( A ) HeLa cells were transfected with vector or ORF6 - Flag for 12 h and treated with 200 μM OA for another 12 h , the medium was removed and replaced with fresh complete culture containing 1 μM DGAT1 inhibitor and 2 μM DGAT2 inhibitor for 24 h . Meanwhile , 50 μM ATGL inhibitor or 100 μM Chloroquine ( CQ ) was added and allowed to incubate for 24 h or 4 h to block lipolysis or lipophagy , respectively . Cells were fixed and stained with anti - Flag ( red ) . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression .

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

(A) HeLa cells were transfected with vector or ORF6-Flag for 12 h and treated with 200 μM OA for another 12 h, the medium was removed and replaced with fresh complete culture containing 1 μM DGAT1 inhibitor and 2 μM DGAT2 inhibitor for 24 h. Meanwhile, 50 μM ATGL inhibitor or 100 μM Chloroquine (CQ) was added and allowed to incubate for 24 h or 4 h to block lipolysis or lipophagy, respectively. Cells were fixed and stained with anti-Flag (red). LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6 and yellow ROIs indicate the cells without ORF6 expression.


( C ) HEK293T cells were transfected with ORF6 - Flag . Protein interactions were detected by immunoprecipitation with anti - Flag beads and immunoblotting analysis with ATGL , HSL , and CGI58 antibodies .

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(C) HEK293T cells were transfected with ORF6-Flag. Protein interactions were detected by immunoprecipitation with anti-Flag beads and immunoblotting analysis with ATGL, HSL, and CGI58 antibodies.


( D ) Purified GST - ORF6 or indicated mutants were incubated with purified His tagged ATGL , and analyzed the interactions by GST pull - down .

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(D) Purified GST-ORF6 or indicated mutants were incubated with purified His tagged ATGL, and analyzed the interactions by GST pull-down.


( H and I ) The effect of ORF6 - Flag or the ΔNTD mutant on the interactions of Flag - ATGL with GFP - Plin2 or GFP - UBXD8 was analyzed by immunoprecipitation .

(H and I) The effect of ORF6-Flag or the ΔNTD mutant on the interactions of Flag-ATGL with GFP-Plin2 or GFP-UBXD8 was analyzed by immunoprecipitation.


( K and L ) Vero - E6 cells were transfected with Flag - ATGL and GFP - Plin2 or GFP - UBXD8 for 24 h and infected or non - infected with SARS - CoV - 2 for 24 h . Protein interactions were analyzed by immunoprecipitation with anti - GFP beads and immunoblotting analysis .

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(K and L) Vero-E6 cells were transfected with Flag-ATGL and GFP-Plin2 or GFP-UBXD8 for 24 h and infected or non-infected with SARS-CoV-2 for 24 h. Protein interactions were analyzed by immunoprecipitation with anti-GFP beads and immunoblotting analysis.


( M ) SARS - CoV - 2 - infected Vero - E6 cells were treated with 50 μM ATGL inhibitor for 24 h . Cell viability was analyzed . Cell lysates were analyzed via WB . Supernatants were determined by plaque assays . Virus titer values represent mean ± SD for three independent replicates . Two - tailed Unpaired Student ' s t - test , * p < 0 . 05 , ns means no significance . Error bars represent as the mean ± SD .

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(M) SARS-CoV-2-infected Vero-E6 cells were treated with 50 μM ATGL inhibitor for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, *p < 0.05, ns means no significance. Error bars represent as the mean ± SD.


( C ) Representative transmission electron micrograph of ORF6 - Flag stable expressing or vector HeLa cells upon OA treatment . Red arrows mark the contact sites between LDs and mitochondria . M , mitochondria . LD , lipid droplets . Scale bar represents 1 μm . ( D ) The number of mitochondria - LD contacts per cell was counted from 20 cells . The number of mitochondrial engaged in mitochondrial - LD contact per LD in ( C ) was counted from 20 LDs . Two independent experiments . Two - tailed Unpaired Student ' s t - test , * * * * p < 0 . 0001 . Error bars represent the mean ± SD .

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(C) Representative transmission electron micrograph of ORF6-Flag stable expressing or vector HeLa cells upon OA treatment. Red arrows mark the contact sites between LDs and mitochondria. M, mitochondria. LD, lipid droplets. Scale bar represents 1 μm. (D) The number of mitochondria-LD contacts per cell was counted from 20 cells. The number of mitochondrial engaged in mitochondrial-LD contact per LD in (C) was counted from 20 LDs. Two independent experiments. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.


( E ) Representative transmission electron micrograph of non - infected or SARS - CoV - 2 infected Vero - E6 cells . Red arrows mark the mitochondria - LD contact . LD , lipid droplets . M , mitochondria . Scale bar represents 1 μm . Quantification of number of mitochondria engaged in mitochondria - LD contact per LD . 20 LDs of two independent experiments were counted . Two - tailed Unpaired Student ' s t - test , * * * * p < 0 . 0001 . Error bars represent the mean ± SD .

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(E) Representative transmission electron micrograph of non-infected or SARS-CoV-2 infected Vero-E6 cells. Red arrows mark the mitochondria-LD contact. LD, lipid droplets. M, mitochondria. Scale bar represents 1 μm. Quantification of number of mitochondria engaged in mitochondria-LD contact per LD. 20 LDs of two independent experiments were counted. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.


( F and G ) SARS - CoV - 2 - infected Vero - E6 cells were fixed and stained with anti - Tom20 ( cyan ) . Tom20 represents mitochondria marker . LDs were labeled with LipidTOX Deep Red ( red ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . Colocalization of LDs and mitochondria ( Pearson ' s Coefficient ) , n = 20 cells , two independent experiments . Two - tailed Unpaired Student ' s t - test , * * p < 0 . 01 . Error bars represent the mean ± SD .

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(F and G) SARS-CoV-2-infected Vero-E6 cells were fixed and stained with anti-Tom20 (cyan). Tom20 represents mitochondria marker. LDs were labeled with LipidTOX Deep Red (red). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. Colocalization of LDs and mitochondria (Pearson's Coefficient), n=20 cells, two independent experiments. Two-tailed Unpaired Student's t-test, **p < 0.01. Error bars represent the mean ± SD.


( H ) Cos7 cells expressing ORF6 - Flag and vector were treated with 200 μM OA for 12 h , and then were fixed and stained with anti - Flag ( white ) and anti - Tom20 ( red ) . Tom20 represents mitochondria marker . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm .

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(H) Cos7 cells expressing ORF6-Flag and vector were treated with 200 μM OA for 12 h, and then were fixed and stained with anti-Flag (white) and anti-Tom20 (red). Tom20 represents mitochondria marker. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.


( L ) Seahorse FAO assays to examine the oxidation of endogenous FAs in vector , or ORF6 - Flag or ORF64Q - Flag over - expressed HeLa cells . Three biological replicates were performed with similar results . Error bars represent the mean ± SD .

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(L) Seahorse FAO assays to examine the oxidation of endogenous FAs in vector, or ORF6-Flag or ORF64Q-Flag over-expressed HeLa cells. Three biological replicates were performed with similar results. Error bars represent the mean ± SD.


( M ) The concentration of ATP in cells was analyzed according to manufacturer ' s instructions . Error bars , mean ± SD of four independent experiments . Two - tailed Unpaired Student ' s t - test , * * * p < 0 . 001 , ns means no significance .

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(M) The concentration of ATP in cells was analyzed according to manufacturer's instructions. Error bars, mean ± SD of four independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ns means no significance.


( N ) Vero - E6 cells stable expressing GFP or GFP - Plin5 or GFP - Plin5Δ443 - 463 were infected with SARS - CoV - 2 for 24 h . Cell viability was analyzed . Cell lysates were analyzed via WB . Supernatants were determined by plaque assays . Virus titer values represent mean ± SD for three independent replicates . Two - tailed Unpaired Student ' s t - test , * * p < 0 . 01 , ns means no significance . Error bars represent the mean ± SD .

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(N) Vero-E6 cells stable expressing GFP or GFP-Plin5 or GFP-Plin5Δ443-463 were infected with SARS-CoV-2 for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, **p < 0.01, ns means no significance. Error bars represent the mean ± SD.


( C ) HeLa cells were transfected with negative , or si - MTX1 , or si - MTX2 , or si - SAMM50 for 24 h and further transfected with ORF6 - GFP 24 h . Subcellular fractions were isolated from cells . VDAC1 and ATP5A1 represent mitochondria markers , Tubulin represents cytosolic marker .

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(C) HeLa cells were transfected with negative, or si-MTX1, or si-MTX2, or si-SAMM50 for 24 h and further transfected with ORF6-GFP 24 h. Subcellular fractions were isolated from cells. VDAC1 and ATP5A1 represent mitochondria markers, Tubulin represents cytosolic marker.


