( A ) In vitro kinase of endogenous ULK1 . MEFs were cultured in complete or starvation medium for 60 min . ULK1 kinase activity was determined as described in Materials methods . Relative kinase activity is shown . Data are mean ± SE of five independent experiments .

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(A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments.


( B C ) NIH3T3 were transiently transfected with HA - ULK1 , HA - ULK2 , or their kinase - dead mutants subjected to immunofluoresence microscopy using monoclonal anti - HA polyclonal anti - Atg16L1 antibodies for Alexa Fluor 488 - conjugated goat anti - mouse IgG Alexa Fluor 568 - conjugated goat anti - rabbit IgG antibodies for secondary antibodies . Transfected are indicated by arrows . Bars , 20 μm . ( D ) NIH3T3 stably expressing GFP - ULK1K46N GFP - ULK2K39T were cultured in starvation medium for 120 min . were subjected to immunofluorescence microscopy using anti - Atg16L1 antibody . Bar , 20 μm .

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(B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm.


( E ) NIH3T3 were transfected with retroviral vectors encoding HA - ULK1K46N or with corresponding empty retrovirus ( mock ) . They were cultured in complete or starvation medium for 120 min . lysates were then analyzed by immunoblot with indicated antibodies .

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(E) NIH3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.


( A ) HEK293T were cotransfected with FLAG - FIP200 HA - ULKs . lysates were subjected to immunoprecipitation ( IP ) using antibodies against FLAG . resulting precipitates were examined by immunoblot ( IB ) with indicated antibodies . asterisk indicates nonspecific band .

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(A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band.


( B ) Lysates of MEFs were immunoprecipitated with anti - ULK1 or anti - FIP200 antibody or preimmune rabbit serum , resulting precipitates were subjected to immunoblot with antibodies against ULK1 FIP200 .

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(B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200.


( D ) HEK293T were cotransfected with FLAG - FIP200 various ULK1 mutants analyzed as in A using anti - HA anti - FLAG antibodies .

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(D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies.


( E ) NIH3T3 stably expressing GFP - ULK1 ( left ) GFP - ULK1ΔC ( right ) were cultured in starvation medium for 120 min . Bar , 20 μm .

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(E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.


( A ) NIH3T3 stably expressing GFP - FIP200 were cultured in complete or starvation medium for 120 min GFP signal was observed .

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(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed.


( B ) NIH3T3 stably expressing GFP - FIP200 were cultured in starvation medium for 120 min then subjected to immunofluorescence microscopy using anti - Atg16L1 antibody Alexa Fluor 568 - conjugated secondary antibody . Bars , 20 μm . More than 90 % of GFP - FIP200 dots were positive for Atg16L1 .

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(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568-conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1.


( C ) NIH3T3 stably expressing GFP - ULK1 were starved for 120 min then subjected to immunofluorescence microscopy using anti - FIP200 antibody Alexa Fluor 568 - conjugated secondary antibody . Black squares indicate enlarged areas shown in insets . Bar , 20 μm .

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(C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568-conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.


( A ) FIP200 + / + , FIP200 − / − , Atg5 + / + , Atg5 − / − MEFs were cultured in complete or starvation medium for 60 min . In recovery experiments , starved MEFs were cultured in fresh complete medium for an additional 60 min ( replenishment ) . lysates were subjected to immunoblot with indicated antibodies .

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(A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) Wild - type FIP200 − / − MEFs were cultured in complete or starvation medium for indicated time with or without 100 nM bafilomycin A1 . lysates were subjected to immunoblot with anti - LC3 antibody .

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(B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( C ) Wild - type FIP200 − / − MEFs were transfected with retroviral vectors encoding GFP - Atg5 or GFP - LC3 . Resulting were cultured in starvation medium for 120 min . were fixed examined by microscopy . Bar , 20 μm .

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(C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( D - G ) Wild - type ( D E ) FIP200 − / − ( F G ) MEFs were cultured in complete ( D F ) or starvation ( E G ) medium for 120 min then fixed subjected to EM . Autophagosome - like structures ( open arrowheads ) , autolysosomes ( closed arrowheads ) are indicated . Bar , 1 μm . ( H ) of total of autophagosomes ( AP ) autolysosomes ( AL ) to total in D - G was determined by morphometric .

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(D-G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D-G was determined by morphometric analysis.


( A ) FAK + / − FAK − / − MEFs were cultured in complete or starvation medium for indicated times with or without 100 nM bafilomycin A1 . lysates were subjected to immunoblot with indicated antibodies .

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(A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) FAK + / − FAK − / − MEFs stably expressing GFP - LC3 were cultured in complete or starvation medium for 120 min . were fixed examined by microscopy .

