining was quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Scale bar: 20 μm. Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


A , B WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . The fold changes in mRNA levels of SNAI1 ( encoding Snail ) ( A ) and SNAI2 ( encoding Slug ) ( B ) in Caco - 2 cells were determined by RT - qPCR . n = 6 independent experiments . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . ns , not significant .

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

A, B WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT-qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ns, not significant.


C WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots .

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C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots.


WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h . quantiﬁed the percentage of Snail ( E ) or Slug ( F ) - positive nuclei respectively . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h. quantiﬁed the percentage of Snail (E) or Slug (F)-positive nuclei respectively. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


H WT and SNX10 KO Caco - 2 cells were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without 100 μg / mL OMVs for 24 h . Proteins from nucleus , cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies .

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H WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/mL OMVs for 24 h. Proteins from nucleus, cytoplasm and total cell lysates were subjected to immunoblots with the indicated antibodies.


C , D Levels of p - Lyn , Lyn , E - cadherin and SNX10 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24h were determined by immunoblots ( C ) and quantified by ImageJ software ( D ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Levels of p-Lyn, Lyn, E-cadherin and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


E , F Caco - 2 cells were transfected with Ad - vector or Ad - SNX10 , followed by treatment with or without OMVs ( 100 μg / mL ) for 24h . Lysates were extracted and subjected to immunoblots with the indicated antibodies ( E ) and quantified by ImageJ software ( F ) ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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E, F Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/mL) for 24h. Lysates were extracted and subjected to immunoblots with the indicated antibodies (E) and quantified by ImageJ software (F) (n = 3 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco - 2 cells treated with or without Tolimidone ( 10 μM ) , and the indicated proteins were analyzed by immunoblots .

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I Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by immunoblots.


D WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24 h and then subjected to IP with anti - caspase - 5 antibody .

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D WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24 h and then subjected to IP with anti-caspase-5 antibody.


E Representative images of co - staining of Lyn and caspase - 5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h . Scale bar : 20 μm . F The co - localization of Lyn and caspase - 5 was quantified by ImageJ software . n = 3 independent experiments , n = 18 fields analysed . Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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E Representative images of co-staining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h. Scale bar: 20 μm. F The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analysed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


H Lysates of Flag - tagged full - length SNX10 or its different truncated mutants transfected Caco - 2 cells were subjected to immunoprecipitation .

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H Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected Caco-2 cells were subjected to immunoprecipitation.


E , WT and SNX10 KO Caco - 2 cells were treated with OMVs ( 100 μg / mL ) for the indicated time and stained with antibodies against LPS and Rab5 ( E ) . Scale bar : 20 μm .

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E, WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/mL) for the indicated time and stained with antibodies against LPS and Rab5 (E). Scale bar: 20 μm.


G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . H LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) with or without chloroquine ( CQ ) ( 200 nM ) or bafilomycin A1 ( Baf A1 ) ( 50 nM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant .

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G LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by LAL assay (n = 6 independent experiments). H LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.


C WT and SNX10 KO Caco - 2 cells were treated with or without OMVs ( 100 μg / mL ) for 24h and then subjected to IP with anti - PIKfyve antibody .

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C WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/mL) for 24h and then subjected to IP with anti-PIKfyve antibody.


D , E Co - staining of PIKfyve and caspase - 5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h ( D ) . Anti - PIKfyve and anti - caspase - 5 antibodies were used to detect endogenous PIKfyve and caspase - 5 respectively . Scale bar : 20 μm . Colocalized PIKfyve and caspase - 5 were quantified by ImageJ software ( E ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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

D, E Co-staining of PIKfyve and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (D). Anti-PIKfyve and anti-caspase-5 antibodies were used to detect endogenous PIKfyve and caspase-5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (E) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


F Caco - 2 cells were stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for 24h . Proteins of lysates extracted from Caco - 2 cells were subjected to immunoblots with the indicated antibodies .

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

F Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for 24h. Proteins of lysates extracted from Caco-2 cells were subjected to immunoblots with the indicated antibodies.