( D ) HeLa cells were transfected with negative or si - MTX1 and si - MTX2 for 24 h and further transfected with ORF6 - GFP 24 h . Subcellular fractions were isolated from cells . VDAC1 and ATP5A1 represent mitochondria markers , Tubulin represents cytosolic marker .

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(D) HeLa cells were transfected with negative or si-MTX1 and si-MTX2 for 24 h and further transfected with ORF6-GFP 24 h. Subcellular fractions were isolated from cells. VDAC1 and ATP5A1 represent mitochondria markers, Tubulin represents cytosolic marker.


( F ) Cos7 cells expressing ORF6 - Flag were transfected with negative or si - MTX1 / si - MTX2 / si - SAMM50 ( Triple KD ) for 36 h and treated with 200 μM OA for another 12 h . Cells were fixed and stained with anti - Flag ( white ) and anti - Tom20 ( red ) . Tom20 represents mitochondria marker . LDs were labeled with BODIPY - 493 / 503 ( green ) . Cells were imaged by confocal microscopy . Scale bar represents 10 μm . ( G ) Quantification of average number of LD - mitochondria contacts per LD from ( F ) . 25 cells ( Negative ) , 50 cells ( Triple KD ) from three independent experiments were calculated . Two - tailed Unpaired Student ' s t - test , * * * * p < 0 . 0001 . Error bars represent the mean ± SD .

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(F) Cos7 cells expressing ORF6-Flag were transfected with negative or si-MTX1/si-MTX2/si-SAMM50 (Triple KD) for 36 h and treated with 200 μM OA for another 12 h. Cells were fixed and stained with anti-Flag (white) and anti-Tom20 (red). Tom20 represents mitochondria marker. LDs were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (G) Quantification of average number of LD-mitochondria contacts per LD from (F). 25 cells (Negative), 50 cells (Triple KD) from three independent experiments were calculated. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.


( H ) Vero - E6 cells were transfected with indicated siRNAs for 24 h and infected with SARS - CoV - 2 for 24 h . Cell viability was analyzed . Cell lysates were analyzed via WB . Supernatants were determined by plaque assays . Virus titer values represent mean ± SD for three independent replicates . Two - tailed Unpaired Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , ns means no significance . Error bars represent the mean ± SD .

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(H) Vero-E6 cells were transfected with indicated siRNAs for 24 h and infected with SARS-CoV-2 for 24 h. Cell viability was analyzed. Cell lysates were analyzed via WB. Supernatants were determined by plaque assays. Virus titer values represent mean ± SD for three independent replicates. Two-tailed Unpaired Student's t-test, *p < 0.05, ***p < 0.001, ns means no significance. Error bars represent the mean ± SD.


( a ) Atg7 + / − mice were generated as described in the Methods . PCR was done employing genomic DNA and primers flanking the floxed Atg7 regions . Arrows indicate wild - type Atg7 band in Atg7 + / + mice , increased size of PCR band due to inserted flox sequence in Atg7F / + mice and decreased size of PCR band due to Cre - mediated deletion in Atg7 + / − mice , respectively .

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(a) Atg7+/− mice were generated as described in the Methods. PCR was done employing genomic DNA and primers flanking the floxed Atg7 regions. Arrows indicate wild-type Atg7 band in Atg7+/+ mice, increased size of PCR band due to inserted flox sequence in Atg7F/+ mice and decreased size of PCR band due to Cre-mediated deletion in Atg7+/− mice, respectively.


( b ) RT - PCR ( left ) and real - time RT - PCR ( right ) were performed using total RNA prepared from tissues of Atg7 + / − or control Atg7 + / + mice , and primers specific for Atg7 or β - actin sequence . Fold changes of the RT - PCR band intensities are shown ( left ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

(b) RT-PCR (left) and real-time RT-PCR (right) were performed using total RNA prepared from tissues of Atg7+/− or control Atg7+/+mice, and primers specific for Atg7 or β-actin sequence. Fold changes of the RT-PCR band intensities are shown (left). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3.


( c ) Tissue lysate from fasted 12 - week - old Atg7 + / − and control Atg7 + / + mice was subjected to immunoblot analysis using anti - LC3 or - p62 antibody . Fold changes of the immunoblot band intensities are shown ( middle and right ) . * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

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(c) Tissue lysate from fasted 12-week-old Atg7+/− and control Atg7+/+mice was subjected to immunoblot analysis using anti-LC3 or -p62 antibody. Fold changes of the immunoblot band intensities are shown (middle and right). **P0.01, ***P0.001; Student's t-test, n=3.


( d ) Cell lysate of Atg7 + / + , Atg7 + / − and Atg7 − / − MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin ( Rap ) or control solvent ( dimethyl sulfoxide , DMSO ) treatment for 3 h . Fold changes of the immunoblot band intensities are shown ( middle and right ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , n = 3 .

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(d) Cell lysate of Atg7+/+, Atg7+/− and Atg7−/− MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin (Rap) or control solvent (dimethyl sulfoxide, DMSO) treatment for 3 h. Fold changes of the immunoblot band intensities are shown (middle and right). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3.


( e ) Sections of the liver and muscle tissues from fasted 12 - week - old GFP - LC3 + - Atg7 + / + and GFP - LC3 + - Atg7 + / − mice were subjected to fluorescent microscopy to examine GFP - LC3 puncta ( left ) . The number of GFP - LC3 puncta was counted ( right ) . Scale bars , 10 μm . * P0 . 05 ; Student ' s t - test , n = 5 .

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(e) Sections of the liver and muscle tissues from fasted 12-week-old GFP-LC3+-Atg7+/+ and GFP-LC3+-Atg7+/− mice were subjected to fluorescent microscopy to examine GFP-LC3 puncta (left). The number of GFP-LC3 puncta was counted (right). Scale bars, 10 μm. *P0.05; Student's t-test, n=5.


( f ) Tissue lysates were prepared from the liver of fasted Atg7 + / + and Atg7 + / − mice 4 h after leupeptin administration , and immunoblotting was done . Fold changes of the immunoblot band intensities are shown ( right ) . * P0 . 05 ; Student ' s t - test , n = 3 .

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(f) Tissue lysates were prepared from the liver of fasted Atg7+/+ and Atg7+/− mice 4 h after leupeptin administration, and immunoblotting was done. Fold changes of the immunoblot band intensities are shown (right). *P0.05; Student's t-test, n=3.


( a ) Non - fasting blood glucose level was monitored weekly in Atg7 + / + , Atg7 + / − , Atg7 + / + - ob / ob and Atg7 + / − - ob / ob mice . * * * P0 . 001 ; two - way ANOVA .

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(a) Non-fasting blood glucose level was monitored weekly in Atg7+/+, Atg7+/−, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice. ***P0.001; two-way ANOVA.


( b ) IPGTT was done by glucose injection at 16 weeks of age as described in the Methods ( left ) , and AUC calculated ( right ) . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

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(b) IPGTT was done by glucose injection at 16 weeks of age as described in the Methods (left), and AUC calculated (right). *P0.05, **P0.01, ***P0.001; Student's t-test (left) and one-way ANOVA (right).


( c ) HOMA - IR index was calculated as described in Methods . * P0 . 05 ; one - way ANOVA .

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(c) HOMA-IR index was calculated as described in Methods. *P0.05; one-way ANOVA.


( d ) ITT was performed by insulin injection at 16 weeks of age as described in the Methods . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test .

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(d) ITT was performed by insulin injection at 16 weeks of age as described in the Methods. *P0.05, **P0.01, ***P0.001; Student's t-test.


( e ) Tissue lysates from Atg7 + / + - ob / w , Atg7 + / − - ob / w , Atg7 + / + - ob / ob and Atg7 + / − - ob / ob mice were prepared before and 7 min after injection of 5 U kg − 1 regular insulin ( Ins ) into the tail vein , and subjected to immunoblot analysis using antibodies specific for phospho - Akt S473 and total Akt . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

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(e) Tissue lysates from Atg7+/+-ob/w, Atg7+/−-ob/w, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice were prepared before and 7 min after injection of 5 U kg−1 regular insulin (Ins) into the tail vein, and subjected to immunoblot analysis using antibodies specific for phospho-Akt S473 and total Akt. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( a ) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK , phospho - JNK , total IRS - 1 and phospho - IRS - 1 S307 .

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(a) Tissue lysates from each type of mouse were subjected to immunoblot analysis using antibodies specific for total JNK, phospho-JNK, total IRS-1 and phospho-IRS-1 S307.


