Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA _ KO mutants versus WT . Of 759 predicted miRNA target genes , 728 ( 96 % ) were detectable in ribosome profiling data appear in plot . Data information : In A , student ' s t - was applied to assess significance for increase in ribosome occupancy of 728 / 759 miRNA targets ( p - value < 1e51 for every mutant ) .

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Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot. Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant).


Top : Immunoblot of CIC in WT , Drosha _ KO , Dicer _ KO Ago2 & 1 _ KO mESCs . TUBULIN was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of CIC , normalized to TUBULIN relative to WT sample in three biological replicates . Data information : Bar graph in Fig 2F shows mean of CIC signal ± SD normalized to TUBULIN . Values are relative to WT , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 .

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Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates. Data information: Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.


MA plot of DGE in miR - 290 - 295 _ KO mESCs . Data information : In A , significant genes in MA plot were determined using an adjusted p - value of 0 . 1 .

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MA plot of the DGE analysis in miR-290-295_KO mESCs. Data information: In A, significant genes in MA plot were determined using an adjusted p-value of 0.1.


F , G . Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR - 290 - 295 . In addition to data shown described in Fig 1D , E , a second AGO2 - binding dataset , on cross - linking immunoprecipitation sequencing ( CLIP - seq ) , has been integrated . Response elements for miR - 290 - 295 cluster members are denoted in red labeled .

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F, G. Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR-290-295. In addition to the data shown and described in Fig 1D, E, a second AGO2-binding dataset, based on cross-linking immunoprecipitation and sequencing (CLIP-seq), has been integrated. Response elements for miR-290-295 cluster members are denoted in red and labeled.


Top : Immunoblot of TFAP4 in WT , Drosha _ KO , Dicer _ KO , Ago2 & 1 _ KO miR - 290 - 295 _ KO mESCs . LAMINB1 was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of CIC , normalized to LAMINB1 relative to WT sample in three biological replicates . Data information : In H bar graphs show mean of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

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Top: Immunoblot analysis of TFAP4 in WT, Drosha_KO, Dicer_KO, Ago2&1_KO and miR-290-295_KO mESCs. LAMINB1 was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to LAMINB1 and relative to the WT sample in three biological replicates. Data information: In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Immunoblot of TFAP4 after transfection of miRNA mimics ( miR - 291a - 5p , miR - 291a - 3p miR - 291a - 5p + miR - 291a - 3p combined ) in miR - 290 - 295 _ KO mESCs . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing of TFAP4 , normalized to Coomassie relative to WT sample in three biological replicates . Data information : In I , bar graphs show mean of TFAP4 signal ± SD normalized to Coomassie . Values are relative to WT or negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - comparing each value to WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

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Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Mature sequences of 5p 3p miRNAs of miR - 290 - 295 family mature of human miR - 302c - 3p . Colors indicate identical seed sequences . Bottom : AGO2 CLIP - seq shows a peak at miR - 302c - 3p binding site on human TFAP4 .

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Top: Mature sequences of 5p and 3p miRNAs of the miR-290-295 family and the mature sequence of the human miR-302c-3p. Colors indicate identical seed sequences. Bottom: AGO2 CLIP-seq shows a peak at the miR-302c-3p binding site on human TFAP4.


Immunoblot validation of siPOOL - mediated knock down of Tfap4 . TFAP4 were compared between untreated WT versus miR - 290 - 295 _ KO treated with a negative control a Tfap4 - targeted siPOOL . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of two biological replicates .

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Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.


Scatterplot of DEG in miR - 290 - 295 _ KO control versus siPOOL - Tfap4 . SiPOOL experiments were performed in miR - 290 - 295 _ KO DE was assessed relative to a negative control siPOOL transfection . Only genes that are statistically significantly differentially expressed in miR - 290 - 295 _ KO are shown . Genes predicted to be targeted by miR - 290 - 295 are marked in blue . Genes are defined as rescued ( orange ) if log2FoldChange - between miR - 290 - 295 _ KO control siPOOL - Tfap4 is in range [ - 2 , - 1 / 2 ] . Tfap4 is marked by a red dot .

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Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.


Representative of NANOG ( red ) OCT4 ( green ) immunofluorescence in WT , Drosha _ KO Drosha & Tfap4 _ KO mESCs . Scale bar = 20 μm . of NANOG signal in WT , Drosha _ KO Drosha & Tfap4 _ KO mESCs show no significant rescue of NANOG in Drosha & Tfap4 _ KO Data information : In E , box plots show of NANOG in ~ 300 per genotype . P - values were generated using ordinary one - way ANOVA . * * * * p - value < 0 . 0001 . In panel E , box plot ' s central band represents median relative for each genotype . boxes represent interquartile range whiskers represent maximum minimum relative values .