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(B) FAK+/− and FAK−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy.


( C ) Wild - type MEFs stably expressing GFP - FAK were cultured in complete or starvation medium for 120 min . were fixed subjected to immunofluorescence microscopy using anti - Atg16 antibody . Black squares indicate enlarged areas shown in insets . Bars , 20 μm .

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(C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.


( A ) Wild - type FIP200 − / − MEFs were treated with 100 ng / ml rapamycin ( rapa ) or vehicle ( DMSO ) for 120 min in presence or absence of 100 nM bafilomycin A1 . lysates were subjected to immunoblot with anti - LC3 antibody .

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(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( B ) Wild - type FIP200 − / − MEFs stably expressing GFP - Atg5 or GFP - LC3 were cultured in presence of 100 ng / ml rapamycin for 120 min . formation of GFP - Atg5 ( top ) GFP - LC3 ( bottom ) was examined by microscopy . Bar , 20 μm .

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(B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm.


( C ) Wild - type FIP200 − / − MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2 - ceramide for 2 h .

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(C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.


( A ) FIP200 + / − FIP200 − / − MEFs stably expressing GFP - ULK1 or - ULK2 were cultured in starvation medium for 120 min . were fixed examined by microscopy . Bar , 20 μm .

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(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( B ) FIP200 + / + FIP200 − / − MEFs were cultured in complete medium . lysates were subjected to immunoblot with antibodies against ULK1 or HSP90 ( loading control ) .

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(B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control).


( C ) Phosphatase sensitivity of ULK1 . FIP200 + / + FIP200 − / − MEFs were cultured in complete medium . ULK1 was immunoprecipitated from lysates treated with λ phosphatase for 30 min at 30 ° C in absence or presence of phosphatase inhibitors ( 1 mM Na2VO4 , 50 mM KF , 15 mM Na4P2O7 , 1 mM EGTA ) .

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(C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).


b , Representative phase of of WT Apc - / - organoids before mRNA isolation .

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b, Representative phase images of the morphology of WT and Apc-/- organoids before mRNA isolation.


c , Heat map illustrating differentially expressed genes ( DEGs ) encoding heat shock between WT Apc - / - organoids .

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c, Heat map illustrating differentially expressed genes (DEGs) encoding heat shock proteins between WT and Apc-/- organoids.


d , Volcano plot of DEGs between normal mouse intestinal tissue adenomas ( GSE65461 ) .

d, Volcano plot of DEGs between normal mouse intestinal tissue and adenomas (GSE65461).


e , f , Relative gene expression of Hspb1 ( * * * P = 0 . 0004 , n = 6 ) ( e ) expression of HSP25 ( f ) in unrecombined versus recombined murine organoids .

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e, f, Relative gene expression of Hspb1 (***P=0.0004, n=6)(e) and protein expression of HSP25 (f) in unrecombined versus recombined murine organoids.


g , Gene expression of Hspb1 in normal ( N ) versus adenoma ( A ) tissue ( * * * P = 0 . 0002 , n = 4 ) .

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g, Gene expression of Hspb1 in normal (N) versus adenoma (A) tissue (***P=0.0002, n=4).


H , - ISH for Hspb1 in murine intestinal adenoma . Scalebar , 100 μm . Zoom panel , 25

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H, RNA-ISH for Hspb1 in murine intestinal adenoma. Scalebar, 100 μm. Zoom panel, 25 μm


i , Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue , ( * * * * P < 0 . 0001 , paired t - , n = 59 pairs ) .

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i, Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue, (****P<0.0001, paired t-test, n=59 pairs).


Relative gene expression of AXIN2 ( * P = 0 . 0129 ) , P21 ( * P = 0 . 0103 ) , HSPB1 ( * P = 0 . 0236 ) ( b ) in Ls174t - dnTCF4 presence or absence of doxycycline .

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Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


expression of HSP27 ( c ) in Ls174t - dnTCF4 presence or absence of doxycycline .

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protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR - 99021 ( * * P = 0 . 0016 , one way ANOVA , n = 4 ) ( d )

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Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d)


expression of HSP25 in WT Apc - / - organoids ( e ) .

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protein expression of HSP25 in WT and Apc-/- organoids (e).


g , h , Representative of single Hspb1 KO clones passaged after loss of Apc ( g ) , of their clonogenic potential ( * P = 0 . 0147 ( # 1 ) , * * P = 0 . 0035 ( # 2 ) , * * P = 0 . 0032 ( # 3 ) ) ( h ) .