( b ) Liver sections from each type of mouse were subjected to immunohistochemistry using anti - nitrotyrosine antibody . Scale bars , 100 μm .

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(b) Liver sections from each type of mouse were subjected to immunohistochemistry using anti-nitrotyrosine antibody. Scale bars, 100 μm.


( c ) Tissue lysates were prepared from 18 - week - old mice of each genotype , and subjected to immunoblot analysis using a kit detecting carbonylated proteins .

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(c) Tissue lysates were prepared from 18-week-old mice of each genotype, and subjected to immunoblot analysis using a kit detecting carbonylated proteins.


( d ) Immunoblot analysis was performed using anti - phospho - p53 and - p53 antibodies . p21 and β - actin mRNA expression was evaluated by RT - PCR . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

(d) Immunoblot analysis was performed using anti-phospho-p53 and -p53 antibodies. p21 and β-actin mRNA expression was evaluated by RT-PCR. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( a ) GFP - LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state , and the numbers of GFP - LC3 puncta counted . Scale bars , 20 μm . * P0 . 05 , * * P0 . 01 ; Student ' s t - test .

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(a) GFP-LC3 puncta were visualized by fluorescent microscopy of tissue from each type of mouse in fed state, and the numbers of GFP-LC3 puncta counted. Scale bars, 20 μm. *P0.05, **P0.01; Student's t-test.


( b ) Tissues of ob / w and ob / ob mice were subjected to EM , and the number of autophagosomes was counted . * * * P0 . 001 ; Student ' s t - test .

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(b) Tissues of ob/w and ob/ob mice were subjected to EM, and the number of autophagosomes was counted. ***P0.001; Student's t-test.


( c ) Immunoblotting using anti - GFP or - p62 antibody .

(c) Immunoblotting using anti-GFP or -p62 antibody.


( d ) Tissue lysates were prepared from the liver of fed ob / w or ob / ob mice 4 h after leupeptin administration , and immunoblotting was performed . Numbers below immunoblot bands indicate the fold changes normalized to control bands .

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(d) Tissue lysates were prepared from the liver of fed ob/w or ob/ob mice 4 h after leupeptin administration, and immunoblotting was performed. Numbers below immunoblot bands indicate the fold changes normalized to control bands.


( e ) After labelling SK - Hep1 cells with C14 - leucine , cells were treated with FFAs ( 600 μM PA or 1 , 200 μM OA ) . Percent release of radioactivity was measured for 3 - 6 h as described in the Methods . The difference between the percent proteolysis without E64d / pepstatin A / NH4Cl and that with E64d / pepstatin A / NH4Cl was regarded as lysosomal proteolysis . * P0 . 05 , * * P0 . 01 ; one - way ANOVA , n = 3 .

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(e) After labelling SK-Hep1 cells with C14-leucine, cells were treated with FFAs (600 μM PA or 1,200 μM OA). Percent release of radioactivity was measured for 3-6 h as described in the Methods. The difference between the percent proteolysis without E64d/pepstatin A/NH4Cl and that with E64d/pepstatin A/NH4Cl was regarded as lysosomal proteolysis. *P0.05, **P0.01; one-way ANOVA, n=3.


( f ) Atg7 + / + and Atg7 + / − primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h , and TG content was estimated by ORO staining . * * P0 . 01 , * * * P0 . 001 ; two - way ANOVA , n = 4 .

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(f) Atg7+/+ and Atg7+/− primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h, and TG content was estimated by ORO staining. **P0.01, ***P0.001; two-way ANOVA, n=4.


( a ) The number of CLSs was counted in WAT of each type of mouse as described in the Methods . * P0 . 05 ; one - way ANOVA , n = 3 .

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(a) The number of CLSs was counted in WAT of each type of mouse as described in the Methods. *P0.05; one-way ANOVA, n=3.


( b ) Total mRNA was extracted from each tissue , and RT - PCR was done using primers specific for Tnfa , Il6 , F4 / 80 or pro - Il1b . The expression of pro - Il1b was determined using SVF fraction of WAT tissue .

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(b) Total mRNA was extracted from each tissue, and RT-PCR was done using primers specific for Tnfa, Il6, F4/80 or pro-Il1b. The expression of pro-Il1b was determined using SVF fraction of WAT tissue.


( c , d ) Lysate of SVF was subjected to immunoblot analysis using anti - IL - 1β ( c ) or anti - caspase - 1 antibody ( d ) .

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(c,d) Lysate of SVF was subjected to immunoblot analysis using anti-IL-1β (c) or anti-caspase-1 antibody (d).


( e ) Primary peritoneal macrophages were isolated , and treated with PA of the indicated concentrations in the presence or absence of LPS . ELISA was conducted to determine IL - 1β concentrations in the culture supernatant . ( BSA , bovine serum albumin ) * * * P0 . 001 ; two - way ANOVA , n = 4 .

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(e) Primary peritoneal macrophages were isolated, and treated with PA of the indicated concentrations in the presence or absence of LPS. ELISA was conducted to determine IL-1β concentrations in the culture supernatant. (BSA, bovine serum albumin) ***P0.001; two-way ANOVA, n=4.


( f ) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h , NAD + / NADH ratio was determined using a commercial kit . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ; two - way ANOVA , n = 5 .

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(f) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h, NAD+/NADH ratio was determined using a commercial kit. *P0.05, **P0.01, ***P0.001; two-way ANOVA, n=5.


( g ) After the same treatment of peritoneal macrophages as in f , cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence - activated cell sorting ( FACS ) analysis .

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(g) After the same treatment of peritoneal macrophages as in f, cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence-activated cell sorting (FACS) analysis.


( h ) After the same treatment of peritoneal macrophages as in f , cells were stained with MitoTracker Red and MitoTracker Green for FACS analysis to determine mitochondrial potential . The numbers in g , h indicate the percentages of cells in the designated gate .

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(h) After the same treatment of peritoneal macrophages as in f, cells were stained with MitoTracker Red and MitoTracker Green for FACS analysis to determine mitochondrial potential. The numbers in g,h indicate the percentages of cells in the designated gate.


( a ) Atg7 + / − and control Atg7 + / + mice were fed HFD or normal chow diet ( NCD ) , and non - fasting blood glucose levels were monitored . * P0 . 05 , * * P0 . 01 ; Student ' s t - test .

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(a) Atg7+/− and control Atg7+/+ mice were fed HFD or normal chow diet (NCD), and non-fasting blood glucose levels were monitored. *P0.05, **P0.01; Student's t-test.


( b ) Fasting bloodglucose levels were determined after 18 weeks of HFD . * P0 . 05 ; one - way ANOVA .

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(b) Fasting bloodglucose levels were determined after 18 weeks of HFD. *P0.05; one-way ANOVA.


( c ) IPGTT was performed after 18 weeks of HFD , ( left ) and AUC calculated ( right ) . * P0 . 05 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

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(c) IPGTT was performed after 18 weeks of HFD, (left) and AUC calculated (right). *P0.05; Student's t-test (left) and one-way ANOVA (right).


( d ) HOMA - IR was calculated after 18 weeks of HFD . * * * P0 . 001 ; one - way ANOVA .

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(d) HOMA-IR was calculated after 18 weeks of HFD. ***P0.001; one-way ANOVA.


( e ) ITT was performed after 18 weeks of HFD ( left ) , and KITT calculated ( right ) . * P0 . 05 ; Student ' s t - test ( left ) and one - way ANOVA ( right ) .

(e) ITT was performed after 18 weeks of HFD (left), and KITT calculated (right). *P0.05; Student's t-test (left) and one-way ANOVA (right).


( a ) One hour after intraperitoneal injection of 30 mg kg − 1 leupeptin to C57BL / 6 mice , 25 mg kg − 1 of imatinib ( Ima ) was injected intraperitoneally . Three hours later , tissue lysate was prepared from the liver and subjected to immunoblot analysis .

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(a) One hour after intraperitoneal injection of 30 mg kg−1 leupeptin to C57BL/6 mice, 25 mg kg−1 of imatinib (Ima) was injected intraperitoneally. Three hours later, tissue lysate was prepared from the liver and subjected to immunoblot analysis.


( b ) Imatinib ( 25 mg kg − 1 ) , trehalose ( 2 g kg − 1 ) or PBS was injected intraperitoneally into 12 - week - old diabetic Atg7 + / − - ob / ob mice 3 times a week , and the blood glucose level was monitored . * * * P0 . 001 , # # # P0 . 001 ; two - way ANOVA .

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(b) Imatinib (25 mg kg−1), trehalose (2 g kg−1) or PBS was injected intraperitoneally into 12-week-old diabetic Atg7+/−-ob/ob mice 3 times a week, and the blood glucose level was monitored. ***P0.001, ###P0.001; two-way ANOVA.