G , H Co - staining of PIKfyve and Rab5 in WT and SNX10 KO Caco - 2 cells treated with or without OMVs ( 100 μg / mL ) for 24 h ( G ) . Anti - PIKfyve and anti - Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively . Scale bar : 20 μm . Colocalized PIKfyve and caspase - 5 were quantified by ImageJ software ( H ) ( n = 3 independent experiments , n = 18 fields analysed ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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

G, H Co-staining of PIKfyve and Rab5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/mL) for 24 h (G). Anti-PIKfyve and anti-Rab5 antibodies were used to detect endogenous PIKfyve and Rab5 respectively. Scale bar: 20 μm. Colocalized PIKfyve and caspase-5 were quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analysed). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


I LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100 μg / mL ) with or without apilimod ( 30 nM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . J Caco - 2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs ( 100 μg / mL ) for the indicated time . LPS levels in the cytosolic and residual fractions of Caco - 2 cells were detected by LAL assay ( n = 6 independent experiments ) . K LPS levels in the cytosolic and residual fractions of Caco - 2 cells stimulated by OMVs ( 100μg / mL ) with or without bafetinib ( 1μM ) for the indicated time were detected by LAL assay ( n = 6 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * * * p < 0 . 001 .

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

I LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by LAL assay (n = 6 independent experiments). J Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time. LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by LAL assay (n = 6 independent experiments). K LPS levels in the cytosolic and residual fractions of Caco-2 cells stimulated by OMVs (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . B , C Length of colons from WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice was measured ( B ) and analysed ( C ) at the 16th week . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). B, C Length of colons from WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice was measured (B) and analysed (C) at the 16th week. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . E , F Representative H & E images of colon tissues of WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice at the 16th week were shown ( E ) and analyzed ( F ) . Scale bar : 100 μm . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). E, F Representative H&E images of colon tissues of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were shown (E) and analyzed (F). Scale bar: 100 μm. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . G The content of TNF - α , IL - 6 , IL - 12 / IL - 23 p40 and IL - 17A in the serum of WT , SNX10 cKO , IL - 10 KO and SNX10 cKO IL - 10 KO mice at the 16th week were measured by ELISA . Data information : data are means ± SD . One - way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). G The content of TNF-α, IL-6, IL-12/IL-23 p40 and IL-17A in the serum of WT, SNX10 cKO, IL-10 KO and SNX10 cKO IL-10 KO mice at the 16th week were measured by ELISA. Data information: data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001.


SNX10 conditional knockout ( SNX10 cKO ) mice with or without IL - 10 KO background and littermate wild type ( WT ) mice with or without IL - 10 KO background were compared ( n = 6 animals , each group ) . J Protein expression of E - cadherin , p - Lyn and Lyn in colon epithelial tissues was determined by immunoblots .

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

SNX10 conditional knockout (SNX10 cKO) mice with or without IL-10 KO background and littermate wild type (WT) mice with or without IL-10 KO background were compared (n = 6 animals, each group). J Protein expression of E-cadherin, p-Lyn and Lyn in colon epithelial tissues was determined by immunoblots.


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

(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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 .

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 B-CONTROLLED_VAR 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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O 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 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 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 .

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

(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 .

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

(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 ) .

O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 .

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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 ) Still frames from time - lapse recordings of an embryo expressing a myosin - II probe ( Sqh : : GFP ) . Embryos were mounted vertically to image the transverse cross - section using two - photon microscopy . At the onset of gastrulation , myosin - II localized to ring structures representing the leading edge of the cellularization front ( lower arrow ) . During ventral furrow formation ( torques open rectangle ) , myosin - II accumulated at the apical side ( upper arrow ) of the cells that invaginate and the basal pool was progressively depleted . Scale bar , 40 µm

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(C) Still frames from time-lapse recordings of an embryo expressing a myosin-II probe (Sqh::GFP). Embryos were mounted vertically to image the transverse cross-section using two-photon microscopy. At the onset of gastrulation, myosin-II localized to ring structures representing the leading edge of the cellularization front (lower arrow). During ventral furrow formation (torques open rectangle), myosin-II accumulated at the apical side (upper arrow) of the cells that invaginate and the basal pool was progressively depleted. Scale bar, 40 µm


( D ) Quantification of basal myosin - II levels ( N = 3 embryos ) in ectodermal and mesodermal cells . In the mesoderm , basal myosin - II levels decreasd 5 - fold over the course of internalization , while in the ectoderm only ~ 3 - fold . Additionally , myosin - II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit ( 3rd degree with r2Ectoderm : 0 . 98 and r2Mesoderm : 0 . 87 ) . Note the logarithmic y - axis