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g, h, Representative images of single cell Hspb1 KO clones passaged after loss of Apc (g), and quantification of their clonogenic potential (*P=0.0147(#1), **P=0.0035(#2), **P=0.0032(#3)) (h).


i , j , Representative of Apc - / - organoids cultured in absence or presence of 60 μM BVDU ( i ) , of their clonogenic potential ( * * P = 0 . 0039 ) ( j ) .

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i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j).


k , l , Relative gene expression of Wnt target genes Axin2 ( * * P = 0 . 0080 ) Lgr5 ( * P = 0 . 0142 ) in Apc - / - organoids cultured in absence or presence of 60 μM BVDU .

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k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.


m , - ISH for Lgr5 in control or BVDU - treated Apc - / - organoids . Scale bar , 50 μm , zoom panel 20 μm .

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m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.


b , c , Visualization of proliferating in tissues incubated with EdU for 2 - hours ( b ) , of hyperproliferative zone ( * * P = 0 . 0057 , n = 3 mice ) ( c ) .

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b, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c).


e , f , - ISH for Notum reveals Apc - mutant clones in crypt bottoms of control or BVDU - treated mice ( e ) , of abundance of mutant crypts ( n = 2 mice per condition , n = 5 technical replicates per mouse ) ( f ) .

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e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f).


g , h , Clone distributions of Notum + crypts in control ( n = 265 crypts ) or BVDU - treated ( n = 260 crypts ) mice , average clone number of fixed clones are included in figure .

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g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.


Survival curves for control BVDU - treated mice ( P = 0 . 0351 , Mantel - Cox ) ( j )

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Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j)


macroscopic of adenoma development in distal small intestine ( k )

macroscopic images of adenoma development in the distal small intestine (k)


boxplots for number of adenomas per intestinal region ( * * P = 0 . 0013 ) ( l ) , n = 8 control , n = 11 treated .

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boxplots for the number of adenomas per intestinal region(**P=0.0013) (l), n=8 control, n=11 treated.


upper panel shows strength of association of Affymetrix 500K SNPs in region , with combined P values shown in red for replicated SNPs . lower panel is a recombination map showing how signals are clearly demarcated by recombination hotspots . Nearby genes are shown in yellow .

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The upper panel shows strength of association of Affymetrix 500K SNPs in the region, with combined P values shown in red for replicated SNPs. The lower panel is a recombination map showing how the signals are clearly demarcated by recombination hotspots. Nearby genes are shown in yellow.


A . Four full - thickness excisional wounds were produced on each of CD18 - / - or WT mice by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or PBS control . Each wound region was digitally photographed at indicated time points , wound areas were analyzed using Adobe Photoshop . Depicted are representative macroscopic pictures of PBS injected or AT - MSCs injected WT or CD18 - / - wounds at days 0 , 3 , 5 , 7 9 post - wounding . B . Quantitative of 20 wound areas per group , expressed as of initial wound at day 0 . in each group represents mean value of 20 wounds from 5 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t - with Welch ' s correction .

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A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding. B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction.


A . AT - MSCs or PBS were intradermally injected to WT or CD18 - / - wounds wound tissues were harvested at days 2 , 5 , 7 post - wounding . Depicted are representative of wound cryosections at days 2 5 post - wounding stained for human - specific β2M ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bars : 100 µm .

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A. AT-MSCs or PBS were intradermally injected to WT or CD18-/- wounds and the wound tissues were harvested at days 2, 5, 7 post-wounding. Depicted are representative photographs of wound cryosections at days 2 and 5 post-wounding stained for human-specific β2M (green). Nuclei were stained with DAPI (blue). Scale bars: 100 µm.


B . Genomic was isolated from wound tissues , amount of human - specific Alu was quantified by real time PCR , then converted to number of AT - MSCs on a standard curve . Data are expressed as mean ± , n = 3 at each time point , * p < 0 . 05 by two - unpaired t - .

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B. Genomic DNA was isolated from the wound tissues, and the amount of human-specific Alu DNA was quantified by real time PCR, and then converted to the number of AT-MSCs based on a standard curve. Data are expressed as mean ± SEM, n = 3 at each time point, *p<0.05 by two-tailed unpaired t-test.