( c ) IPGTT were performed after treatment of Atg7 + / − - ob / ob mice with imatinib or trehalose for 8 weeks . # P0 . 05 ; # # P or * * P0 . 01 ; # # # P or * * * P0 . 001 ; Student ' s t - test .

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(c) IPGTT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.


( d ) ITT were performed after treatment of Atg7 + / − - ob / ob mice with imatinib or trehalose for 8 weeks . # P0 . 05 ; # # P or * * P0 . 01 ; # # # P or * * * P0 . 001 ; Student ' s t - test .

(d) ITT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.


( e ) Regular insulin ( Ins ) was injected into the tail vein of Atg7 + / − - ob / ob mice that were treated with imatinib or PBS for 8 weeks . Seven minutes later , tissue lysates were prepared and subjected to immunoblotting .

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(e) Regular insulin (Ins) was injected into the tail vein of Atg7+/−-ob/ob mice that were treated with imatinib or PBS for 8 weeks. Seven minutes later, tissue lysates were prepared and subjected to immunoblotting.


( f ) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting . Numbers below immunoblot bands indicate the fold changes normalized to control bands . ( ' * ' indicates comparison between imatinib and control : ' # ' indicates comparison between trehalose and control ) .

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(f) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting. Numbers below immunoblot bands indicate the fold changes normalized to control bands. ('*' indicates comparison between imatinib and control: '#' indicates comparison between trehalose and control).


B . Rif1 - ∆ C594 retains function to control DNA replication . Growth of a cdc7 - 1 rif1 - ∆ C594 mutant was compared with growth of cdc7 - 1 RIF1 and cdc7 - 1 rif1 ∆ strains at 23ºC ( permissive temperature for cdc7 - 1 allele ) , 26ºC ( mild restrictive temperature ) and 30ºC ( strict restrictive temperature )

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B. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature)


A . Specimen overview of Chromosome VI - left region showing results of ChIP - Seq analysis of Rif1 and Rif1 - ∆ C594 proteins . ChIP sequence reads were normalised against sequence reads from corresponding input samples , and relative enrichment is plotted for Chromosome VI coordinates 1 - 80 , 000 . Y - axis shows enrichment values ( linear scale , range is 0 to 3 . 5 ) . Values below 1 are shown in grey , and values above 1 ( i . e . sequences enriched in ChIP samples ) are coloured blue ( Rif1 ) and red ( Rif1 - ∆ C594 ) . Plots show ChIP analysis results from cells arrested by α - factor ( G1 ) , released from α - factor at 16ºC for 60 min and 90 min , or released from α - factor into 0 . 2 M HU for 60 min at 23ºC

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A. Specimen overview of Chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for Chromosome VI coordinates 1-80,000. Y-axis shows enrichment values (linear scale, range is 0 to 3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16ºC for 60 min and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23ºC


B . Rap1 - dependent association of Rif1 with the promoter regions of Rap1 - controlled genes . ChIP enrichment around PAU3 ( left ) and MAM3 ( right ) , both genes whose transcription is controlled by Rap1 . Values above 1 ( i . e . enriched ) shown by overlaid blue and red histograms for Rif1 and Rif1 - ∆ C594 , respectively . Values below 1 shown in grey

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B. Rap1-dependent association of Rif1 with the promoter regions of Rap1-controlled genes. ChIP enrichment around PAU3 (left) and MAM3 (right), both genes whose transcription is controlled by Rap1. Values above 1 (i.e. enriched) shown by overlaid blue and red histograms for Rif1 and Rif1-∆C594, respectively. Values below 1 shown in grey


A . ChIP - Seq analysis of Rif1 and Rif1 - ∆ C594 proteins shows tRNA gene and origin binding , with widened peaks at early origins ARS606 and ARS607 in HU block . Plots show Chromosome VI genome coordinates 160 , 000 - 210 , 000 . Plot colours here and in following Figures are as in Fig 2B . Widened peaks are not observed in unperturbed S phase samples , or for Rif1 - ∆ C594

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A. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594


B . Association of Rif1 - ∆ C594 at replication origins is enhanced in HU block . ChIP enrichment of Rif1 and Rif1 - ∆ C594 around early origin ARS1426 ( left ) and late origin ARS1412 ( right )

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B. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right)


C . Differential association of Rif1 and Rif1 - ∆ C594 to centromeres . ChIP enrichment of Rif1 and Rif1 - ∆ C594 around the CEN4 locus

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C. Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus


A . ChIP - qPCR confirmation of Rif1 and Rif1 - ∆ C594 association with replication origins . ChIP was performed using cells arrested in α - factor ( open bars ) or HU ( grey bars ) , and qPCR analysis performed for early origin ARS1426 ( left ) and late origin ARS1412 ( right ) . Values shown are ' normalised ChIP efficiencies ' obtained by subtracting the value obtained at a control locus ( see Materials and Methods ) . Bars indicate the averages of two biological replicates , with values from each replicate shown by open circles

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A. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles


B . Numbers of early and late origins associated with Rif1 and Rif1 - ∆ C594 peaks . Plot showing numbers of early and late origins bound by Rif1 and Rif1 - ∆ C594 in G1 and HU - blocked cells , based on peak - calling results

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B. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results


C . Replication timing programme is intact in rif1 - ∆ C594 cells . Replication of selected origins at an HU block analysed by BrdU incorporation . Cells were synchronised by α - factor and released into the medium containing 0 . 2 M HU and 1 . 13 mM BrdU . Plots show the percentage of total ARS607 , ARS422 . 5 , or ARS1412 DNA pulled down by IP with anti - BrdU , calculated from two biological replicate samples . Bars indicate the average of two biological replicates and open circles the values from each replicate . Insets in ARS422 , 5 and ARS1412 panels show the same data with Y - axis scales adjusted to 0 - 0 . 03 %

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C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03%


( B & Northern blot validation of new sRNAs . ( B ) Samples were taken in early exponential ( EE ) , mid - exponential ( ME ) , early late exponential ( eLE ) , late exponential ( LE ) , early stationary ( eST ) and stationary ( ST ) phase of growth in TY medium . The error bars represent the mean ± SD of four biological

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(B& Northern blot validation of new sRNAs. (B) Samples were taken in early exponential (EE), mid-exponential (ME), early late exponential (eLE), late exponential (LE), early stationary (eST) and stationary (ST) phase of growth in TY medium. The error bars represent the mean ± SD of four biological replicates


( D ) Coverage plots of several sRNA - IGR chimeras , where the IGR is located within poly - cistronic operons . Chimeric reads are highlighted in red ; the RBS is depicted as a yellow box . The number of chimeras covering each interaction is provided and the respective interacting sRNA is highlighted in bold .

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(D) Coverage plots of several sRNA-IGR chimeras, where the IGR is located within poly-cistronic operons. Chimeric reads are highlighted in red; the RBS is depicted as a yellow box. The number of chimeras covering each interaction is provided and the respective interacting sRNA is highlighted in bold.


( C ) Northern blot validation of SpoY , SpoX and nc037 expression in early exponential ( EE ) , mid - exponential ( ME ) , early late exponential ( eLE ) , late exponential ( LE ) , early stationary ( eST ) and stationary ( ST ) phase of growth in TY medium . 5S rRNA served as a loading ctl . A representative image of three independent experiments is shown .

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(C) Northern blot validation of SpoY, SpoX and nc037 expression in early exponential (EE), mid-exponential (ME), early late exponential (eLE), late exponential (LE), early stationary (eST) and stationary (ST) phase of growth in TY medium. 5S rRNA served as a loading ctl. A representative image of three independent experiments is shown.


( A ) Quantification of EMSAs ( error bars represent the mean ± SD of n = 3 biological replicates performed with either 32P - labeled SpoY or SpoX ( short isoform ) with increasing concentrations of the spo0A target region , respectively . Purified Hfq was added to facilitate SpoY - spo0A complex formation . Mutating the respective sRNA seed region ( Appendix Figure S4A , SpoY * / SpoX * ) abolished the interaction , while introducing compensatory mutations into the spo0A target region ( spo0A * C ) slightly rescued the complex formation .

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(A) Quantification of EMSAs (error bars represent the mean ± SD of n=3 biological replicates performed with either 32P-labeled SpoY or SpoX (short isoform) with increasing concentrations of the spo0A target region, respectively. Purified Hfq was added to facilitate SpoY-spo0A complex formation. Mutating the respective sRNA seed region (Appendix Figure S4A, SpoY*/SpoX*) abolished the interaction, while introducing compensatory mutations into the spo0A target region (spo0A*C) slightly rescued the complex formation.