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(D) Quantification of basal myosin-II levels (N=3 embryos) in ectodermal and mesodermal cells. In the mesoderm, basal myosin-II levels decreasd 5-fold over the course of internalization, while in the ectoderm only ~3-fold. Additionally, myosin-II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit (3rd degree with r2Ectoderm: 0.98 and r2Mesoderm: 0.87). Note the logarithmic y-axis


( E - H ) Embryos at the end of cellularization co - expressing RhoGEF2 - CRY2 ; CIBN : : pmGFP , and Sqh : : mCherry were mounted with the dorsal epithelium facing the objective and a region of interest ( red dashed line ) was defined to specifically illuminate the cell base using two - photon microscopy . A representative embryo ( N = 3 ) showing basal myosin - II from top view ( sum - of - slice projection of 6 focal planes ) at the initial stage of the experiment ( E ) and 6 min after activation ( F ) . Z - projection of the same embryo showing the cross section of the activated region ( AR ) ( G ) and non - activated region ( NR ) ( H ) with the superimposed plasma membrane signal ( CIBN : : GFPpm in magenta ) recorded immediately after the final Sqh : : mCherry acquisition ( displayed in white ) . The contrast has been adjusted to better visualize the distribution of myosin - II along the apicobasal axis in the respective area of the tissue . Scale bars , 20 µm

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(E-H) Embryos at the end of cellularization co-expressing RhoGEF2-CRY2; CIBN::pmGFP, and Sqh::mCherry were mounted with the dorsal epithelium facing the objective and a region of interest (red dashed line) was defined to specifically illuminate the cell base using two-photon microscopy. A representative embryo (N=3) showing basal myosin-II from top view (sum-of-slice projection of 6 focal planes) at the initial stage of the experiment (E) and 6 min after activation (F). Z-projection of the same embryo showing the cross section of the activated region (AR) (G) and non-activated region (NR) (H) with the superimposed plasma membrane signal (CIBN::GFPpm in magenta) recorded immediately after the final Sqh::mCherry acquisition (displayed in white). The contrast has been adjusted to better visualize the distribution of myosin-II along the apicobasal axis in the respective area of the tissue. Scale bars, 20 µm


( I ) Quantification of myosin - II levels ( N = 3 ) within the region of activation ( AR , red ) relative to the non - activated region ( NR , green ) over the course of 6 min . The y - axis represents the fold - change of myosin - II signal intensity in the activated region versus non - activated region . A polynomial function ( 2nd degree , r2AR = 0 . 88 ) was fitted to the data ( straight line ) indicating the level of saturation that is reached at about 1 . 6 - fold change of myosin - II in the activated area after 6 min

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(I) Quantification of myosin-II levels (N=3) within the region of activation (AR, red) relative to the non-activated region (NR, green) over the course of 6 min. The y-axis represents the fold-change of myosin-II signal intensity in the activated region versus non-activated region. A polynomial function (2nd degree, r2AR = 0.88) was fitted to the data (straight line) indicating the level of saturation that is reached at about 1.6-fold change of myosin-II in the activated area after 6 min


( J Analysis of the extent of signal spreading in the z - dimension ( apico - basla ) of basal myosin - II . Myosin - II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak . The width of the Gaussian fit was used as a parameter to estimate myosin - II signal spreading in the z - dimension , which was limited to 4 µm ( N = 3 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data

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(J Analysis of the extent of signal spreading in the z-dimension (apico-basla) of basal myosin-II. Myosin-II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak. The width of the Gaussian fit was used as a parameter to estimate myosin-II signal spreading in the z-dimension, which was limited to 4 µm (N=3). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point


( K ) Quantification of the diameter of basal actomyosin rings in the non - activated region ( NR , green ) and activated region ( AR , red ) showing increased speed ( 4 . 8 - fold change ) and extent of ring constrictions in the photo - activated region ( n = > 41 cells ) . The ring diameter was normalized to the mean value of the first time point . The constriction speed was estimated by the slope of a fitted linear function to the data . ( L ) Light activation of the cell base resulted in a compaction of the tissue in the activated ( AR ) compared to the non - activated region ( NR ) . Actomyosin rings were counted and normalized to the analyzed area to score ring density , which increased 1 . 35 times in the activated region after 5 min of photo - activation , showing that cells were still interconnected at the base and that photo - activation did not affect tissue integrity ( N = 3 embryos ; nNR : 431 , nAR : 302 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point