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Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm. Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO Data information: In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.


Confocal of mPS - PI stained 6 - day - old seedlings of Col - 0 ( A ) , bravo - 2 ( B ) , wox5 - 1 ( C ) bravo - 2 wox5 - 1 ( D ) mutants . Left black arrows indicate QC right arrows indicate CSC . Scale bar : 50 µm .

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Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.


of QC divisions in 6 - day - old roots expressed in ( n > 50 , 3 replicates ) . D : QC divided ; ND : QC non divided .

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Quantification of the QC divisions in 6-day-old roots expressed in percentage (n>50, 3 replicates). D: QC divided; ND: QC non divided.


of CSC layers in 6 - day - old roots expressed in ( n > 50 , 3 replicates ) .

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Quantification of CSC layers in 6-day-old roots expressed in percentage (n>50, 3 replicates).


Lateral root density ( number of lateral roots per mm of root ) of 7 - day - olf WT , bravo - 2 , wox5 - 1 bravo - 2 wox5 - 1 mutants ( n > 40 , 2 replicates ) . Different letters indicate statistically significant differences ( p - value < 0 . 05 Student ´ s t - ) . In boxplot , box width represents interquartile range ( IQR = Q3 - Q1 ) , with horizontal denoting median , while whiskers extend from Q1 - 1 . 5IQR to Q3 + 1 . 5IQR . dots are outliers .

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Lateral root density (number of lateral roots per mm of root length) of 7-day-olf WT, bravo-2, wox5-1 and bravo-2 wox5-1 mutants (n>40, 2 replicates). Different letters indicate statistically significant differences (p- value < 0.05 Student´s t-test). 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.


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

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

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

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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.


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

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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 showing BRAVO interacting with WOX5 , BES1 - D TPL ; BES1 - D interacting with TPL . In left column yeast were grown on control media , in right column yeast were grown on control media lacking Leu , Trp His , indicating an interaction between .

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

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

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

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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 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 infected with 50 MOI S . Typhimurium for 2 h , lysates , culture supernatants ( SN ) were collected immunoblotted with indicated antibodies .

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

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(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 - / - Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) lysates were collected immunoblotted with indicated antibodies .

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

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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 - / - NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) lysates were collected immunoblotted with 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 - / - 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 mean ± ; determined by Student ' s t - ; * * , P < 0 . 01 .

B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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 - / - NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium burden was detected in spleens ( D ) , PCF ( E ) , blood ( F ) of WT , Vdr - / - , NLRP3 - / - , NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as mean ± ; determined by Student ' s t - ; * * , P < 0 . 01 .

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

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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 were immunoblotted with indicated antibodies ( A ) . Whole - lysates were shown as input . Data are representative of three independent experiments .

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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 ; were immunoprecipitated analyzed by immunoblotting with indicated antibodies . Whole - lysates were shown as 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 subjected to immunoblot . 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 ) ) HA - VDR were expressed in HEK293T , immunoprecipitated , 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 ) Myc - NLRC4 were expressed in HEK293T , immunoprecipitated , analyzed by immunoblotting with indicated antibodies . Data are representative of three independent experiments .

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

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

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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 ) Myc - NLRC4 were expressed in HEK293T , immunoprecipitated with anti - Flag antibody , analyzed by immunoblotting with indicated antibodies .

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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 ) Flag - NLRC4 , Myc - PrgJ , HA - NAIP2 in HEK293T , immunoprecipitated with anti - HA antibody , 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 , VDR - K123A , infected with S . Typhimurium at an MOI of 50 for 2 h , lysates ( Lysate ) culture supernatants ( SN ) were collected immunoblotted with 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 ) TNF - ɑ secretion ( H ) in supernatants . Data are shown as means ± ; determined by Student ' s t - ; * * , 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 of four histone in each tissue replicate ( order of appearance per replicate : H2A , H2B , H3 . 3 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 from Human Atlas ( www . proteinatlas . org ) for ANXA1 with ( 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 four histone peptides , with decreasing amount of QPrEST standard spiked into a U2OS 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 in kidney tissue human embryonal kidney ( HEK293 ) , liver tissue liver cancer ( HepG2 ) , lung tissue lung cancer ( A549 ) , breast tissue breast cancer ( MCF7 ) . order of are same in tissue corresponding have been order according to abundance in 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 . direct correlation between ( TPM ) abundances ( copy number ) for quantified genes in same tissues lines . Spearman ( ρ ) Pearson ( r ) correlation between two values across quantified genes are shown . other seven tissues five 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 immunohistochemistry in tissue sections by three different antibodies ( SELENBP1 , HPA011731 ; STOM , HPA010961 ; ASS1 , HPA020896 ) . Microtubule - probes are visualized in red blue respectively . Antibody is shown in green . to across nine lines 11 tissues with Spearman rho , Pearson r R2 for each gene . 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 ATP - treated msEGCs .