( B ) In - line probing of 0 . 2 pmol of 32P - labeled SpoY and SpoX ( short isoform ) in the absence ( lane 4 ) or presence of increasing concentrations ( lane 5 - 7 ) of the spo0A long 5 ′ UTR ( starting from pTSS ) and first 69 nt of the CDS . RNase T1 and alkali - digested ( OH ) SpoY and SpoX serve as ladders respectively . Secondary structure and predicted seed region are highlighted . A representative image of three independent experiments is shown .

(B) In-line probing of 0.2 pmol of 32P-labeled SpoY and SpoX (short isoform) in the absence (lane 4) or presence of increasing concentrations (lane 5-7) of the spo0A long 5′UTR (starting from pTSS) and first 69 nt of the CDS. RNase T1 and alkali-digested (OH) SpoY and SpoX serve as ladders respectively. Secondary structure and predicted seed region are highlighted. A representative image of three independent experiments is shown.


( C ) mCherry fluorescence of translational fusion constructs ( error bars represent the mean ± SD of n = 5 biological replicates , expressed in the respective sRNA knock - out background . Fluorescence intensity was normalized to that of the respective p [ spo0A ] ctl . Mutating the sRNA seed regions ( Appendix Figure S4A , SpoY * / SpoX * ) abolished the impact on spo0A translation , while introducing compensatory mutations into the spo0A target region ( spo0A * C ) partially rescued the effect . The long SpoX isoform used in reporter assays . Ordinary one - way ANOVA with Dunnett ′ s multiple comparison test was used to calculate statistical significance . Not significant ( ns ) P > 0 . 05 ; ( ∗ ) P ≤ 0 . 05 ; ( ∗ ∗ ∗ ∗ ) P ≤ 0 . 0001 .

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(C) mCherry fluorescence of translational fusion constructs (error bars represent the mean ± SD of n=5 biological replicates, expressed in the respective sRNA knock-out background. Fluorescence intensity was normalized to that of the respective p[spo0A] ctl. Mutating the sRNA seed regions (Appendix Figure S4A, SpoY*/SpoX*) abolished the impact on spo0A translation, while introducing compensatory mutations into the spo0A target region (spo0A*C) partially rescued the effect. The long SpoX isoform used in reporter assays. Ordinary one-way ANOVA with Dunnett′s multiple comparison test was used to calculate statistical significance. Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗∗∗) P ≤ 0.0001.


( A ) Western blot analysis comparing Spo0A protein levels in a WT strain ( p [ ctl ] ) , sRNA knock - out mutants ( ΔSpoY / ΔSpoX - p [ ctl ] ) and strains constitutively expressing the respective sRNA ( ΔSpoY / ΔSpoX - p [ ΔSpoY / ΔSpoX ] ) . Equal optical density ( OD ) units of total cell lysates ( error bars represent the mean ± SD of n = 3 biological replicates ) were loaded from strains gown till mid - exponential ( ME ) or stationary ( ST ) phase of growth in TY . Band intensities were measured and Log2 fold changes were calculated relative to the respective WT . Western blot membranes were incubated with anti - Spo0A antibody . To calculate statistical significance , an ordinary one - way ANOVA with Dunnett ′ s multiple comparison test was applied in . Not significant ( ns ) P > 0 . 05 ; ( ∗ ) P ≤ 0 . 05 ; ( ∗ ∗ ∗ ) P ≤ 0 . 001 . For western blot analysis Ponceau S staining of the blotting membrane served as loading

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis comparing Spo0A protein levels in a WT strain (p[ctl]), sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]). Equal optical density (OD) units of total cell lysates (error bars represent the mean ± SD of n=3 biological replicates) were loaded from strains gown till mid-exponential (ME) or stationary (ST) phase of growth in TY. Band intensities were measured and Log2 fold changes were calculated relative to the respective WT. Western blot membranes were incubated with anti-Spo0A antibody. To calculate statistical significance, an ordinary one-way ANOVA with Dunnett′s multiple comparison test was applied in. Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗∗) P ≤ 0.001. For western blot analysis Ponceau S staining of the blotting membrane served as loading control


( B ) Northern blot analysis validating sRNA expression in the respective growth conditions .

(B) Northern blot analysis validating sRNA expression in the respective growth conditions.


Transcript levels of genes encoding sporulation - specific sigma factors and their respective regulon in sRNA knock - out mutants ( ΔSpoY / ΔSpoX - p [ ctl ] ) and strains constitutively expressing the respective sRNA ( ΔSpoY / ΔSpoX - p [ ΔSpoY / ΔSpoX ] ) relative to C . difficile 630 WT ( p [ ctl ] ) . A schematic representation of the first four stages of sporulation in C . difficile is given on the left . RNA was extracted from samples ( n = 3 biological replicates ) taken at 9 h and 12 h post induction of sporulation on 70 : 30 sporulation plates . Log2 fold changes were calculated relative to the WT . Statistical significance was determined using two - way ANOVA with Dunnett ′ s multiple comparison test . P > 0 . 05 ; ( ∗ ) P ≤ 0 . 05 ; ( ∗ ∗ ) P ≤ 0 . 01 ; ( ∗ ∗ ∗ ) P ≤ 0 . 001 ; ( ∗ ∗ ∗ ∗ ) P ≤ 0 . 0001 . Error bars represent the mean ± SD .

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

Transcript levels of genes encoding sporulation-specific sigma factors and their respective regulon in sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]) relative to C. difficile 630 WT (p[ctl]). A schematic representation of the first four stages of sporulation in C. difficile is given on the left. RNA was extracted from samples (n = 3 biological replicates) taken at 9 h and 12 h post induction of sporulation on 70:30 sporulation plates. Log2 fold changes were calculated relative to the WT. Statistical significance was determined using two-way ANOVA with Dunnett′s multiple comparison test. P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001. Error bars represent the mean ± SD.


( A ) Representative phase - contrast images ( n = 4 ) of a WT strain ( p [ ctl ] ) , sRNA knock - out mutants ( ΔSpoY / ΔSpoX - p [ ctl ] ) and strains constitutively expressing the respective sRNA ( ΔSpoY / ΔSpoX - p [ ΔSpoY / ΔSpoX ] ) at 6 h , 12 h , 24 h and 48 h post inoculation of 70 : 30 liquid sporulation medium . ( B ) Sporulation frequencies calculated based on phase - contrast microscopy . Error bars represent the mean ± SD of n = 4 biological replicates ( A ) . 2 - way ANOVA with Dunnett ′ s multiple comparison test was used to calculate statistical significance . Not significant ( ns ) P > 0 . 05 ; ( ∗ ∗ ) P ≤ 0 . 01 ; ( ∗ ∗ ∗ ) P ≤ 0 . 001 ; ( ∗ ∗ ∗ ∗ ) P ≤ 0 . 0001 .

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

(A) Representative phase-contrast images (n=4) of a WT strain (p[ctl]), sRNA knock-out mutants (ΔSpoY/ΔSpoX-p[ctl]) and strains constitutively expressing the respective sRNA (ΔSpoY/ΔSpoX-p[ΔSpoY/ΔSpoX]) at 6 h, 12 h, 24 h and 48 h post inoculation of 70:30 liquid sporulation medium. (B) Sporulation frequencies calculated based on phase-contrast microscopy. Error bars represent the mean ± SD of n=4 biological replicates (A). 2-way ANOVA with Dunnett′s multiple comparison test was used to calculate statistical significance. Not significant (ns) P > 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.


( B ) Body weight loss over the course of 7 days ( data points and error bars represent mean ± standard error of the mean ; the dotted lines serve as a visual guideline ) of mice infected with C . difficile 630 WT ( n = 15 ) , ΔSpoY ( n = 15 ) or ΔSpoX ( n = 15 ) are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body weight loss over the course of 7 days (data points and error bars represent mean ± standard error of the mean; the dotted lines serve as a visual guideline) of mice infected with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15)are indicated


( C ) final colon length at day 7 post infection of uninfected mice ( n = 10 ) and mice infected with C . difficile 630 WT ( n = 15 ) , ΔSpoY ( n = 15 ) or ΔSpoX ( n = 15 ) are indicated ( box - and - whisker plots show minimum , first quartile , median , third quartile , and maximum ) . Data information : Kruskal - Wallis was performed with Dunnett ′ s multiple comparison test to calculate statistical significance in ( C ) Not significant ( ns ) P > 0 . 05 ; ( ∗ ) P ≤ 0 . 05 ; ( ∗ ∗ ) P ≤ 0 . 01 ; ( ∗ ∗ ∗ ) P ≤ 0 . 001 ; ( ∗ ∗ ∗ ∗ ) P ≤ 0 . 0001 .

