Confocal images of PI - stained 6 - day - old roots . GFP - tagged expression is shown in yellow . A - C ) pBRAVO : GFP in WT ( A ) , bravo - 2 ( B ) and wox5 - 1 ( C ) knockout backgrounds . D - G ) pWOX5 : GFP in the WT ( D ) , bravo - 2 ( E ) , wox5 - 1 ( F ) and bravo - 2 wox5 - 1 ( G ) knockout backgrounds . Scale bar : 50 µm . Quantification of the GFP fluorescent signal of the roots in A - C ( H ) and D - G ( I ) . Boxplot indicating the average pixel intensity of the GFP in the stem cell niche ( n > 25 , 3 biological replicates , * p - value < 0 . 05 Student ´ s t - test for each genotype versus the WT in the same condition ) . Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN ( Appendix Figure S7 ) . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers and black dots are the experimental observations .

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

Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.


Parameter space exploration of ( C ) the alleviation and the ( D ) activation models . Boxplots showing the distribution of stationary promoter pBRAVO ( pB ) and pWOX5 ( pW ) fold - changes in each mutant and overexpression lines over the WT obtained for different parameter values . Superindexes denote the genotype , standing bravo for bravo mutant , wox5 for wox5 mutant , dm for the bravo wox5 double mutant , Woe for WOX5 overexpression and Boe for BRAVO overexpression . Box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . The results are obtained by solving N = 1000 different runs of each model at the stationary state ( red and blue stripplots ) for the WT , mutants and overexpressor scenarios . These N runs differ in the parameter values , which are chosen at random from a uniform distribution

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

Parameter space exploration of (C) the alleviation and the (D) activation models. Boxplots showing the distribution of stationary promoter pBRAVO (pB) and pWOX5 (pW) fold-changes in each mutant and overexpression lines over the WT obtained for different parameter values. Superindexes denote the genotype, standing bravo for bravo mutant, wox5 for wox5 mutant, dm for the bravo wox5 double mutant, Woe for WOX5 overexpression and Boe for BRAVO overexpression. Box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. The results are obtained by solving N=1000 different runs of each model at the stationary state (red and blue stripplots) for the WT, mutants and overexpressor scenarios. These N runs differ in the parameter values, which are chosen at random from a uniform distribution between


Interaction of BRAVO with WOX5 ( B ) , BES1 ( C ) , BES1D ( D ) and TPL ( E ) ; and interaction of WOX5 with BRAVO ( G ) , BES1 ( H ) , BES1D ( I ) and TPL ( J ) measured by FRET - FLIM . GFP fluorescence lifetime τ [ ns ] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells . GFP fluorescence lifetime fitted pixel - wise with a mono - exponential model of BRAVO and WOX5 interactions . mV , mVenus ; mCh , mCherry . Scale bar : 5 µm .

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

Interaction of BRAVO with WOX5 (B), BES1 (C), BES1D (D) and TPL (E); and interaction of WOX5 with BRAVO (G), BES1 (H), BES1D (I) and TPL (J) measured by FRET-FLIM. GFP fluorescence lifetime τ [ns] was measured in transiently expressing Nicotiana benthamiana leaf epidermal cells. GFP fluorescence lifetime fitted pixel-wise with a mono-exponential model of BRAVO and WOX5 interactions. mV, mVenus; mCh, mCherry. Scale bar: 5 µm.


Fluorescence - weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double - exponential model of the indicated samples are summarized in box plots . Statistical significance was tested by one - way ANOVA with a Sidakholm post - hoc test . Different letters indicate statistically significant differences ( p - value < 0 . 01 ) . For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured . In the boxplot , box width represents the interquartile range ( IQR = Q3 - Q1 ) , with the horizontal line denoting the median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . White dots are the outliers .

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

Fluorescence-weighted average lifetimes of BRAVO and WOX5 interactions fitted with a double-exponential model of the indicated samples are summarized in box plots. Statistical significance was tested by one-way ANOVA with a Sidakholm post-hoc test. Different letters indicate statistically significant differences (p-value < 0.01). For each combination two to three independent experiments were carried out and in total more than 20 biological replicates were measured. In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers.


Yeast two - hybrid assay showing BRAVO interacting with WOX5 , BES1 - D and TPL ; and BES1 - D interacting with TPL . In the left column yeast cells were grown on control media , and in the right column yeast cells were grown on control media lacking Leu , Trp and His , indicating an interaction between the proteins .

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

Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.


Expression of the 380 common BRAVO and WOX5 regulated genes from ( B ) . Expression of the 53 genes from ( C ) showing similar expression in bravo and wox5 mutants . Expression of the 41 genes from ( C ) showing opposite expression levels in bravo and wox5 mutants .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

Expression of the 380 common BRAVO and WOX5 regulated genes from (B). Expression of the 53 genes from (C) showing similar expression in bravo and wox5 mutants. Expression of the 41 genes from (C) showing opposite expression levels in bravo and wox5 mutants.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) and cell lysates ( Lysate ) were collected and immunoblotted with the indicated antibodies （ A ） .

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

LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected and ( B - E ) IL - 1β secretion ( B ) , IL - 18 secretion ( C ) and TNF - ɑ secretion ( D ) and LDH release ( E ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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

LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . ( F ) HBVLV - Vdr expression in Vdr - / - BMDMs by lentivirus - mediated transduction primed with LPS and infected with 50 MOI S . Typhimurium for 2 h , cell lysates , and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies .

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

LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies.


( G ) LPS - primed WT and Vdr - / - BMDMs were treated with 1 µg / ml FLIC ( LFn - Flagellin and PA ) or PrgJ （ 1 µg / ml LFn - PrgJ and PA ） for 1 h , total mixtures ( culture supernatants and cell lysates ) were collected and immunoblotted with the indicated antibodies .

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

(G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies.


( H ) LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) and cell lysates were collected and immunoblotted with the indicated antibodies .

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

(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies.


LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) were collected and ( I - L ) IL - 1β secretion ( I ) , IL - 18 secretion ( J ) and TNF - ɑ secretion ( K ) and LDH release ( L ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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

LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) and Cell lysates were collected and immunoblotted with the indicated antibodies ( A ) .