C . Total TGF - β1 concentrations in wound lysates were measured by TGF - β1 specific ELISA . Results are expressed as mean ± , n = 3 at each time

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C. Total TGF-β1 concentrations in wound lysates were measured by TGF-β1 specific ELISA. Results are expressed as mean ± SEM, n = 3 at each time point


D . expression of human TGF - β1 in cultured AT - MSCs wound tissues of WT or CD18 - / - wounds received AT - MSCs injection was quantified by qPCR with primers specifically amplify human TGF - β1 , normalized on numbers of MSCs in each condition Data are expressed as mean ± , n = 8 wounds ( 2 wounds x 4 mice ) per time

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D. The expression of human TGF-β1 in cultured AT-MSCs and wound tissues of WT or CD18-/- wounds received AT-MSCs injection was quantified by qPCR with primers specifically amplify human TGF-β1, and normalized on the numbers of MSCs in each condition Data are expressed as mean ± SEM, n = 8 wounds (2 wounds x 4 mice) per time point


A . Cryosections of PBS or AT - MSCs injected CD18 - / - wounds at days 5 7 post - wounding were immunostained for α - SMA ( red ) . Nuclei were stained with DAPI ( blue ) . dashed lines separate epidermis wound bed . e , epidermis ; wb , wound bed . Scale bars : 100 µm . B . of α - SMA immunofluorescence shown in ( A )

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A. Cryosections of PBS or AT-MSCs injected CD18-/- wounds at days 5 and 7 post-wounding were immunostained for α-SMA (red). Nuclei were stained with DAPI (blue). The dashed lines separate epidermis and wound bed. e, epidermis; wb, wound bed. Scale bars: 100 µm. B. Quantification of α-SMA immunofluorescence shown in (A)


C . Cryosections of AT - MSCs injected CD18 - / - wounds at day 5 post - wounding were co - immunostained for human β2M ( green ) TGF - β1 ( red , upper panel ) or α - SMA ( red , lower panel ) .

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C. Cryosections of AT-MSCs injected CD18-/- wounds at day 5 post-wounding were co-immunostained for human β2M (green) and TGF-β1 (red, upper panel) or α-SMA (red, lower panel).


A - F . Representative of immunofluorescent for α - SMA ( red ) human β2M ( green ) on cultured human AT - MSCs ( A ) , murine dermal fibroblasts ( mFB ) ( B ) , mFB treated with 2 ng / ml recombinant human TGF - β1 ( C ) , co - cultures of mFB with AT - MSCs ( D ) or TGF - β1 siRNA transfected AT - MSCs ( E ) or control siRNA transfected AT - MSCs ( F ) after 48 h culture . Nuclei were counterstained with DAPI ( blue ) . G . of α - SMA immunofluorescence shown in ( A - F )

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A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). G. Quantification of α-SMA immunofluorescence shown in (A-F)


H . Four full - thickness excisional wounds were produced on each of CD18 - / - mouse by 6 mm biopsy punches . Each wound received intradermal injection of 2 . 5x105 AT - MSCs or human dermal fibroblasts ( HDF ) or TGF - β1 siRNA transfected AT - MSCs or control siRNA transfected AT - MSCs or PBS . Each wound region was digitally photographed at days 0 , 3 , 5 , 7 10 post - wounding , wound areas were analyzed using Adobe Photoshop . depicted result is quantitative of wound areas per group , expressed as of initial wound at day 0 .

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H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0.


A - C . 2 . 5x105 of TGF - β1 siRNA or control siRNA transfected AT - MSCs were intradermally injected around each of CD18 - / - murine wound . PBS mock injection served as negative control . Wound tissue were harvested at day 2 , 5 , 7 post - wounding for of human TGF - β1 mRNA ( A ) at day 2 by qPCR , total TGF - β1 ( B ) active TGF - β1 ( C ) at day 5 by ELISA .

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A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA.


Expression of α - SMA ( D , E ) at day 5 7 by immunostaining on tissue sections . dashed lines indicate border of wound bed epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . data are expressed as mean ± , n = 3 wounds per

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Expression of α-SMA (D, E) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group


Expression of CD31 ( F , G ) at day 5 7 by immunostaining on tissue sections . dashed lines indicate border of wound bed epidermis or eschar . e , epidermis ; es , eschar ; wb , wound bed . data are expressed as mean ± , n = 3 wounds per

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Expression of CD31 (F, G) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Quantification data are expressed as mean ± SEM, n = 3 wounds per group


A . AT - MSCs were cultured in labelling media containing [ 35S ] - Met / Cys increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for overnight . Supernatants were harvested incubated with TGF - β1 capture antibody . After washing captured TGF - β1 was transferred to glass filter , radioactivity was counted with a scintillation counter ( black bars ) . supernatants were incubated with new TGF - β1 capture antibody , radioactivity of captured TGF - β1 was counted again ( bars ) . data is given as mean ± SD of 5 repeated measurements . CPM , per minute .

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A. AT-MSCs were cultured in the labelling media containing [35S]-Met/Cys and increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for overnight. Supernatants were harvested and incubated with TGF-β1 capture antibody. After washing the captured TGF-β1 was transferred to glass filter membrane, and the radioactivity was counted with a scintillation counter (black bars). The supernatants were incubated with new TGF-β1 capture antibody, and the radioactivity of captured TGF-β1 was counted again (white bars). The data is given as mean ± SD of 5 repeated measurements. CPM, count per minute.