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(C) final colon length at day 7 post infection of uninfected mice (n=10) and mice infected with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15)are indicated (box-and-whisker plots show minimum, first quartile, median, third quartile, and maximum). Data information: Kruskal-Wallis was performed with Dunnett′s multiple comparison test to calculate statistical significance in (C) Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.


( D ) Comparison of CFUs of C . difficile vegetative cells and spores in fecal pellets of mice at different time points during infection with C . difficile 630 WT ( n = 15 ) , ΔSpoY ( n = 15 ) or ΔSpoX ( n = 15 ) . Replicates with CFUs below the detection limit were set to 100 . Box - and - whisker plots show minimum , first quartile , median , third quartile , and maximum . Data information : Kruskal - Wallis was performed with Dunnett ′ s multiple comparison test to calculate statistical significance in ( D ) . Not significant ( ns ) P > 0 . 05 ; ( ∗ ) P ≤ 0 . 05 ; ( ∗ ∗ ) P ≤ 0 . 01 ; ( ∗ ∗ ∗ ) P ≤ 0 . 001 ; ( ∗ ∗ ∗ ∗ ) P ≤ 0 . 0001 .

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

(D) Comparison of CFUs of C. difficile vegetative cells and spores in fecal pellets of mice at different time points during infection with C. difficile 630 WT (n=15), ΔSpoY (n=15) or ΔSpoX (n=15). Replicates with CFUs below the detection limit were set to 100. Box-and-whisker plots show minimum, first quartile, median, third quartile, and maximum. Data information: Kruskal-Wallis was performed with Dunnett′s multiple comparison test to calculate statistical significance in (D). Not significant (ns) P > 0.05; (∗) P ≤ 0.05; (∗∗) P ≤ 0.01; (∗∗∗) P ≤ 0.001; (∗∗∗∗) P ≤ 0.0001.


A S . cerevisiae wt or hsp42Δ cells expressing mCherry ‐ VHL ( red ) and GFP ‐ luciferase ‐ DM ‐ NLS ( green ) were grown at 30 ° C and shifted to 37 ° C for 90 min . MG132 was added prior to temperature upshift . Changes in protein localizations were recorded . DNA was stained by DAPI ( blue ) .

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

A S. cerevisiae wt or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐luciferase‐DM‐NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue).


B S . cerevisiae wt cells or hsp42Δ cells expressing GFP ‐ VHL ( green ) were treated as in ( A ) . Nuclear membranes were visualized by Nsp1immunofluorescence microscopy labeling ( red ) .

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B S. cerevisiae wt cells or hsp42Δ cells expressing GFP‐VHL (green) were treated as in (A). Nuclear membranes were visualized by Nsp1immunofluorescence microscopy labeling (red).


C S . cerevisiae wt cells or hsp42Δ cells expressing mCherry ‐ VHL ( red ) and GFP ‐ Nup49 ( green ) were treated as in ( A ) . Three ‐ dimensional reconstructions of respective cells are given .

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C S. cerevisiae wt cells or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐Nup49 (green) were treated as in (A). Three‐dimensional reconstructions of respective cells are given.


D Time ‐ lapse microscopy of wt cells expressing mCherry ‐ VHL ( green ) and GFP ‐ Nup49 ( red ) upon temperature upshift from 30 ° C to 37 ° C . * indicates collision of peripheral foci leading to fusion , and an arrow indicates apparent encounters of peripheral foci and nuclear INQ , followed by foci separation .

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D Time‐lapse microscopy of wt cells expressing mCherry‐VHL (green) and GFP‐Nup49 (red) upon temperature upshift from 30°C to 37°C. * indicates collision of peripheral foci leading to fusion, and an arrow indicates apparent encounters of peripheral foci and nuclear INQ, followed by foci separation.


A - D Cryo ‐ sections of ( A ) S . cerevisiae wt cells expressing GFP ‐ luciferase ‐ DM ‐ NLS , ( B ) hsp42Δ cells expressing GFP ‐ VHL . Sections were immunogold ‐ labeled with GFP ‐ specific antibodies . Gold particles are marked ( black arrows ) . Electron ‐ dense regions represent protein aggregates ( A ) . Locations of cytosol ( C ) , nuclear envelope ( NE , orange arrows ) , and nucleus ( N ) are given . Scale bars , 200 nm . E , F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined .

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A-D Cryo‐sections of (A) S. cerevisiae wt cells expressing GFP‐luciferase‐DM‐NLS, (B) hsp42Δ cells expressing GFP‐VHL. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined.


A - D Cryo ‐ sections of S . cerevisiae ( C , D ) wt or hsp42Δ cells expressing Hsp104 ‐ GFP . Sections were immunogold ‐ labeled with GFP ‐ specific antibodies . Gold particles are marked ( black arrows ) . Electron ‐ dense regions represent protein aggregates ( A ) . Locations of cytosol ( C ) , nuclear envelope ( NE , orange arrows ) , and nucleus ( N ) are given . Scale bars , 200 nm . E , F Average numbers of gold particles associated with nuclear and cytosolicprotein aggregates or distributed throughout the cytosol or nucleus were determined .

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

A-D Cryo‐sections of S. cerevisiae (C, D) wt or hsp42Δ cells expressing Hsp104‐GFP. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolicprotein aggregates or distributed throughout the cytosol or nucleus were determined.


A S . cerevisiae wt cells expressing GFP ‐ VHL ( green ) were grown at 30 ° C and shifted to 37 ° C for 90 min in the presence of MG132 . Ubiquitin ( red ) was stained by immunofluorescence microscopy using specific antibodies . DNA was stained by DAPI ( blue ) . Changes in protein localizations were recorded . Scale bars , 2 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 B-CONTROLLED_VAR 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

A S. cerevisiae wt cells expressing GFP‐VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence microscopy using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm.


B tGnd1 ‐ GFP is stabilized in ubr1Δ san1Δ cells . S . cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1 ‐ GFP were grown at 30 ° C . Cycloheximide was added , and protein levels were determined at the indicated time points by Western blot using GFP ‐ specific antibodies . Zwf1 levels are given as a loading control .

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B tGnd1‐GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1‐GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP‐specific antibodies. Zwf1 levels are given as a loading control.


C Ubiquitination status of tGnd1 ‐ GFP expressed in S . cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull ‐ down and Western blot analysis using ubiquitin ‐ specific antibodies . Levels of isolated tGnd1 ‐ GFP after GFPpull ‐ down were determined by Western blot using GFP ‐ specific antibodies and are given as a control .

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

C Ubiquitination status of tGnd1‐GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFPpull‐down and Western blot analysis using ubiquitin‐specific antibodies. Levels of isolated tGnd1‐GFP after GFPpull‐down were determined by Western blot using GFP‐specific antibodies and are given as a control.


D S . cerevisisae wt , ubr1Δ san1Δ , and ubr1Δ san1Δ btn2Δ cells expressing tGnd1 ‐ GFP ( green ) were grown at 30 ° C . DNA and nuclear envelopes were visualized by coexpressing Htb1 ‐ mCherry or Nic96 ‐ mCherry , respectively ( red ) . The fraction of cells showing specific tGnd1 ‐ GFP foci numbers per cell was determined . INQ formation in cells containing tGnd1 ‐ GFP foci was determined ( % cells with INQ ) based on close vicinity of tGnd1 ‐ GFP foci to Htb1 ‐ mCherry or localization inside Nic96 ‐ mCherry signals , yielding comparable results . Scale bars , 2 μm .

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D S. cerevisisae wt, ubr1Δ san1Δ, and ubr1Δ san1Δ btn2Δ cells expressing tGnd1‐GFP (green) were grown at 30°C. DNA and nuclear envelopes were visualized by coexpressing Htb1‐mCherry or Nic96‐mCherry, respectively (red). The fraction of cells showing specific tGnd1‐GFP foci numbers per cell was determined. INQ formation in cells containing tGnd1‐GFP foci was determined (% cells with INQ) based on close vicinity of tGnd1‐GFP foci to Htb1‐mCherry or localization inside Nic96‐mCherry signals, yielding comparable results. Scale bars, 2 μm.


A , B S . cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry ‐ VHL ( red ) were grown at 30 ° C and shifted to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . Scale bars , 2 μm . The total number of mCherry ‐ VHL foci per cell was determined .

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A, B S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry‐VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry‐VHL foci per cell was determined.


C Dynamic exchange of a misfolded model protein between cytosol and nucleus . FLIP measurements of mCherry ‐ VHL were performed in S . cerevisiae wild ‐ type cells at 30 ° C . Nuclear or cytosolic areas were bleached , and loss of fluorescence intensities in non ‐ bleached compartments was determined . Bleaching and acquisition controls are given . Error bars : SEM .