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LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISA


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected antibodies ( A ) . ELISA detected TNF - ɑ ( B ) andIL - 1β ( C ) in supernatants . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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

LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) were collected antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium Bacterial burden was detected in spleens ( D ) , PCF ( E ) , and blood ( F ) of WT , Vdr - / - , NLRP3 - / - , and NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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

LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


A LPS - primed WT and Vdr - / - BMDMs were infected with S . Typhimurium at an MOI of 50 for 2 h . Endogenous ASC specks ( arrows ) were represented and quantified by immunofluorescence images . The data show representative results from three independent experiments : scale bar , 20 μm . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * * , p < 0 . 001 .

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A LPS-primed WT and Vdr-/- BMDMs were infected with S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC specks (arrows) were represented and quantified by immunofluorescence images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.


B ASC oligomerization induced by the indicated stimuli in WT and Vdr − / − BMDMs primed with LPS .

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

B ASC oligomerization induced by the indicated stimuli in WT and Vdr −/− BMDMs primed with LPS.


LPS - primed BMDMs were infected with S . Typhimurium at an MOI of 50 for 2h . Lysates were immunoprecipitated ( IP ) using an anti - VDR antibody and were immunoblotted with the indicated antibodies ( A ) . Whole - cell lysates were shown as the input . Data are representative of three independent experiments .

O O O O 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

LPS-primed BMDMs were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated (IP) using an anti-VDR antibody and were immunoblotted with the indicated antibodies(A). Whole-cell lysates were shown as the input. Data are representative of three independent experiments.


( B - C ) Flag - VDR was co - expressed with Myc - NLRC4 in HEK293T cells ; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies . Whole - cell lysates were shown as the input . Data are representative of three independent experiments .

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(B-C) Flag-VDR was co-expressed with Myc-NLRC4 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Whole-cell lysates were shown as the input. Data are representative of three independent experiments.


( D ) Purified GST - VDR was incubated with purified His - NLRC4 for 2 h . His - NLRC4 - bound to GST - VDR was pulled down by glutathione beads and subjected to immunoblot analysis . Data are representative of three independent experiments .

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(D) Purified GST-VDR was incubated with purified His-NLRC4 for 2 h. His-NLRC4- bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. Data are representative of three independent experiments.


( E ) WT or mutant NLRC4 ( CARD , NACHT , or LRR ( leucine - rich repeat ) ) and HA - VDR were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . * no specific band . Data are representative of three independent experiments .

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(E) WT or mutant NLRC4 (CARD, NACHT, or LRR (leucine-rich repeat)) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies.*no specific band. Data are representative of three independent experiments.


( F ) WT or mutant VDR ( DBD or LBD ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . Data are representative of three independent experiments .

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

(F) WT or mutant VDR (DBD or LBD) and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies. Data are representative of three independent experiments.


A - B HEK293T cells were transfected with Flag - NLRC4 , VDR , Myc - FLagellin , and HA - NAIP5 ( A ) or Flag - NLRC4 , VDR , Myc - PrgJ and HA - NAIP2 ( B ) Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies .

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 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

A-B HEK293T cells were transfected with Flag-NLRC4, VDR, Myc-FLagellin, and HA-NAIP5 (A) or Flag-NLRC4, VDR, Myc- PrgJ and HA-NAIP2(B) Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


HEK293T cells were transfected with Flag - NLRC4 , LBD - VDR , Myc - FLagellin , and HA - NAIP5 ( C ) or Flag - NLRC4 , LBD - VDR , Myc - PrgJ , and HA - NAIP2 ( D ) . Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies .

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

HEK293T cells were transfected with Flag-NLRC4, LBD-VDR, Myc-FLagellin, and HA-NAIP5 (C) or Flag-NLRC4, LBD-VDR, Myc-PrgJ, and HA-NAIP2 (D). Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


B VDR or mutant VDR ( K123A ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated with the anti - Flag antibody , and analyzed by immunoblotting with indicated antibodies .

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

B VDR or mutant VDR(K123A)and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated with the anti-Flag antibody, and analyzed by immunoblotting with indicated antibodies.


C Transfected VDR or mutant VDR ( K123A ) and Flag - NLRC4 , Myc - PrgJ , and HA - NAIP2 in HEK293T cells , immunoprecipitated with the anti - HA antibody , and analyzed by immunoblotting with indicated antibodies .

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C Transfected VDR or mutant VDR(K123A) and Flag-NLRC4, Myc- PrgJ, and HA-NAIP2 in HEK293T cells, immunoprecipitated with the anti-HA antibody, and analyzed by immunoblotting with indicated antibodies.


D Vdr - / - BMDMs were infected with lentivirus , which expressed Vector ( NC ) , VDR , and VDR - K123A , infected with S . Typhimurium at an MOI of 50 for 2 h , cell lysates ( Lysate ) and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies .

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D Vdr-/- BMDMs were infected with lentivirus, which expressed Vector(NC), VDR, and VDR-K123A, infected with S. Typhimurium at an MOI of 50 for 2 h, cell lysates(Lysate) and culture supernatants(SN) were collected and immunoblotted with the indicated antibodies.


E - H LDH release ( E ) , IL - 1β secretion ( F ) , IL - 18 secretion ( G ) and TNF - ɑ secretion ( H ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; In each panel , data are representative of at least three independent experiments .

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E-H LDH release(E), IL-1β secretion(F), IL-18 secretion (G) and TNF-ɑ secretion (H) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; **, P < 0.01; ***, P <0.001; In each panel, data are representative of at least three independent experiments.


B . Relative quantification of all four histone proteins in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 and H4 ) .

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B. Relative quantification of all four histone proteins in each tissue replicate (order of appearance per replicate: H2A, H2B, H3.3 and H4).


C . Immunohistochemistry images from the Human Protein Atlas ( www . proteinatlas . org ) for protein ANXA1 with nuclear staining ( blue ) for three selected tissues .

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C. Immunohistochemistry images from the Human Protein Atlas (www.proteinatlas.org) for protein ANXA1 with nuclear staining (blue) for three selected tissues.