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


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

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


J : qPCR 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 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 . were treated with P2 - antagonist suramin ( 1 , 10 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 . were treated with indicated concentrations of ATP - 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 . were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 P2X7 - antagonists PSB - 1011 , 5 - BD - BD 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 RCAN by qPCR in msEGCs . were treated with 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 of P2X2 ( green ) GFAP ( violet ) positive msEGCs in vivo in vitro . arrows mark double positive ( ) . 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 dampens 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 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 naïve mice ; n = 3 for each group .

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


F - H : qPCR 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 of EGC proliferation in vivo . Representative confocal show Sox10 ( green ) Ki67 ( violet ) positive EGCs ( arrows ) in small bowel muscularis externa at naïve , IM6h 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 of indicated gliosis markers in td + glia from naïve mice 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 ME .

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


E : Histological of EGC proliferation by of Ki67 ( violet ) 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 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 of infiltrating in ME of mice treated with ambroxol or vehicle . CD45 , Ly6C CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) 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 .

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


E : IL - 6 release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h 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 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 human colon . arrows mark double positive glia 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 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 transfected with P2X2 . Data are represented as ΔF / F0 + ; n = 102 HEK . J : P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished ATP - triggered calcium response in HEK transfected with P2X2 . n = 219 HEK .

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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 ; release measurement by ELISA of IL - 6 in hEGCs . 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 of several gliosis ATP - target genes in mechanically manipulated surgical specimens incubated in presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human ) .

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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 of CasTA candidates using different gRNAs targeting to different locations of GFP reporter gene compared to a without CasTA . dCas9 - ω was verified in ∆ rpoZ background , of candidates were verified in wild - type ( 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 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 dCas9 - ω to profile optimal gRNA binding . dCas9 - AsiA was verified in wild - type ( BW25113 ) , dCas9 - ω was examined in ∆ rpoZ 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 multiple gRNA with dCas9 - AsiA .

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


( C ) Distribution of signal of 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 . CRISPRa - , basal expression of promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 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 activation fold change 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 target gene . A non - specific gRNA is used as negative control .

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(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 CRISPRi using CasTA2 . 1 on a reporter containing GFP mScarlet . Parental had low basal GFP 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 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 ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) induced fold changes ( gray bars ) are shown . Promoters were first ranked grouped by basal expression . 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 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 K . oxytoca using CasTA1 . 0 ( dCas9 - AsiA _ wt ) , CasTA2 . 1 ( dCas9 - AsiA _ m2 . 1 ) or ancestral 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 Drp1 - KO MEFs with or without FCCP treatment ( 30 min ) using 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 ) of band . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

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(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 , Drp1 - KO MEFs carrying Drp1 , of which express L2 - HA , using indicated antibodies . expression of L2 - HA was induced for 16 h ( 0 . 1 µg / ml doxycycline ) . 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 ) of band . Values are average ± SD ( n = 3 ) .

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


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

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

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


( A ) mitochondrial potential was measured in WT Drp1 - KO MEFs using a potential - dependent dye ( MitoLite NIR ) 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 viewed by scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . arrows indicate mitochondria that showed flickering . Three frames from time - lapse 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.


of that showed flickering in 30 min . Values are average ± SD ( n = 3 experiments ) . In each experiment , 25 - 89 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 . mitochondrial potential was analyzed using MitoLite NIR 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 indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( H ) of band . 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 indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies .

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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.


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

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(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 . asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(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 viewed by scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . arrows indicate mitochondria that showed flickering . Three frames from time - lapse are shown ( please also see Movie EV1 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 indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( M ) of band . Values are average ± SD ( n = 3 ) .

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(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 indicated antibodies . asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(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 Drp1Opa1 - KO MEFs . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) of band . Values are average ± SD ( n = 3 ) .

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(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 Drp1Opa1 - KO MEFs . asterisk indicates non - specific bands of anti - Oma1 antibodies .