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C Dynamic exchange of a misfolded model protein between cytosol and nucleus. FLIP measurements of mCherry‐VHL were performed in S. cerevisiae wild‐type cells at 30°C. Nuclear or cytosolic areas were bleached, and loss of fluorescence intensities in non‐bleached compartments was determined. Bleaching and acquisition controls are given. Error bars: SEM.


S . cerevisiae hsp42Δ and hsp42Δ tet ‐ off sis1 cells expressing GFP ‐ VHL were grown for 20 h in the absence ( − Dox ) or presence ( + Dox ) of doxycycline at 30 ° C and shifted for 90 min to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 μm .

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S. cerevisiae hsp42Δ and hsp42Δ tet‐off sis1 cells expressing GFP‐VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


B S . cerevisiae wt , hsp42Δ , sti1Δ , and hsp42Δ sti1Δ cells expressing GFP ‐ VHL were grown at 30 ° C and shifted to 37 ° C in the presence of MG132 . Changes in protein localization were monitored . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 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 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 S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP‐VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


A , B S . cerevisiae wt , hsp42Δ , btn2Δ , and hsp42Δ btn2Δ cells expressing GFP ‐ VHL ( green , A ) or GFP ‐ luciferase ‐ DM ‐ NLS ( green , B ) were grown at 30 ° C and shifted to 37 ° C for 90 min in the presence of MG132 , and protein localizations were recorded . DNA was stained by DAPI ( blue ) , and the nuclear envelope was stained by Nsp1immunofluorescence microscopy ( red ) . Solubilities of GFP ‐ VHL and GFP ‐ luciferase ‐ DM ‐ NLS were determined after stress application by Western blot using GFP ‐ specific antibodies . Solubility of actin was determined as a control . T , total fraction ; S , soluble fraction ; P , pellet fraction . Scale bars , 2 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 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

A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1immunofluorescence microscopy (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.


C S . cerevisiae wt , hsp42Δ , btn2Δ , and hsp42Δ btn2Δ cells expressing mCherry ‐ VHL were heat ‐ shocked to 38 ° C . Upon return to 30 ° C , protein synthesis was stopped by cycloheximide ( CHX ) addition and degradation of mCherry ‐ VHL was monitored by Western blot analysis . The stability of GFP ‐ luciferase ‐ DM ‐ NLS expressed in S . cerevisiae wt and btn2Δ cells was determined accordingly . Ccs1 or Zwf1 levels are given as loading controls .

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C S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry‐VHL were heat‐shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry‐VHL was monitored by Western blot analysis. The stability of GFP‐luciferase‐DM‐NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls.


D S . cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM ‐ GFP and Htb1 ‐ mCherry were grown at 30 ° C and heat ‐ shocked to 37 ° C for 30 min . Line intensity plots of luciferase ‐ DM ‐ GFP and Htb1 ‐ mCherry before and after heat shock are given . The ratio of nuclear and cytosolic luciferase ‐ DM ‐ GFP fluorescence intensity was determined at 30 ° C and 37 ° C ( n > 25 ) . Scale bars , 2 μm .

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D S. cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM‐GFP and Htb1‐mCherry were grown at 30°C and heat‐shocked to 37°C for 30 min. Line intensity plots of luciferase‐DM‐GFP and Htb1‐mCherry before and after heat shock are given. The ratio of nuclear and cytosolic luciferase‐DM‐GFP fluorescence intensity was determined at 30°C and 37°C (n > 25). Scale bars, 2 μm.


A , B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy . DNA was stained by either Htb1 ‐ mCherry or DAPI . Scale bars , 2 μm . In case of Hsp42 immunofluorescence , a line intensity plot of a deconvoluted widefield image is given .

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

A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy. DNA was stained by either Htb1‐mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given.


C S . cerevisiae btn2Δ hsp42Δ Hsp42 ‐ NLS cells expressing GFP ‐ VHL ( green ) were grown at 30 ° C and heat ‐ shocked to 37 ° C for 30 min in the presence of MG132 . Changes in protein localizations were recorded . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined . Scale bars , 2 μm .

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C S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing GFP‐VHL (green) were grown at 30°C and heat‐shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.


D S . cerevisiaebtn2Δ hsp42Δ Hsp42 ‐ NLS cells expressing mCherry ‐ VHL ( red ) were treated as described in ( C ) . The nuclear membrane was visualized by Nsp1 immunofluorescence ( green ) and colocalization of mCherry ‐ VHL foci with Hsp42 was probed by Hsp42 ( green ) immunofluorescence microscopy . DNA was stained with DAPI ( blue ) . Scale bars , 2 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 B-MEASURED_VAR O O O O O O O O O O O O O O

D S. cerevisiaebtn2Δ hsp42Δ Hsp42‐NLS cells expressing mCherry‐VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry‐VHL foci with Hsp42 was probed by Hsp42 (green) immunofluorescence microscopy. DNA was stained with DAPI (blue). Scale bars, 2 μm.


E Hsp42 and Btn2 levels were determined prior and post ‐ heat shock at the indicated time points by Western blot . The numbers of molecules / cell were calculated using purified proteins as standard .

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E Hsp42 and Btn2 levels were determined prior and post‐heat shock at the indicated time points by Western blot. The numbers of molecules/cell were calculated using purified proteins as standard.


A S . cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points . Zwf1 levels are given as a loading control .

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

A S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control.


B , C S . cerevisiae wt and btn2Δ cells expressing Hos2 ‐ GFP ( green , B ) or GFP ‐ VHL ( green , C ) were grown at 30 ° C and treated with MMS . Changes in protein localizations were recorded at the indicated time points . DNA was stained by DAPI ( blue ) . The total number of GFP ‐ VHL foci per cell and frequencies ( % ) of INQ formation were determined .

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

B, C S. cerevisiae wt and btn2Δ cells expressing Hos2‐GFP (green, B) or GFP‐VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined.


( A ) Relative mRNA expression of Foxc1 and Foxc2 in isolated blood endothelial cells ( BECs ) and lymphatic endothelial cells ( LECs ) from Foxc1fl / fl ; Foxc2fl / fl mouse distal jejuna in sham and I / R - 4h groups . Isolated epithelial cells ( Epis ) were used as a relative control , Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 5 ~ 9 , * P < 0 . 05 , * * P < 0 . 01 , n . s . = not significant . The box - and - whisker plots in ( A ) display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

(A) Relative mRNA expression of Foxc1 and Foxc2 in isolated blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) from Foxc1fl/fl;Foxc2fl/fl mouse distal jejuna in sham and I/R-4h groups. Isolated epithelial cells (Epis) were used as a relative control, Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~9, *P<0.05, **P<0.01, n.s. = not significant. The box-and-whisker plots in (A) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( B - C ) Representative confocal images of the whole - mount intestine stained with CD31 / LYVE1 ( B ) , FOXC1 / CD31 / LYVE1 ( C , left ) and FOXC1 / LYVE1 ( C , right ) in Foxc1fl / fl ; Foxc2fl / fl mice . Images of maximum intensity projections ( B ) show the intestinal blood ( green ) and lymphatic ( white ) vasculatures from villous to submucosa . The regions in pink box 1 ~ 8 in Figure 1B are chosen for the specific images in Figure 1C . Scale bars = 50 μm . ( C ) Images of optical sections with high magnification chosen from specific regions shown in Figure 1B ( B1 ~ B8 ) , with an additional channel of FOXC1 staining ( red ) , show the up - regulation of FOXC1 in BECs ( white arrows in blood vessels ) and LECs ( violet arrows in lymphatic vessels ) at the level of villus and submucosa 4 h after I / R . Scale bars = 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

(B-C) Representative confocal images of the whole-mount intestine stained with CD31/LYVE1 (B), FOXC1/CD31/LYVE1 (C, left) and FOXC1/LYVE1 (C, right) in Foxc1fl/fl;Foxc2fl/fl mice. Images of maximum intensity projections (B) show the intestinal blood (green) and lymphatic (white) vasculatures from villous to submucosa. The regions in pink box 1~8 in Figure 1B are chosen for the specific images in Figure 1C. Scale bars = 50 μm. (C) Images of optical sections with high magnification chosen from specific regions shown in Figure 1B (B1~B8), with an additional channel of FOXC1 staining (red), show the up-regulation of FOXC1 in BECs (white arrows in blood vessels) and LECs (violet arrows in lymphatic vessels) at the level of villus and submucosa 4 h after I/R. Scale bars = 20 μm.