D . Calibration curves for two of the four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS cell lysate .

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D. Calibration curves for two of the four histone peptides, with decreasing amount of QPrEST standard spiked into a U2OS cell lysate.


A . Absolute copy number of protein in kidney tissue and human embryonal kidney cells ( HEK293 ) , liver tissue and liver cancer cell line ( HepG2 ) , lung tissue and lung cancer cell line ( A549 ) , and breast tissue and breast cancer cell line ( MCF7 ) . The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue .

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A. Absolute copy number of protein in kidney tissue and human embryonal kidney cells (HEK293), liver tissue and liver cancer cell line (HepG2), lung tissue and lung cancer cell line (A549), and breast tissue and breast cancer cell line (MCF7). The order of proteins are the same in the tissue and corresponding cell line and the proteins have been order according to the abundance in the respective tissue.


B . The direct correlation between RNA ( TPM ) and protein abundances ( copy number ) for all quantified genes in the same tissues and cell lines . Spearman ( ρ ) and Pearson ( r ) correlation between the two values across the quantified genes are shown . The other seven tissues and five cell lines are shown in Figure EV4 .

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B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.


Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - and nuclear probes are visualized in red and blue respectively . Antibody staining is shown in green . RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho , Pearson r and R2 for each gene . All other genes can be found in Figure EV1 .

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Subcellular localization by immunofluorescence staining and immunohistochemistry staining in tissue sections by three different antibodies (SELENBP1, HPA011731; STOM, HPA010961; ASS1, HPA020896). Microtubule- and nuclear probes are visualized in red and blue respectively. Antibody staining is shown in green. RNA to protein ratio across nine cell lines and 11 tissues with Spearman rho, Pearson r and R2 for each gene. All other genes can be found in Figure EV1.


A : Volcano plot showing significantly regulated genes between control and ATP - treated msEGCs .

A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.


G - I : qPCR analysis of indicated gliosis genes in ATP - treated EGCs .

G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.


J : qPCR analysis of IL - 6 in msEGCs that were treated for 6h with ATP .

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J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.


K : IL - 6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP .

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K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.


A : Effect of P2 receptor antagonism on ATP induced IL - 6 release . Cells were treated with P2 - antagonist suramin ( 1 , 10 and 100µM ) alone or together with ATP ( 100µM ) for 24h .

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A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.


B : ATP induced IL - 6 release in msEGCs measured by ELISA . Cells were treated with the indicated concentrations of ATP - and ATPɣS for 24h .

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B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.


C : Effects of P2X antagonists on ATP induced IL - 6 release . Cells were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 and P2X7 - antagonists PSB - 1011 , 5 - BD - BD and A740003 , respectively .

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C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.


D : P2X2 antagonism of ATP induced mRNA expression of IL - 6 , GFAP and RCAN by qPCR in msEGCs . Cells were treated with the P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 10µM ) for 6h .

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D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.


E : Representative confocal images of P2X2 ( green ) and GFAP ( violet ) positive msEGCs in vivo and in vitro . White arrows mark double positive ( white ) cells . Scale bar 50µm .

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E: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.


F : P2X2 - siRNA reduces P2X2 - mRNA and dampens the gliosis gene expression after ATPɣS ( 100µM ) treatment for 6h .

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F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.


G : P2X2 - siRNA reduces IL - 6 release after ATPɣS treatment ( 10µM , 100µM ) for 6h .

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G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.


A : ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation ( IM ) or were naïve .

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A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.


B : PCA plot of gene expression from POI mice at different disease stages and naïve mice ; n = 3 for each group .

B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.


F - H : qPCR analysis of indicated gliosis marker in mice that underwent IM ( n = 7 ) .

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F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).


I : Histological analysis of EGC proliferation in vivo . Representative confocal images and quantification show Sox10 ( green ) and Ki67 ( violet ) positive EGCs ( white arrows ) in the small bowel muscularis externa at naïve , IM6h and 24h . Scale bar 50µm .

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I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.


K - M : qPCR analysis of indicated gliosis markers in td + glia cells from naïve mice and mice that underwent IM ( n = 5 - 7 , POI mice ) .

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K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).


Postoperative gene expression analyses of indicated gliosis marker in the ME .

Postoperative gene expression analyses of indicated gliosis marker in the ME.


E : Histological analysis of EGC proliferation by quantification of Ki67 ( violet ) and Sox10 positive EGCs at IM24h . Mice were treated as seen in ( A ) .

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E: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).


F : Histological counting of infiltrating myeloperoxidase ( MPO ) - positive leukocytes in the postoperative ME ( n = 6 - 8 ) .

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F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).


G : FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle . CD45 , Ly6C and CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) and infiltrated monocyte - derived macrophages ( CD45 + / Ly6C + / CX3CR1 + ) .

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G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).


H : Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle .

H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.


E : IL - 6 protein release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h and 24h treatment ( n = 4 - 6 , hEGCs ) .

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E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).


F : IL - 6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations ; n = 4 - 6 , hEGCs .

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F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.


G : Immunofluorescence microscopy revealed P2X2 expression ( green ) in a majority of s100β + ( violet ) hEGCs in intact myenteric ganglia of the human colon . White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons . Scale bar , 50µm .

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G: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.


H : Effect of the P2X2 antagonism on ATP - induced IL - 6 release . ELISA measurement of IL - 6 in hEGCs upon treatment with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 200µM ) treated for 24h ( n = 6 , hEGCs ) .

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H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).


I : PSB - 1011 ( 20μM ) treatment inhibited ATP - triggered calcium responses in HEK cells transfected with P2X2 . Data are represented as ΔF / F0 + SEM ; n = 102 HEK cells . J : The P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished the ATP - triggered calcium response in HEK cells transfected with P2X2 . n = 219 HEK cells .

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I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.


K : Ambroxol blocks ATP - induced IL - 6 release in hEGCs ; Protein release measurement by ELISA of IL - 6 in hEGCs . Cells were treated with ATP ( 200 µM ) alone or together with ambroxol ( 20 µM ) for 24h ; n = 6 , hEGCs .