AT - MSCs ( B ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for 24 h , before lysates or total RNAs were harvested . Western blot of Smad7 was performed with lysates semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± of three independent blots .

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AT-MSCs (B) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots.


HDFs ( C ) were cultured in media containing increasing concentrations of r . h . TGF - β1 at 0 , 0 . 1 , 0 . 3 , 1 , 3 10 ng / ml for 24 h , before lysates or total RNAs were harvested . Western blot of Smad7 was performed with lysates semi - quantitatively analyzed with densitometry . β - actin served as loading control . Data is given as mean ± of three independent blots . *

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HDFs (C) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots. *


D . From total , mature miR - 21 endogenous control RNU6B were converted to cDNA , quantified by qPCR TaqMan microRNA assays . expression of miR - 21 was normalized on RNU6B . Data is given as mean ± of three independent experiments .

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D. From total RNA, the mature miR-21 and endogenous control RNU6B were converted to cDNA, and quantified by qPCR based TaqMan microRNA assays. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.


A . AT - MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM , or control DMSO for 1 h , subsequently exposed to r . h . TGF - β1 at indicated concentrations . were cultured for another 24 h harvested for miR - 21 . expression of miR - 21 was normalized on RNU6B . Data is given as mean ± of three independent experiments .

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A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.


B - D . AT - MSCs were transfected with 30 nM Smad7 - siRNAs or control siRNA . Untransfected AT - MSCs served as control . were isolated 2 days after transfection for Smad7 expression by qPCR ( B ) western blot ( C ) . remaining were treated with r . h . TGF - β1 at indicated concentrations for another 24 h . expression of human TGF - β1 was analyzed by qPCR normalized on human GAPDH ( D ) . Data is given as mean ± of three independent experiments .

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B-D. AT-MSCs were transfected with 30 nM Smad7-siRNAs or control siRNA. Untransfected AT-MSCs served as control. RNA and protein were isolated 2 days after transfection for Smad7 expression by qPCR (B) and western blot (C). The remaining cells were treated with r.h. TGF-β1 at indicated concentrations for another 24 h. The expression of human TGF-β1 was analyzed by qPCR and normalized on human GAPDH (D). Data is given as mean ± SEM of three independent experiments.


E - G . AT - MSCs were transfected with 5 nM miR - 21 mimic or control mimic , were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h . expression miR - 21 was analyzed by qPCR - miR - 21 normalized on RNU6B ( E ) , Samd7 level was shown by western blot ( F ) , human TGF - β1 expression was monitored by qPCR normalized on human GAPDH ( G ) . Data is given as mean ± of three independent experiments .

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E-G. AT-MSCs were transfected with 5 nM miR-21 mimic or control mimic, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h. The expression miR-21 was analyzed by qPCR-based miR-21 assay normalized on RNU6B (E), Samd7 protein level was shown by western blot (F), and human TGF-β1 expression was monitored by qPCR normalized on human GAPDH (G). Data is given as mean ± SEM of three independent experiments.


H - J . AT - MSCs were transfected with 50 nM miR - 21 inhibitor or control inhibitor , were exposed to r . h . TGF - β1 2 days after transfection at indicated concentrations for another 24 h , subsequently subjected to of expression of miR - 21 ( H ) , Smad7 ( I ) human TGF - β1 mRNA ( J ) . Data is given as mean ± of three independent experiments .

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H-J. AT-MSCs were transfected with 50 nM miR-21 inhibitor or control inhibitor, and were exposed to r.h. TGF-β1 2 days after transfection at indicated concentrations for another 24 h, and subsequently subjected to the analysis of expression of miR-21 (H), Smad7 protein (I) and human TGF-β1 mRNA (J). Data is given as mean ± SEM of three independent experiments.


K - L . Full - thickness excisional wounds were produced on CD18 - / - mice by 6 mm biopsy punches . One day after wounding , each wound received intradermal injection of 2 . 5x105 AT - MSCs that had been transfected with 50 nM miR - 21 inhibitor or control inhibitor 2 days after transfection . CD18 - / - wounds received mock injection with PBS served as control . Each wound region was digitally photographed at days 0 , 3 , 6 post - wounding , expressed as of initial wound at day 0 ( K ) . Wound tissues were harvested on day 6 post - wounding for of total TGF - β1 by ELISA ( L ) . Data is given as mean ± ; n = 5 wounds per group ; * , p < 0 . 05 ; * * , p < 0 . 01 , by one - way ANOVA with Tukey ' s . MSC _ miR - 21 - IN , miR - 21 inhibitor transfected AT - MSCs ; MSC _ ctrl - IN , control inhibitor transfected AT - MSCs .