( F ) Relative mRNA expression of Foxc1 and Foxc2 in isolated CD45 - CD31 + ECs from distal jejuna 12d after Tm treatment . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 4 ~ 5 , * P < 0 . 05 . Data information : The box - and - whisker plots in ( F ) display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA expression of Foxc1 and Foxc2 in isolated CD45-CD31+ ECs from distal jejuna 12d after Tm treatment. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~5, *P<0.05. Data information: The box-and-whisker plots in (F) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( C - E ) Quantification of Chiu Score for Control and EC - Foxc - DKO ( C ) , Control and EC - Foxc1 - KO ( D ) , and Control and EC - Foxc2 - KO groups ( E ) at I / R - 24h . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 3 in C sham groups , N = 7 ~ 12 in C , D , E I / R - 24h groups , * P < 0 . 05 , * * * * P < 0 . 0001 . The box - and - whisker plots in display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

(C-E) Quantification of Chiu Score for Control and EC-Foxc-DKO (C), Control and EC-Foxc1-KO (D), and Control and EC-Foxc2-KO groups (E) at I/R-24h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 3 in C sham groups, N = 7~12 in C, D, E I/R-24h groups, *P<0.05, ****P<0.0001. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( G ) Representative immunostaining images of intestinal crypts labeled with BrdU ( proliferative marker , injection performed 2h before tissue collection ) and EpCAM ( epithelial marker ) show the proliferation of epithelial cells in crypts . Paraffin sections ( 4 µm ) , ( H ) Quantification of the number of BrdU + epithelial cells per crypt based on Figure 2G . Data are box - and - whisker plots , Kruskal - Wallis One - way ANOVA test , each symbol represents one mouse , N = 3 ~ 6 , * P < 0 . 05 , n . s . = not significant . The box - and - whisker plots in display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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(G) Representative immunostaining images of intestinal crypts labeled with BrdU (proliferative marker, injection performed 2h before tissue collection) and EpCAM (epithelial marker) show the proliferation of epithelial cells in crypts. Paraffin sections (4 µm), (H) Quantification of the number of BrdU+ epithelial cells per crypt based on Figure 2G. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~6, *P<0.05, n.s. = not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( I ) Representative images of intestinal crypts immunostained with β - catenin and the intestinal epithelial stem cell ( ISC ) marker OLFM4 . At I / R - 48h , the nuclear translocation of β - catenin in ISCs ( dotted circles ) was found in the control , whereas it was seldom found in EC - Foxc - DKO mice . Paraffin sections ( 4 µm ) , ( J , K ) ( J ) Quantification of relative fluorescent intensity ( FI ) of β - catenin immunostaining within ISC and ( K ) quantification of number of OLFM4 + ISCs per crypt were performed based on Figure 2I . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 4 ~ 5 , * P < 0 . 05 , n . s . = not significant . The box - and - whisker plots in display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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(I) Representative images of intestinal crypts immunostained with β-catenin and the intestinal epithelial stem cell (ISC) marker OLFM4. At I/R-48h, the nuclear translocation of β-catenin in ISCs (dotted circles) was found in the control, whereas it was seldom found in EC-Foxc-DKO mice. Paraffin sections (4 µm), (J, K) (J) Quantification of relative fluorescent intensity (FI) of β-catenin immunostaining within ISC and (K) quantification of number of OLFM4+ ISCs per crypt were performed based on Figure 2I. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~5, *P<0.05, n.s. = not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( L ) Representative images of intestinal mucosa labeled with Cyclin D1 ( CCND1 ) in EC - Foxc - DKO mice compared with Control group in sham , 24h and 48h after I / R . Paraffin sections ( 4 µm ) , ( M ) Quantification of the number of CCND1 + epithelial cells per crypt based on Figure 2L . Data are box - and - whisker plots , Kruskal - Wallis One - way ANOVA test , each symbol represents one mouse , N = 4 ~ 6 , * * P < 0 . 01 , * * * * P < 0 . 0001 , n . s . = not significant . The box - and - whisker plots in display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

(L) Representative images of intestinal mucosa labeled with Cyclin D1 (CCND1) in EC-Foxc-DKO mice compared with Control group in sham, 24h and 48h after I/R. Paraffin sections (4 µm), (M) Quantification of the number of CCND1+ epithelial cells per crypt based on Figure 2L. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 4~6, **P<0.01, ****P<0.0001, n.s.=not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


Chiu Score analysis from H & E stained distal jejunum 24h after I / R for Control ( Foxc1f / f ; Foxc2f / f ) and LEC - Foxc - DKO ( Vegfr3 - CreERT2 ; Foxc1f / f ; Foxc2f / f ) mice ( B ) , Control ( Foxc1f / f ) and LEC - Foxc1 - KO ( Vegfr3 - CreERT2 ; Foxc1f / f ) mice ( C ) Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 9 ~ 13 , * P < 0 . 05 , * * P < 0 . 01 . display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

Chiu Score analysis from H&E stained distal jejunum 24h after I/R for Control (Foxc1f/f;Foxc2f/f) and LEC-Foxc-DKO (Vegfr3-CreERT2;Foxc1f/f;Foxc2f/f) mice (B), Control (Foxc1f/f) and LEC-Foxc1-KO (Vegfr3-CreERT2;Foxc1f/f) mice (C) Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N=9~13, *P<0.05, **P<0.01. display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( F ) Representative images of crypts immunostained with OLFM4 and β - catenin in control and LEC - Foxc - DKO mice 24h after I / R . The accumulation of β - catenin in the nuclei of ISCs ( dotted circles ) was found in Control mice but inhibited in LEC - Foxc - DKO mice . Paraffin sections ( 4 µm ) , scale bars = 20 μm .

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(F) Representative images of crypts immunostained with OLFM4 and β-catenin in control and LEC-Foxc-DKO mice 24h after I/R. The accumulation of β-catenin in the nuclei of ISCs (dotted circles) was found in Control mice but inhibited in LEC-Foxc-DKO mice. Paraffin sections (4 µm), scale bars = 20 μm.


( A ) Representative images of intestinal whole - mount VEGFR2 / CD31 ( green / red ) immunostaining show increased VEGFR2 ( green ) expression at the angiogenic front of villous blood capillaries in control mice at I / R - 18 . 5h . In EC - Foxc - DKO mice , the increase of VEGFR2 is inhibited in villous blood vessels and the blood vasculatures are damaged ( arrow ) . ( B ) Representative images of intestinal whole - mount VEGFR3 / LYVE1 ( red / green ) immunostaining . 18 . 5h after I / R , VEGFR3 ( red ) is increased in lacteals especially at the lacteal tips in the control but is inhibited in the EC - Foxc - DKO lacteals ( arrow ) .

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(A) Representative images of intestinal whole-mount VEGFR2/CD31 (green/red) immunostaining show increased VEGFR2 (green) expression at the angiogenic front of villous blood capillaries in control mice at I/R-18.5h. In EC-Foxc-DKO mice, the increase of VEGFR2 is inhibited in villous blood vessels and the blood vasculatures are damaged (arrow). (B) Representative images of intestinal whole-mount VEGFR3/LYVE1 (red/green) immunostaining. 18.5h after I/R, VEGFR3 (red) is increased in lacteals especially at the lacteal tips in the control but is inhibited in the EC-Foxc-DKO lacteals (arrow).


( A ) Representative BrdU / CD31 / LYVE1 / DAPI immunostaining images of intestinal paraffin sections ( 15 µm ) from mice injected with BrdU 18 . 5h before euthanasia for the analysis of proliferative BECs ( B in 1 , 3 , 5 , 7 ) and LECs ( L in 2 , 4 , 6 , 8 ) in intestines . Arrow heads show BrdU + BECs or LECs . White / yellow bars = 100 or 10 µm , respectively . ( B ) The numbers of BrdU + BECs and LECs per 0 . 1 mm2 blood vessel ( CD31 + LYVE1 - ) and lymphatic vessel ( CD31 + LYVE1 + ) area in intestinal mucosa were quantified respectively based on Figure 5A . Data are box - and - whisker plots , Kruskal - Wallis One - way ANOVA test , each symbol represents one mouse , N = 3 ~ 8 , * P < 0 . 05 , * * P < 0 . 01 , n . s . = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative BrdU/CD31/LYVE1/DAPI immunostaining images of intestinal paraffin sections (15 µm) from mice injected with BrdU 18.5h before euthanasia for the analysis of proliferative BECs (B in 1, 3, 5, 7) and LECs (L in 2, 4, 6, 8) in intestines. Arrow heads show BrdU+ BECs or LECs. White/yellow bars = 100 or 10 µm, respectively. (B) The numbers of BrdU+ BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5A. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~8, *P<0.05, **P<0.01, n.s. = not significant.