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K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.


L - N : qPCR analysis of several gliosis and ATP - target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human patients ) .

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L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).


( C ) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA . The dCas9 - ω was verified in the ∆ rpoZ strain background , and the rest of candidates were verified in the wild - type strain ( BW25113 ) .

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(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).


( D ) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9 - AsiA with gRNA - H3 under kanamycin selection ( 2 . 5 μg / ml ) .

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(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).


( E ) Different gRNAs were paired with dCas9 - AsiA and dCas9 - ω to profile the optimal gRNA binding distance . The dCas9 - AsiA was verified in the wild - type strain ( BW25113 ) , and dCas9 - ω was examined in the ∆ rpoZ strain background .

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(E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background.


( F ) Comparing single and multiple gRNA with dCas9 - AsiA .

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(F) Comparing single and multiple gRNA with dCas9-AsiA.


( C ) Distribution of fluorescence signal of the GFP reporter induced by different dCas9 - AsiA variants ( top ) . Fold induction by different dCas9 - AsiA variants is shown ( bottom ) .

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(C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).


( D ) CRISPRa induction of promoters with varying basal expression levels . CRISPRa - , basal expression of the promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 and associated gRNA - H4 .

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(D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.


( A ) CasTA2 . 1 upregulates a genomically inserted GFP reporter .

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(A) CasTA2.1 upregulates a genomically inserted GFP reporter.


( B ) Chromosomal gene targets activated by CasTA2 . 1 with bars showing the activation fold change and dots showing basal expression of each gene . Expression was quantified using RT - qPCR .

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(B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR.


( C ) CasTA2 . 1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene . A non - specific gRNA is used as the negative control .

(C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control.


( D ) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2 . 1 on a reporter containing GFP and mScarlet . Parental cells had low basal GFP and high basal mScarlet expression .

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(D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.


( B ) Promoters significantly activated by CasTA2 . 1 using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted with basal expression level on x - axis against fold activation by CRISPRa on y - axis . N is the total number of promoters shown . Red box corresponds to highly activated promoters ( fold change > 10 ) .

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(B) Promoters significantly activated by CasTA2.1 using gRNA-H23 (left) or gRNA-H22 (right) are plotted with basal expression level on x-axis against fold activation by CRISPRa on y-axis. N is the total number of promoters shown. Red box corresponds to highly activated promoters (fold change >10).


( C ) Highly activated promoters ( > 10 - fold ) using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted . Basal expression levels ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) and induced fold changes ( gray bars ) are shown . Promoters were first ranked and grouped by basal expression levels . Within each group , promoters were ranked by activation fold changes .

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(C) Highly activated promoters (>10-fold) using gRNA-H23 (left) or gRNA-H22 (right) are plotted. Basal expression levels (purple lines), induced expression level (orange lines on left or red lines on right, activated with gRNA-H23 or gRNA-H22, respectively) and induced fold changes (gray bars) are shown. Promoters were first ranked and grouped by basal expression levels. Within each group, promoters were ranked by activation fold changes.


( B ) CRISPRi in S . enterica and K . oxytoca using CasTA2 . 1 .

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(B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1.


( C ) CRISPRa in S . enterica and K . oxytoca using CasTA1 . 0 ( dCas9 - AsiA _ wt ) , CasTA2 . 1 ( dCas9 - AsiA _ m2 . 1 ) or ancestral strain with basal promoter expression ( none ) .

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(C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).


( B ) Western blotting of WT and Drp1 - KO MEFs with or without FCCP treatment ( 30 min ) using the indicated antibodies .

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(B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies.


( C ) Quantification of band intensity . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * 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

(C) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( B ) Western blotting of WT MEFs , Drp1 - KO MEFs , and Drp1 - KO MEFs carrying Drp1 , all of which express L2 - HA , using the indicated antibodies . The expression of L2 - HA was induced for 16 h ( 0 . 1 µg / ml doxycycline ) . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(B) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(C) Quantification of band intensity. Values are average ± SD (n = 3).


( E ) WT and Drp1 - KO MEFs , both of which express L2 - HA , were transduced with lentiviruses carrying either scramble or Oma1 - targeted shRNAs . Whole - cell lysates were analyzed by Western blotting using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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 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

(E) WT and Drp1-KO MEFs, both of which express L2-HA, were transduced with lentiviruses carrying either scramble or Oma1-targeted shRNAs. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( F ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(F) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) The mitochondrial membrane potential was measured in WT and Drp1 - KO MEFs using a membrane potential - dependent dye ( MitoLite NIR ) and flow cytometry .

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(A) The mitochondrial membrane potential was measured in WT and Drp1-KO MEFs using a membrane potential-dependent dye (MitoLite NIR) and flow cytometry.


( B ) Drp1 - KO MEFs carrying Su9 - GFP were treated with DMSO ( control ) or 10 nM antimycin A or 10 ng / ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The arrows indicate mitochondria that showed flickering . Three frames from the time - lapse analysis are shown ( please also see Movie EV1 - EV4 ) . Scale bar , 10 µm .

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(B) Drp1-KO MEFs carrying Su9-GFP were treated with DMSO (control) or 10 nM antimycin A or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1-EV4). Scale bar, 10 µm.


The percentage of cells that showed flickering in 30 min . Values are average ± SD ( n = 3 experiments ) . In each experiment , 25 - 89 cells were analyzed .

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The percentage of cells that showed flickering in 30 min. Values are average ± SD (n = 3 experiments). In each experiment, 25-89 cells were analyzed.


( F ) Drp1 - KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng / ml oligomycin or 10 µM FCCP for 1 h . The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry .

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(F) Drp1-KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng/ml oligomycin or 10 µM FCCP for 1 h. The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry.


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( H ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

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(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (H) Quantification of band intensity. Values are average ± SD (n = 3).


( G ) Drp1 - KO MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 nM antimycin A along with 10 µM FCCP . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 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 O O O O O B-MEASURED_VAR O O

(G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( J ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

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

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (J) Quantification of band intensity. Values are average ± SD (n = 3).