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K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.


a , b , THP1 were stimulated with rotenone ( 10 µM ) , TTFA ( 10 µM ) or antimycin ( 40 µg ml − 1 ) for 6 h then stained with Mitotracker green Mitotracker deep red ( a ) or MitoSOX ( b ) for 30 min analysed by flow cytometry .

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a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.


c , THP1 were stimulated for 6 h with indicated amounts of rotenone , TTFA or antimycin . Supernatants ( SN ) extracts ( input ) were analysed by western blotting as indicated .

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c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated.


d , LPS - primed bone - marrow - derived macrophages ( BMDMs ) from wild - type ( WT ) , Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU ( 150 µg ml − 1 ) , rotenone ( 10 µM for THP1 40 µM for BMDMs ) or antimycin ( 40 µg ml − 1 for THP1 10 µg ml − 1 for BMDMs ) . release of caspase - 1 ( western blot ) was then determined . Data shown are representative of three independent experiments .

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d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.


a , BMDMs expressing GFP - LC3 were stimulated with rotenone ( 40 µM ) for 3 h co - localization of mitochondria GFP - LC3 dots were analysed using confocal microscopy .

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a, BMDMs expressing GFP-LC3 were stimulated with rotenone (40 µM) for 3 h and the co-localization of mitochondria and GFP-LC3 dots were analysed using confocal microscopy.


b , THP1 stimulated with 3 - methyladenine ( 3 - MA , 10 mM ) for 24 h or THP1 stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green Mitotracker deep red for 30 min , analysed by flow cytometry .

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b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry.


c , LPS - primed BMDMs from wild - type , Nlrp3 − / − , Asc − / − or Ipaf − / − knockout mice were stimulated with MSU ( 150 µg ml − 1 ) or 3 - MA ( 10 mM ) for 6 h release of active caspase - 1 IL - 1b was determined .

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c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 µg ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1b was determined.


d , THP1 stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h media supernatants ( SN ) inputs were analysed by western blotting .

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d, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants (SN) and inputs were analysed by western blotting.


e , THP1 stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h . Data shown are representative of three independent experiments .

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e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.


a - d , THP1 expressing Flag - NLRP3 ( a , b ) or Flag - ASC ( c , d ) were analysed for co - localization of Flag - NLRP3 with endoplasmic reticulum ( ER ) marker calreticulin or mitochondria ( Mitotracker ) using confocal microscopy .

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a-d, THP1 cells expressing Flag-NLRP3 (a, b) or Flag-ASC (c, d) were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy.


b , THP1 expressing Flag - NLRP3 were analysed for co - localization of Flag - NLRP3 with endoplasmic reticulum ( ER ) marker calreticulin using confocal microscopy .

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b, THP1 cells expressing Flag-NLRP3 were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin using confocal microscopy.


e , THP1 were stimulated with MSU or nigericin then fractionated . , ER , MAM mitochondrial fractions were analysed by western blotting as indicated in text .

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e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text.


f , THP1 stably expressing shRNA against NLRP3 were stimulated with nigericin subcellular fractions analysed as in e .

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f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e.


g , TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS - dependent manner . THP1 were stimulated with MSU nigericin , TXNIP localization was investigated in presence or absence of antioxidant APDC . Data shown are representative of two or three independent experiments . Scale bars : a - g , 20 µm .

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g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.


a , THP1 stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators then analysed for caspase - 1 activation IL - 1b secretion .

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a, THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase-1 activation and IL-1b secretion.


b , THP1 expressing shRNA against VDAC2 were stimulated with MSU , R837 , silica , alum or nigericin analysed for IL - 1b secretion .

b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.


c , THP1 expressing shRNA against VDAC1 were stimulated with MSU nigericin , NLRP3 localization was analysed by confocal microscopy . Scale bars : 20 µm .

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c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 µm.


B PCR performed on genomic with indicated primers as shown : o1 : oPAP8 _ rtpF , o2 : oPAP8 _ E3R , o3 : oPAP8 _ rtpR , oLB : oLBb1 . 3 , EF1α : ELONGATION FACTOR 1α , WT : wild type , pap8 - 1 : Homozygous albino , Ht : Heterozygous green ; T : T - , arrowhead : 670 - bp contaminant amplification product used as loading control .

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B PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: Homozygous albino plant, Ht: Heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control.


C RT - PCR on wild type pap8 - 1 homozygous grown in for 3 days followed with 72 - h growth under to allow greening of wild type ; EF1α used as control .

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C RT-PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control.