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(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 Drp1Opa1 - KO MEFs were transduced with indicated Opa1 constructs . were observed using scanning confocal microscopy for 30 min with 10 - s intervals in presence of TMRE . of that exhibited flickering is shown . Values are average ± SD ( n = 3 experiments ) . In each experiment , 48 - 62 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 Drp1Opa1 - KO MEFs carrying 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 ) of mitochondrial is shown ( n = 3 experiments ) . In each experiment , 30 were analyzed .

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(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 Drp1Opa1 - KO MEFs , both of which carry indicated Opa1 constructs . asterisk indicates non - specific bands of anti - Oma1 antibodies .

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

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


( B ) Three frames from time - lapse during artificial flickering are shown . 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 ) of Su9 - GFP 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 indicated FCCP treatments . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( E ) of band . Values are average ± SD ( n = 3 ) . Significance was calculated using ANOVA with post - hoc Tukey : * * p < 0 . 01 , * * * p < 0 . 001 .

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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. (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 indicated FCCP treatments . 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 Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) / or ectopic Mfn1 . asterisk indicates non - specific bands of anti - Oma1 antibodies . ( B ) of band 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 Drp1 - KO MEFs , both of which carry shRNAs ( Oma1 or scramble ) / or ectopic Mfn1 . 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 E ) Drp1 - KO MEFs ( D F ) were transduced with Su9 - Eos , along with Oma1 shRNAs ectopic Mfn1 . Su9 - Eos in small region of mitochondria ( indicated by asterisks ) was photoconverted using a 405 - nm on confocal microscope . Within 1 s after photoconversion , were obtained for both photoconverted unconverted Su9 - Eos signals . before photoconversion were also taken to detect background signals . Scale bar , 10 µm . ( E F ) To determine connectivity of mitochondria , containing photoconverted Su9 - Eos signals was divided by containing photoconverted unconverted Su9 - Eos signals ( total mitochondria ) . Values are average ± SD ( n = 30 ) .

O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR 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 ) ectopic Mfn1 , were stained with TMRE . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 were analyzed in each experiment .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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 ) ectopic Mfn1 , were stained with TMRE . arrows indicate that lost mitochondrial potential in ( C ) . Scale bar , 10 µm . of that maintained potential is shown . Values are average ± SD ( n = 3 ) . 100 - 150 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 same set of MEFs . of basal OCRs respiratory capacity is shown . Values are average ± SD ( n = 3 ) .

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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 indicated mice at 3 months of using indicated antibodies . ( B ) of band . 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western 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 lines stably expressing GFP , GFP - WIPI1a , 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 treated with DSP , at 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 labeled with an anti - WIPI2 antibody . Scale bars , 10 μm . ( D ) WIPI2 in ( C ) were counted , a statistical of WIPI2 was performed using an unpaired Student ' s t . ∗ p < 0 . 05 . 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 electron microscopy ) of endogenous WIPI2 in Atg16L1Δ / Δ MEFs . ( E ) Merged phase confocal section of Atg16L1Δ / Δ labeled with anti - WIPI2 antibody . Scale bar , 10 μm . ( F ) Low - magnification of in ( E ) . Scale bar 10μm . ( G H ) High magnification of boxed regions in ( E ) ( F ) . Top box bottom box indicate panels ( G ) ( H ) , respectively . Arrows indicate open phagophores . M , mitochondria ; ER , endoplasmic reticulum . Scale bars , 1 μm ( G ) 0 . 5 μm ( H ) .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

(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 J ) 2GL9 ( GFP - LC3 HEK293 ; 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 - imaging demonstrates that Atg16L1 translocates to WIPI2b - positive in starvation . HEK293 expressing GFP - WIPI2b mCherry - Atg16L1 were starved in EBSS imaged every 2 s using a spinning disk microscope . See also Figure S1 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 - ( FL ) , 79 - 623 , or 1 - 265 constructs were expressed in HEK293A stably expressing GFP - WIPI2b 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 , GFP - WIPI2b were transiently expressed in HEK293A . GFP - tagged were isolated using GFP - TRAP incubated with in vitro translated 35S - labeled FLAG - Atg16L1 constructs 1 - 265 , 1 - 242 , 1 - 230 , 1 - 207 before washing by autoradiography . expression was validated by immunoblot ( bottom panel ) .

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

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

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

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O 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 Atg12 - 5 were detected by immunoblotting .

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

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(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 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 of ( A ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 3 . ∗ p < 0 . 05 .

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(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 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 analyzed by autoradiography .

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(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 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 of ( E ) was performed by one - way ANOVA with Tukey ' s posttest . for n = 2 . ∗ p < 0 . 05 .

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

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O 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.