( I ) Western blotting of Drp1 - KO MEFs with or without 10 ng / ml oligomycin treatment . The asterisk indicates non - specific bands of anti - Oma1 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 B-MEASURED_VAR O O

(I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( K ) WT MEFs carrying Su9 - GFP were treated with DMSO or 10 ng / ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The arrows indicate mitochondria that showed flickering . Three frames from the time - lapse analysis are shown ( please also see Movie EV1 and EV5 ) . Scale bar , 10 µm .

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(K) WT MEFs carrying Su9-GFP were treated with DMSO or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1 and EV5). Scale bar, 10 µm.


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( M ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

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

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (M) Quantification of band intensity. Values are average ± SD (n = 3).


( L ) WT MEFs expressing L2 - HA ( induced for 16 h ) were treated with 10 ng / ml oligomycin . Western blotting was performed using the indicated antibodies . The asterisk indicates non - specific bands of anti - Oma1 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 B-MEASURED_VAR O O

(L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of Drp1flox / floxOpa1flox / flox and Drp1Opa1 - KO MEFs . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3).


( A ) Western blotting of Drp1flox / floxOpa1flox / flox and Drp1Opa1 - KO MEFs . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(A) Western blotting of Drp1flox/floxOpa1flox/flox and Drp1Opa1-KO MEFs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C ) Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs were transduced with the indicated Opa1 constructs . The cells were observed using laser scanning confocal microscopy for 30 min with 10 - s intervals in the presence of TMRE . The percentage of cells that exhibited flickering is shown . Values are average ± SD ( n = 3 experiments ) . In each experiment , 48 - 62 cells were analyzed .

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(C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed.


( D ) Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti - PDH antibodies . Scale bar , 10 µm .

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(D) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs carrying the indicated Opa1 constructs were subjected to confocal immunofluorescence microscopy using anti-PDH antibodies. Scale bar, 10 µm.


( E ) Quantification of mitochondrial morphology is shown ( n = 3 experiments ) . In each experiment , 30 cells were analyzed .

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

(E) Quantification of mitochondrial morphology is shown (n = 3 experiments). In each experiment, 30 cells were analyzed.


( G ) Western blotting of Drp1flox / floxOpa1flox / flox MEFs and Drp1Opa1 - KO MEFs , both of which carry the indicated Opa1 constructs . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

(G) Western blotting of Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs, both of which carry the indicated Opa1 constructs. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( H ) Quantification of band intensity . Values are average ± SD ( n = 3 ) .

(H) Quantification of band intensity. Values are average ± SD (n = 3).


( B ) Three frames from the time - lapse analysis during artificial flickering are shown . The boxed regions are magnified . Scale bar , 5 µm .

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(B) Three frames from the time-lapse analysis during artificial flickering are shown. The boxed regions are magnified. Scale bar, 5 µm.


( C ) The fluorescence intensity of Su9 - GFP and TMRE in mitochondria was quantified .

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(C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified.


( D ) Western blotting of WT MEFs after the indicated FCCP treatments . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( E ) Quantification of band intensity . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * 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 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

(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (E) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001.


( D ) Western blotting of WT MEFs after the indicated FCCP treatments . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( A ) Western blotting of WT MEFs and Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) and / or ectopic Mfn1 . The asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) Quantification of band intensity is shown . Values are average ± SD ( n = 3 ) .

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(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (B) Quantification of band intensity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of WT MEFs and Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) and / or ectopic Mfn1 . The asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(A) Western blotting of WT MEFs and Drp1-KO MEFs, both of which carry shRNAs (Oma1 or scramble) and/or ectopic Mfn1. The asterisk indicates non-specific bands of anti-Oma1 antibodies.


( C - F ) WT MEFs ( C and E ) and Drp1 - KO MEFs ( D and F ) were transduced with Su9 - Eos , along with Oma1 shRNAs and ectopic Mfn1 . Su9 - Eos in the small region of mitochondria ( indicated by the asterisks ) was photoconverted using a 405 - nm laser on confocal microscope . Within 1 s after photoconversion , images were obtained for both photoconverted and unconverted Su9 - Eos signals . Images before the photoconversion were also taken to detect background signals . Scale bar , 10 µm . ( E and F ) To determine the connectivity of mitochondria , the area containing photoconverted Su9 - Eos signals was divided by the area containing the photoconverted and unconverted Su9 - Eos signals ( total mitochondria ) . Values are average ± SD ( n = 30 cells ) .

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

(C-F) WT MEFs (C and E) and Drp1-KO MEFs (D and F) were transduced with Su9-Eos, along with Oma1 shRNAs and ectopic Mfn1. Su9-Eos in the small region of mitochondria (indicated by the asterisks) was photoconverted using a 405-nm laser on confocal microscope. Within 1 s after photoconversion, images were obtained for both photoconverted and unconverted Su9-Eos signals. Images before the photoconversion were also taken to detect background signals. Scale bar, 10 µm. (E and F) To determine the connectivity of mitochondria, the area containing photoconverted Su9-Eos signals was divided by the area containing the photoconverted and unconverted Su9-Eos signals (total mitochondria). Values are average ± SD (n = 30 cells).


WT which carry shRNAs ( Oma1 or scramble ) and ectopic Mfn1 , were stained with TMRE . The percentage of cells that maintained the membrane potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 cells were analyzed in each experiment .

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WT which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


Drp1 - KO MEFs which carry shRNAs ( Oma1 or scramble ) and ectopic Mfn1 , were stained with TMRE . The arrows indicate cells that lost the mitochondrial membrane potential in ( C ) . Scale bar , 10 µm . The percentage of cells that maintained the membrane potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 cells were analyzed in each experiment .

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Drp1-KO MEFs which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment.


OCRs were analyzed in the same set of MEFs . Quantification of the basal OCRs and respiratory capacity is shown . Values are average ± SD ( n = 3 ) .

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

OCRs were analyzed in the same set of MEFs. Quantification of the basal OCRs and respiratory capacity is shown. Values are average ± SD (n = 3).