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

(K) Quantification of the diameter of basal actomyosin rings in the non-activated region (NR, green) and activated region (AR, red) showing increased speed (4.8-fold change) and extent of ring constrictions in the photo-activated region (n= >41 cells). The ring diameter was normalized to the mean value of the first time point. The constriction speed was estimated by the slope of a fitted linear function to the data. (L) Light activation of the cell base resulted in a compaction of the tissue in the activated (AR) compared to the non-activated region (NR). Actomyosin rings were counted and normalized to the analyzed area to score ring density, which increased 1.35 times in the activated region after 5 min of photo-activation, showing that cells were still interconnected at the base and that photo-activation did not affect tissue integrity (N = 3 embryos; nNR: 431, nAR: 302). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II levels at the cell base . Embryos co - expressing the optogenetic module and the myosin - II probe Sqh : : mCh were mounted with the ventral tissue facing the objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at the cell base in the anterior half of the embryo , and whole - cell myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm . ( A ) Top view showing apical myosin - II 10 min after the initial activation . While cells in the non - activated region have internalized and disappeared from the apical focal planes ( right ) , cells in the activated region failed to internalize despite showing myosin - II accumulation at the apical surface ( left ) . Red and Blue dotted lines indicate the location of the cross - section panels displayed in ( C ) and ( D ) . ( B ) Quantification of apical myosin - II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin - II accumulation as the average apical myosin - II level per cell did not significantly differ between the non - activated ( NR ) and activated region ( AR ) . The intensity values were normalized using the median value of the combined cell population in the NR and AR . ( N = 3 embryos ; nNR : 94 , nAR : 78 ) . Significances ( by two - sample t - Test ) : n . s . indicates no statistically significant differences . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point and the ' + ' symbol indicates an outlier . ( C

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

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm. (A) Top view showing apical myosin-II 10 min after the initial activation. While cells in the non-activated region have internalized and disappeared from the apical focal planes (right), cells in the activated region failed to internalize despite showing myosin-II accumulation at the apical surface (left). Red and Blue dotted lines indicate the location of the cross-section panels displayed in (C) and (D). (B) Quantification of apical myosin-II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin-II accumulation as the average apical myosin-II level per cell did not significantly differ between the non-activated (NR) and activated region (AR). The intensity values were normalized using the median value of the combined cell population in the NR and AR. (N = 3 embryos; nNR: 94, nAR: 78). Significances (by two-sample t-Test): n.s. indicates no statistically significant differences. In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point and the '+' symbol indicates an outlier. (C


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II levels at the cell base . Embryos co - expressing the optogenetic module and the myosin - II probe Sqh : : mCh were mounted with the ventral tissue facing the objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at the cell base in the anterior half of the embryo , and whole - cell myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm ( C , D ) z - projections ( transverse cross section ) of myosin - II time - lapse recordings of the non - activated ( C ) and activated region ( D ) . While cells in the non - activated region internalized , resulting in a complete closure of the furrow ( C ) , stabilization of basal myosin - II levels prevented invagination ( D ) . Scale bars , 20

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Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm (C, D) z-projections (transverse cross section) of myosin-II time-lapse recordings of the non-activated (C) and activated region (D). While cells in the non-activated region internalized, resulting in a complete closure of the furrow (C), stabilization of basal myosin-II levels prevented invagination (D). Scale bars, 20 µm


( A - C ) Embryos co - expressing the optogenetic module and Sqh : : mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo - activation of the cell base in the anterior ( left ) half of the embryo and confocal recording of the whole embryo . The experiment was started at the end of cellularization when initial accumulation of apical myosin - II was detected and terminated at different time points to record the plasma membrane signal ( CIBN : : GFP - pm ) in the whole embryo using two - photon illumination . Still frames visualizing the plasma membrane ( CIBN : : GFP - pm ) at a sub - apical plane at the initial time point of the experiment ( A ) , 3 min ( B ) and 10 min ( C ) after initial photo - activation .