E Half - open siliques of a heterozygous showing greening ; scale bar equals 250 µm . given number is position rank of silique from top to bottom of inflorescence presenting segregation of homozygous heterozygous seeds on their ability to transiently develop chloroplasts .

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E Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 µm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts.


F Mutant Rescue : WT two representative pap8 - 1 were grown in vitro using sucrose low ( of 10 µmol . m - 2 . s - 1 ) ; scale bar equals 20 mm .

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F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.


B Two or six ( - 97m3 ) representative transformants expressing GUS under given PAP8 promoter version ; FC + , corresponding promoters were tested positive in functional complementation of mutant pap8 - 1 . Scale bar equals 3 mm .

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B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.


D Dual luciferase reporter ; Renilla luciferase ( Rluc ) used as internal control GFPer used as control for transfected ; promoters driving Firefly luciferase ( Fluc ) were transfected in onion epidermis without or with constitutively expressed HY5 . Fluc / Rluc activity was set to 1 for minus - HY5 control ; mean ± standard error corresponding to 3 replicates ; photon are given in source data . * , ε - = 3 , 43 > 1 . 96 corresponding to p - value < 0 , 001 .

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D Dual luciferase reporter assay; Renilla luciferase (Rluc) used as internal control and GFPer used as control for the transfected area; the promoters driving Firefly luciferase (Fluc) were transfected in onion epidermis cells without or with constitutively expressed HY5. The Fluc/Rluc activity was set to 1 for the minus-HY5 control; mean ± standard error corresponding to 3 replicates; photon counts are given in source data. *, ε-test = 3,43 > 1.96 corresponding to p-value < 0,001.


E Electro - mobility shift of a probe corresponding to near palindromic PAP8 element ( GAcGCTC ) with recombinant HY5 ; a probe containing a canonical G - box element ( CACGTG ) recognized by HY5 was used as cold competitor .

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E Electro-mobility shift assay of a probe corresponding to the near palindromic PAP8 element (GAcGCTC) with recombinant HY5 protein; a probe containing a canonical G-box element (CACGTG) recognized by HY5 was used as cold competitor.


F Integrative genomics viewer ( IGV ) of immunoprecipitation ( ChIP ) sequencing data30 at PAP8 locus ; TAIR10 , annotation according to Arabidopsis thaliana information resource orange box indicates PAP8 locus . ChIP on hy5 - ks50 ; 35S : HY5 - YFP exposed to blue or red using GFP antibody compared to mock corresponding to ChIP control experiment done without antibody . Each treatment is presented as track overlay of triplicates : read is given within " group autoscale " range in brackets . Close up on 5 ' - UTR region centred on - 95 - promoter element in yellow .

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F Integrative genomics viewer (IGV) images of the Chromatin immunoprecipitation (ChIP) sequencing data30 at the PAP8 locus; TAIR10, annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus. ChIP on hy5-ks50; 35S:HY5-YFP exposed to blue light or red light using GFP antibody and compared to mock corresponding to ChIP control experiment done without antibody. Each treatment is presented as track overlay of triplicates: the read count is given within the "group autoscale" range in brackets. Close up on the 5'-UTR region centred on the -95-promoter element in yellow.


B Transiently expressed PAP8FL - GFP ( full - coding of PAP8 fused to GFP ) in onion epidermal displays a dual localization in nucleus ( arrowhead ) in plastids ( yellow arrowheads ) .

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B Transiently expressed PAP8FL-GFP (full-length coding sequence of PAP8 fused to GFP) in onion epidermal cells displays a dual localization in the nucleus (white arrowhead) and in plastids (yellow arrowheads).


C Immuno - blots for detection of PAP8 in total extracts ( Col - 0 Tot ) fraction ( N ) plastidic fractions ( EP , etioplast ; CP , chloroplast ) of etiolated ( ) or photomorphogenic ( ) Arabidopsis seedlings by immuno - Western blotting using a PAP8 antiserum ( PAP8 ) . As control , a mixture of antisera raised against Histone 3 ( H3 ) large subunit of ribulose 1 , 5 - bisphosphate carboxylase / oxygenase ( RbcL ) was used to evaluate reciprocal contaminations . lanes are extracted from same blots provided in Source Data file .

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C Immuno-blots for the detection of PAP8 in total protein extracts (Col-0 Tot) the nuclear fraction (N) and the plastidic fractions (EP, etioplast; CP, chloroplast) of etiolated (Dark) or photomorphogenic (Light) Arabidopsis seedlings by immuno-Western blotting using a PAP8 antiserum (PAP8). As control, a mixture of antisera raised against Histone 3 (H3) and the large subunit of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RbcL) was used to evaluate reciprocal contaminations. The lanes are extracted from the same blots provided in the Source Data file.