( A ) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies . ( B ) Quantification of band intensity . Values are average ± SD ( n = 3 mice for each genotype ) . Significance was calculated using ANOVA with post - hoc Tukey : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) Western blotting of livers isolated from the indicated mice at 3 months of age using the indicated antibodies. (B) Quantification of band intensity. Values are average ± SD (n = 3 mice for each genotype). Significance was calculated using ANOVA with post-hoc Tukey: *p<0.05, **p<0.01, ***p<0.001.


( A ) HEK293A cell lines stably expressing GFP , GFP - WIPI1a , and GFP - WIPI2b were incubated in full medium ( F ) or starvation medium ( EBSS ) ( S ) for 2 hr before being used for GFP - TRAP pull - down .

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(A) HEK293A cell lines stably expressing GFP, GFP-WIPI1a, and GFP-WIPI2b were incubated in full medium (F) or starvation medium (EBSS) (S) for 2 hr before being used for GFP-TRAP pull-down.


( B ) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP , at the indicated concentrations , before immunoblotting .

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(B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting.


( C ) WT or Atg16L1Δ / Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti - WIPI2 antibody . Scale bars , 10 μm . ( D ) WIPI2 puncta in ( C ) were counted , and a statistical analysis of WIPI2 puncta was performed using an unpaired Student ' s t test . ∗ p < 0 . 05 . SEM for n = 3 .

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(C) WT or Atg16L1Δ/Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti-WIPI2 antibody. Scale bars, 10 μm.(D) WIPI2 puncta in (C) were counted, and a statistical analysis of WIPI2 puncta was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( E - H ) CLEM ( correlative light and electron microscopy ) of endogenous WIPI2 in Atg16L1Δ / Δ MEFs . ( E ) Merged phase and confocal section of Atg16L1Δ / Δ cells labeled with anti - WIPI2 antibody . Scale bar , 10 μm . ( F ) Low - magnification TEM of cell in ( E ) . Scale bar 10μm . ( G and H ) High magnification of boxed regions in ( E ) and ( F ) . Top box and bottom box indicate panels ( G ) and ( H ) , respectively . Arrows indicate open phagophores . M , mitochondria ; ER , endoplasmic reticulum . Scale bars , 1 μm ( G ) and 0 . 5 μm ( H ) .

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(E-H) CLEM (correlative light and electron microscopy) of endogenous WIPI2 in Atg16L1Δ/Δ MEFs.(E) Merged phase and confocal section of Atg16L1Δ/Δ cells labeled with anti-WIPI2 antibody. Scale bar, 10 μm.(F) Low-magnification TEM of cell in (E). Scale bar 10μm.(G and H) High magnification of boxed regions in (E) and (F). Top box and bottom box indicate panels (G) and (H), respectively. Arrows indicate open phagophores. M, mitochondria; ER, endoplasmic reticulum. Scale bars, 1 μm (G) and 0.5 μm (H).


( I and J ) 2GL9 cells ( GFP - LC3 HEK293 cells ; Chan et al . , 2007 ) were starved for 2 hr before visualization using indicated antibodies . Scale bars , 10 μm .

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(I and J) 2GL9 cells (GFP-LC3 HEK293 cells; Chan et al., 2007) were starved for 2 hr before visualization using indicated antibodies. Scale bars, 10 μm.


( K ) Live - cell imaging demonstrates that Atg16L1 translocates to WIPI2b - positive puncta in starvation . HEK293 cells expressing GFP - WIPI2b and mCherry - Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope . See also Figure S1 and Movie S1 .

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(K) Live-cell imaging demonstrates that Atg16L1 translocates to WIPI2b-positive puncta in starvation. HEK293 cells expressing GFP-WIPI2b and mCherry-Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope. See also Figure S1 and Movie S1.


( B ) Untransfected ( UN ) or FLAG - Atg16L1 full - length ( FL ) , 79 - 623 , or 1 - 265 constructs were expressed in HEK293A cells stably expressing GFP - WIPI2b and immunoprecipitated using GFP - TRAP . Tags were visualized by immunoblotting . I , input ; UB , unbound .

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(B) Untransfected (UN) or FLAG-Atg16L1 full-length (FL), 79-623, or 1-265 constructs were expressed in HEK293A cells stably expressing GFP-WIPI2b and immunoprecipitated using GFP-TRAP. Tags were visualized by immunoblotting. I, input; UB, unbound.


( C ) GFP , CFP - Atg5 , GFP - WIPI1a , and GFP - WIPI2b were transiently expressed in HEK293A cells . GFP - tagged proteins were isolated using GFP - TRAP and incubated with in vitro translated 35S - labeled FLAG - Atg16L1 constructs 1 - 265 , 1 - 242 , 1 - 230 , and 1 - 207 before washing and analysis by autoradiography . Protein expression was validated by immunoblot ( bottom panel ) .

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

(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1-265, 1-242, 1-230, and 1-207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).


( D ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI1a , or GFP - WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG - Atg16L1 or Atg16L2 . Protein complexes were immunoprecipitated using GFP - TRAP , followed by immunoblot . See also Figure S2 and Table S1 .

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

(D) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, or GFP-WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 or Atg16L2. Protein complexes were immunoprecipitated using GFP-TRAP, followed by immunoblot. See also Figure S2 and Table S1.


( B ) Lysates from HEK293A cells transfected with GFP - WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG - Atg16L2 , FLAG - Atg16L1 , or Flag - Atg16L1 mutants , immunoprecipitated , and analyzed by immunoblotting . ( C ) Statistical analysis of FLAG - Atg16L1 binding in ( B ) was performed by Student ' s t test . SEM for n = 4 . ∗ p < 0 . 05 .

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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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) Lysates from HEK293A cells transfected with GFP-WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG-Atg16L2, FLAG-Atg16L1, or Flag-Atg16L1 mutants, immunoprecipitated, and analyzed by immunoblotting.(C) Statistical analysis of FLAG-Atg16L1 binding in (B) was performed by Student's t test. SEM for n = 4. ∗p < 0.05.


( D ) Cell lysates from HEK293A cells transiently expressing FLAG - Atg16L2 , FLAG - Atg16L1 , or FLAG - Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads . Protein complexes were analyzed using immunoblotting . ( E ) Statistical analysis of FIP200 binding in ( D ) was performed by Student ' s t test . SEM for n = 3 . ∗ p < 0 . 05 .