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

(A-C) Embryos co-expressing the optogenetic module and Sqh::mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo-activation of the cell base in the anterior (left) half of the embryo and confocal recording of the whole embryo. The experiment was started at the end of cellularization when initial accumulation of apical myosin-II was detected and terminated at different time points to record the plasma membrane signal (CIBN::GFP-pm) in the whole embryo using two-photon illumination. Still frames visualizing the plasma membrane (CIBN::GFP-pm) at a sub-apical plane at the initial time point of the experiment (A), 3 min (B) and 10 min (C) after initial photo-activation.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( A ) Still frames of the time lapse membrane ( GAP43 : : mCherry ) recording are shown at the initial time point ( top ) , after 6 . 5 min ( middle ) , and after 13 min ( bottom ) of photo - activation . Cells in the non - activated region ( green ) underwent normal apical constriction . In the activated region , two populations of cells were observed , one that constricted ( purple ) and one that did not constrict ( red ) . Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells . Cells that constricted less were considered to be defective in apical constriction and are referred to as non - constricting . Scale bar 20 µm .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (A) Still frames of the time lapse membrane (GAP43::mCherry) recording are shown at the initial time point (top), after 6.5 min (middle), and after 13 min (bottom) of photo-activation. Cells in the non-activated region (green) underwent normal apical constriction. In the activated region, two populations of cells were observed, one that constricted (purple) and one that did not constrict (red). Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells. Cells that constricted less were considered to be defective in apical constriction and are referred to as non-constricting. Scale bar 20 µm.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( B ) All cells show pulsatile behavior . Graphs illustrate the area of three representative cells in each category ( non - activated region : top ; activated region with constricting cells : middle ; activated region with non - constricting cells : bottom ) over the course of one experiment . Yellow circles indicate the position of automatically identified local maxima used for further analyses .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (B) All cells show pulsatile behavior. Graphs illustrate the area of three representative cells in each category (non-activated region: top; activated region with constricting cells: middle; activated region with non-constricting cells: bottom) over the course of one experiment. Yellow circles indicate the position of automatically identified local maxima used for further analyses.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( C , D ) ( C ) Diagram showing the extent of ratchet - like constrictions . The mean difference in apical area between adjacent peaks ( relaxed cell surface ) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse . Cells in the non - activated region ( NR ) constricted on average more than 5 µm2 , which is characteristic for ratchet - like constriction . Non - constricting cells in the activated region ( AR , non - con . ) displayed a mean difference in apical area per pulse of about 0 , reflecting non - ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size . Constricting cells in the activated region ( AR , con . ) show an intermediate behavior with less efficient constrictions than in the NR . Data points are significantly different as determined by ANOVA ( F ( 2 , 539 ) = 72 . 04 , p = 1 . 9e - 28 ) and post - hoc Tukey test . ( D ) The pulsation period ( T ) . All cells pulse with a period of about 90 s . There were no statistically significant differences between group means as determined by one - way ANOVA ( F ( 2 , 540 ) = 1 . 438 , p = 0 . 24 ) . ( C and D ) Red dots indicate the mean values and the error bars represent the standard deviation . More than 500 pulses ( nNR = 119 , nAR , non - con . = 128 , nAR , con = 295 ) of more than 100 cells were analyzed . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (C, D) (C) Diagram showing the extent of ratchet-like constrictions. The mean difference in apical area between adjacent peaks (relaxed cell surface) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse. Cells in the non-activated region (NR) constricted on average more than 5 µm2, which is characteristic for ratchet-like constriction. Non-constricting cells in the activated region (AR, non-con.) displayed a mean difference in apical area per pulse of about 0, reflecting non-ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size. Constricting cells in the activated region (AR, con.) show an intermediate behavior with less efficient constrictions than in the NR. Data points are significantly different as determined by ANOVA (F(2,539)=72.04, p=1.9e-28) and post-hoc Tukey test. (D) The pulsation period (T). All cells pulse with a period of about 90 s. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,540)=1.438, p=0.24). (C and D) Red dots indicate the mean values and the error bars represent the standard deviation. More than 500 pulses (nNR = 119, nAR, non-con. = 128, nAR, con = 295) of more than 100 cells were analyzed. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( E ) Diagram showing the prevalence of cells in the photo - activated region that constricted ( con . ) and that did not constrict ( Non - con . ) . More than 4 / 5 of the cells did not constrict to half of the initial area ( N = 5 embryos ; n : 407 cells ; Cohen ' s D : d = 10 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (E) Diagram showing the prevalence of cells in the photo-activated region that constricted (con.) and that did not constrict (Non-con.). More than 4/5 of the cells did not constrict to half of the initial area (N = 5 embryos; n: 407 cells; Cohen's D: d = 10). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( F , G ) Graphs showing the average apical area ( F ) and anisotropy ( G ) of cells in the non - activated region ( green ) , in the activated region that constricted ( purple ) and that did not constrict ( red ) . The straight line indicates the mean value at a given time - point , the semi - transparent colored area indicated the respective standard deviation . Cells in the non - activated region constricted efficiently and progressively acquired anisotropy . Constricting cells in the activated region constricted slower in an isotropic manner . Non - constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy ( nNR = 34 , nAR , non - con . = 20 , nAR , con = 59 ) .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (F,G) Graphs showing the average apical area (F) and anisotropy (G) of cells in the non-activated region (green), in the activated region that constricted (purple) and that did not constrict (red). The straight line indicates the mean value at a given time-point, the semi-transparent colored area indicated the respective standard deviation. Cells in the non-activated region constricted efficiently and progressively acquired anisotropy. Constricting cells in the activated region constricted slower in an isotropic manner. Non-constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy (nNR = 34, nAR, non-con. = 20, nAR, con = 59).