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

(D) Cell lysates from HEK293A cells transiently expressing FLAG-Atg16L2, FLAG-Atg16L1, or FLAG-Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads. Protein complexes were analyzed using immunoblotting.(E) Statistical analysis of FIP200 binding in (D) was performed by Student's t test. SEM for n = 3. ∗p < 0.05.


( F ) WIPI2 was immunoprecipitated from lysates from WT or FIP200 − / − MEFS after treatment with 0 . 5 mM DSP . Bound Atg16 and Atg12 - 5 were detected by immunoblotting .

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

(F) WIPI2 was immunoprecipitated from lysates from WT or FIP200−/− MEFS after treatment with 0.5 mM DSP. Bound Atg16 and Atg12-5 were detected by immunoblotting.


( G ) HEK293 cells stably expressing GFP - WIPI2b were and transiently transfected either FLAG - Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) . Bound and input ( bottom ) were analyzed by immunoblotting .

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

(G) HEK293 cells stably expressing GFP-WIPI2b were and transiently transfected either FLAG-Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR). Bound and input (bottom) were analyzed by immunoblotting.


( A ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2a , GFP - WIPI2b , or GFP - WIPI2b FTTG mutant were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( B ) Statistical analysis of ( A ) was performed by one - way ANOVA with Tukey ' s posttest . SEM for n = 3 . ∗ p < 0 . 05 .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2a, GFP-WIPI2b, or GFP-WIPI2b FTTG mutant were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 3. ∗p < 0.05.


( D ) GFP - Trap from HEK293A cells transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2b , GFP - WIPI2b R108E , GFP - WIPI2b R125E , or GFP - WIPI2b R108E R125E was mixed with in vitro translated 35S - labeled FLAG - Atg16L1 and analyzed by autoradiography .

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

(D) GFP-Trap from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2b, GFP-WIPI2b R108E, GFP-WIPI2b R125E, or GFP-WIPI2b R108E R125E was mixed with in vitro translated 35S-labeled FLAG-Atg16L1 and analyzed by autoradiography.


( E ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI2b , GFP - WIPI2b ΔCT , GFP - WIPI2a , GFP - WIPI1a , or GFP - WIPI1a ΔCT were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( F ) Statistical analysis of ( E ) was performed by one - way ANOVA with Tukey ' s posttest . SEM for n = 2 . ∗ p < 0 . 05 .

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

(E) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI2b, GFP-WIPI2b ΔCT, GFP-WIPI2a, GFP-WIPI1a, or GFP-WIPI1a ΔCT were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(F) Statistical analysis of (E) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 2. ∗p < 0.05.


( G ) Lysates from HEK293A cells transiently expressing GFP - WIPI2b WT , GFP - WIPI2b R108E , R125E , or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG - Atg16L1 WT , E226R , E230R , or E226R E230R in all possible permutations . Protein complexes from mixed lysates were immunoprecipitated using GFP - Trap , followed by immunoblot analysis . ( H ) Statistical analysis of ( G ) was performed by one - way ANOVA with Tukey ' s posttest . SEM from n = 3 . ∗ p < 0 . 05 .

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

(G) Lysates from HEK293A cells transiently expressing GFP-WIPI2b WT, GFP-WIPI2b R108E, R125E, or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 WT, E226R, E230R, or E226R E230R in all possible permutations. Protein complexes from mixed lysates were immunoprecipitated using GFP-Trap, followed by immunoblot analysis.(H) Statistical analysis of (G) was performed by one-way ANOVA with Tukey's posttest. SEM from n = 3. ∗p < 0.05.


( I ) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP - WIPI2b RERE and starved in EBSS . Bright - field image ( top ) , GFP - WIPI2 RERE signal ( middle ) , TEM of selected cell ( bottom ) . Boxed area shown in ( J ) . Scale bars represent 10 μM for bright - field and confocal and 5 μM for TEM . ( J ) High - magnification TEM of boxed area in ( I ) . Arrows indicate open phagophores in the vicinity of ER and mitochondria ( M ) , which contain GFP - WIPI2b RERE . See also Figures S4 and S5 and Table S1 .

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

(I) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP-WIPI2b RERE and starved in EBSS. Bright-field image (top), GFP-WIPI2 RERE signal (middle), TEM of selected cell (bottom). Boxed area shown in (J). Scale bars represent 10 μM for bright-field and confocal and 5 μM for TEM.(J) High-magnification TEM of boxed area in (I). Arrows indicate open phagophores in the vicinity of ER and mitochondria (M), which contain GFP-WIPI2b RERE. See also Figures S4 and S5 and Table S1.


( A ) GFP , siRNA - resistant GFP - WIPI2b , or GFP - WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC - free ( RF ) or WIPI2 siRNA . Cells were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot analysis . ( B ) Statistical analysis of ( A ) . SEM for n = 3 . Statistical analysis was performed by one - way ANOVA with Tukey ' s posttest . ∗ p < 0 . 05 .

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

(A) GFP, siRNA-resistant GFP-WIPI2b, or GFP-WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(B) Statistical analysis of (A). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Tukey's posttest. ∗p < 0.05.


( C ) GFP , siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA . Cells were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot analysis . ( D ) Statistical analysis of ( C ) was performed by one - way ANOVA with Tukey ' s posttest . The SEM for LC3 ( n = 2 ) and p62 ( n = 4 ) are shown . ∗ p < 0 . 05 .

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

(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.


( E ) siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA . Cells were starved in EBSS for 2 hr without or with BafA , fixed , and labeled , and LC3 was visualized by confocal microscopy . Scale bars , 10 μm . ( F ) Quantification of WIPI2 puncta per cell from ( E ) . SEM from 10 cells per condition ; ∗ ∗ ∗ p < 0 . 001 using one - way ANOVA . ( G ) Statistical analysis of LC3 puncta from ( E ) with GFP control ( not shown in E ) . SEM for n = 3 . Statistical analysis was performed by one - way ANOVA with Dunn ' s posttest t test . ∗ p < 0 . 05 . See also Table S1 .