( C - E ) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2 - CRY2 : : mCherry were imaged during late cellularization . ( C ) z - projections of confocal image stacks show the localization of the respective GFP - tagged optogenetic anchor . Please note that the construct PatJ - CIBN ( indicated by asterisk ) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2 - CRY2 : : mCh . All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation . Scale bars , 20 μm . ( D ) A region of interest ( right side of the dashed line ) in a 10 µm - sized stack was photo - activated using two - photon illumination for 30 s before acquiring the RhoGEF2 - CRY2 : : mCherry signal using confocal microscopy . All anchor proteins were able to recruit RhoGEF2 - CRY2 : : mCherry specifically to the activated region . Scale bars , 10 μm . ( E ) Although all basal anchors were effective at recruiting RhoGEF2 - CRY2 : : mCherry to the cell base , only PatJ - CIBN : : GFP - pm resulted in effective myosin - II recruitment and actomyosin ring constriction

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

(C-E) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2-CRY2::mCherry were imaged during late cellularization. (C) z-projections of confocal image stacks show the localization of the respective GFP-tagged optogenetic anchor. Please note that the construct PatJ-CIBN (indicated by asterisk) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2-CRY2::mCh. All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation. Scale bars, 20 μm. (D) A region of interest (right side of the dashed line) in a 10 µm-sized stack was photo-activated using two-photon illumination for 30 s before acquiring the RhoGEF2-CRY2::mCherry signal using confocal microscopy. All anchor proteins were able to recruit RhoGEF2-CRY2::mCherry specifically to the activated region. Scale bars, 10 μm. (E) Although all basal anchors were effective at recruiting RhoGEF2-CRY2::mCherry to the cell base, only PatJ-CIBN::GFP-pm resulted in effective myosin-II recruitment and actomyosin ring constriction


( A ) Localization of the basal - specific optogenetic anchor CIBN - PatJ : : GFP - pm during ventral furrow formation . Embryos expressing CIBN - PatJ : : GFP - pm were imaged using two - photon excitation during ventral furrow formation . Transverse cross section ( z - projection ) at a stage when cells were already apically constricted and elongated , and the ventral tissue already internalized .

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

(A) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized.


Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY2 and Sqh : : mCherry ( B , C were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation . ( B ) myosin - II distribution in a cross section ( z - projection ) 3 min after photo - activation . myosin - II was locally recruited to the base as indicated by the red arrowheads in the activated region ( red line ) . ( C ) Top view ( sum of slice of 4 µm - sized image stack ) showing basal myosin - II recruitment in the photo - activated region ( red dashed square ) Data information : Scale bars , 20

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

Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square) Data information: Scale bars, 20 µm