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

(E) siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were starved in EBSS for 2 hr without or with BafA, fixed, and labeled, and LC3 was visualized by confocal microscopy. Scale bars, 10 μm.(F) Quantification of WIPI2 puncta per cell from (E). SEM from 10 cells per condition; ∗∗∗p < 0.001 using one-way ANOVA.(G) Statistical analysis of LC3 puncta from (E) with GFP control (not shown in E). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Dunn's posttest t test. ∗p < 0.05. See also Table S1.


( A ) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium ( F ) , starvation medium ( S ) , or starvation medium with wortmannin ( W ) for 2 hr before immunoblot analysis . ( B ) Statistical analysis of ( A ) was performed by one - way ANOVA with Tukey ' s post hoc test . SEM for n = 3 . ∗ p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( C ) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP - Trap before analysis by immunoblotting .

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) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP-Trap before analysis by immunoblotting.


( D ) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in ( A ) were fixed and visualized by confocal microscopy .

O 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) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in (A) were fixed and visualized by confocal microscopy.


( E ) CLEM of MCF7 cells expressing GFP - LC3 and mCherry - WIPI2b CAAX . Bright - field and confocal image merged ( left ) , confocal of expressing cell ( middle ) , low - magnification TEM of cell of interest ( right ) . Boxed area indicates plasma membrane region showing colocalization of GFP - LC3 and mCherry - WIPI2b . Scale bar , 10 μm ( n = 3 ) . ( F ) High - magnification TEM showing plasma membrane region boxed in ( E ) . Magnified insets , in top right , show small vesicular clusters under the plasma membrane detected in mCherry - WIPI2b CAAX , GFP - LC3 expressing cells but not in untransfected cells from a control experiment .

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

(E) CLEM of MCF7 cells expressing GFP-LC3 and mCherry-WIPI2b CAAX. Bright-field and confocal image merged (left), confocal of expressing cell (middle), low-magnification TEM of cell of interest (right). Boxed area indicates plasma membrane region showing colocalization of GFP-LC3 and mCherry-WIPI2b. Scale bar, 10 μm (n = 3).(F) High-magnification TEM showing plasma membrane region boxed in (E). Magnified insets, in top right, show small vesicular clusters under the plasma membrane detected in mCherry-WIPI2b CAAX, GFP-LC3 expressing cells but not in untransfected cells from a control experiment.


( G ) HEK293A cells treated with either RISC - free ( RF ) , Atg16L1 siRNA , or FIP200 siRNA for 72 hr before transfection with HA - WIPI2b - CAAX were incubated in full medium ( F ) , EBSS ( S ) , or EBSS with wortmannin ( W ) for 2 hr before immunoblot analysis . Please note that all WIPI2 constructs are FTTG mutants . See also Figure S6 and Table S1 .

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

(G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.


( A ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , ERER , or DRDR . After 2 hr in EBSS , cells were fixed and labeled with anti - LC3 and anti - FLAG antibodies and visualized by confocal microscopy . Scale bars , 20 μm . ( B ) Statistical analysis of ( A ) was performed using one - way ANOVA with Dunn ' s posttest . ∗ p < 0 . 05 . SEM for n = 3 .

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

(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3.


( C ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) before analysis by immunoblotting . ( D ) Statistical analysis of ( C ) was performed by one - way ANOVA with Tukey ' s post hoc test . SEM for n = 3 . ∗ p < 0 . 05 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( E ) HEK293A cells were treated with either RISC - free control or WIPI2 siRNA before infection with Salmonella ( moi = 100 ) for 1 hr , labeled with anti - LC3 , WIPI2 , and p62 antibodies , and followed by confocal analysis . Scale bars , 5 μm . ( F ) Statistical analysis for ( E ) was performed using an unpaired Student ' s t test . ∗ p < 0 . 05 . SEM for n = 3 .

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

(E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.(F) Statistical analysis for (E) was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( G ) Atg16L1Δ / Δ MEFs were transiently transfected with FLAG - Atg16L1 WT , FLAG - Atg16L1 ERER , or FLAG - Atg16L2 DRDR 24 hr before being infected with Salmonella ( moi = 25 ) for 1 hr , labeled with anti - LC3 , anti - p62 , and anti - FLAG antibodies , and followed by analysis by confocal microscopy . Scale bars , 5 μm . ( H ) Statistical analysis of ( G ) was performed by one - way ANOVA with Dunn ' s post hoc test . SEM for n = 4 . ∗ p < 0 . 05 . See also Figure S7 .

O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn's post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.


A RT - qPCR reveals that miR - 210 was increased in lungs of mice with PH triggered by various conditions : VHL − / − as compared with VHL + / + mice ( N = 4 / group ) , * * * P < 0 . 0001 ( first graph ) ; hypoxia + SU5416 ( Hyp + SU5416 ) ( N = 6 / group ) as compared with normoxia + SU5416 ( Norm + SU5416 ) ( N = 7 / group ) , * * P = 0 . 0015 ( second graph ) ; Il6 transgenic versus littermate control mice ( N = 4 / group ) , * * P = 0 . 0097 ( third graph ) ; and S . mansoni - infected mice ( N = 4 ) compared with non - infected control mice ( N = 5 ) , * * * P < 0 . 0001 ( fourth graph ) .

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 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 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

A RT-qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).


B From animal subjects in ( A ) , in situ hybridization ( ISH , purple stain ) revealed increased miR - 210 in < 100 - μm pulmonary vessels of mice suffering from PH , * P = 0 . 0493 for first graph , * * P = 0 . 0015 for second graph , * P = 0 . 0391 for third graph .

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B From animal subjects in (A), in situ hybridization (ISH, purple stain) revealed increased miR-210 in < 100-μm pulmonary vessels of mice suffering from PH, *P = 0.0493 for first graph, **P = 0.0015 for second graph, *P = 0.0391 for third graph.


C Representative ISH stain of miR - 210 in < 100 μm pulmonary vessels of mice ( bottom micrographs ) exposed to Hyp + SU5416 compared with Norm + SU5416 ( α - smooth muscle actin stain from serial sections , top row of micrographs ) .

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C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).