D - G Co - expression of PAP8FL - GFP ( D ) with PAP8ΔcTP - RFP ( E ) in onion epidermal ; ( F ) green red channels merged ; yellow arrowheads show plastids ; arrowheads show nuclei as observed with DIC , differential interference contrast in ( G ) ; FL , PAP8 full ORF ; ΔcTP , deletion of cTP ; scale bars equal 20 µm .

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D-G Co-expression analysis of PAP8FL-GFP (D) with PAP8ΔcTP-RFP (E) in onion epidermal cells; (F) green and red channels merged; yellow arrowheads show plastids; white arrowheads show nuclei as observed with DIC, differential interference contrast in (G); FL, PAP8 full length ORF; ΔcTP, deletion of the cTP; scale bars equal 20 µm.


H Confocal imaging on Arabidopsis cotyledons expressing PAP8ΔcTP - GFP , details of a few palisade ( see Fig . S7B for a view in a cross section between two cotyledons ) ; magenta , auto - of chloroplast ; arrowhead shows a nucleus ; scale bar equals 5 µm .

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H Confocal imaging on Arabidopsis cotyledons expressing PAP8ΔcTP-GFP, details of a few palisade cells (see Fig. S7B for a view in a cross section between the two cotyledons); magenta, auto-fluorescence of the chloroplast; the white arrowhead shows a nucleus; scale bar equals 5 µm.


I , J Confocal imaging on Arabidopsis cotyledons stably expressing pP8 : : PAP8ΔNLS - GFP ; ΔNLS , deletion of NLS ( I ) during skotomorphogenesis ( J ) after 24h ; Yellow arrowheads show GFP signal ; picture is a merge of different channels : GFP in green , Pchl , : protochlorophyllide , or Chl : chlorophyll in magenta marked with arrowheads , propidium iodide , showing waxy cuticle in red , empty space correspond to layer of highly vacuolated epidermal .

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I,J Confocal imaging on Arabidopsis cotyledons stably expressing pP8::PAP8ΔNLS-GFP; ΔNLS, deletion of the NLS (I) during skotomorphogenesis and (J) after 24h light; Yellow arrowheads show the GFP signal; the picture is a merge of different channels: GFP in green, Pchl,: protochlorophyllide, or Chl: chlorophyll in magenta marked with arrowheads, and propidium iodide, showing the waxy cuticle in red, the empty space correspond to the layer of highly vacuolated epidermal cells.


( A , B ) ( A ) Transient expression of PAP8NLSm5 - GFP mutated in NLS as described in Fig . EV4A . circle marks position of nucleus ( N ) as observed with DIC in ( B ) ; yellow arrowheads show stromules ( str ) . Scale bars equal 20 µm .

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(A,B) (A) Transient expression of PAP8NLSm5-GFP mutated in the NLS as described in Fig. EV4A. The circle marks the position of the nucleus (N) as observed with DIC in (B); yellow arrowheads show stromules (str). Scale bars equal 20 µm.


C - G Pictures of representative genotypes obtained in functional complementation of pap8 - 1 using pP8 : : PAP8 - NLSm5 ( C , F ) or pP8 : : PAP8ΔcTP ( G ) both constructions without GFP tags ; ⌀ indicates pap8 - 1 without any PAP8 transgene . ( D - G ) Pictures using Keyence technology of with genotypes as labelled ; transgenes expressed using 1 . 1 - kb PAP8 promoter . Scale bars equal 1 mm .

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C-G Pictures of representative genotypes obtained in the functional complementation test of pap8-1 using pP8::PAP8-NLSm5 (C, F) or pP8::PAP8ΔcTP (G) both constructions without GFP tags; ⌀ indicates pap8-1 without any PAP8 transgene. (D-G) Pictures using Keyence technology of plants with genotypes as labelled; transgenes expressed using the 1.1-kb PAP8 promoter. Scale bars equal 1 mm.


A of given genotypes subjected to 5 days of illumination at 8 µmol . m - 2 . s - 1 660 - nm red . PBG , pCaMV35S : : PHYB - GFP transformed in pap8 - 1 / + ( among 25 lines selected for GFP expression see data source ; 2 doubly heterozygous pap8 - 1 / + ; PBG / - lines # 6 # 7 segregated photobodies alteration with albinism ) .

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A Phenotypes of given genotypes subjected to 5 days of illumination at 8 µmol.m-2.s-1 660-nm red light. PBG, pCaMV35S::PHYB-GFP transformed in pap8-1/+ (among 25 lines selected for GFP expression see data source; 2 doubly heterozygous pap8-1/+; PBG/- lines #6 and #7 segregated the photobodies alteration with the albinism).


B Hypocotyl , given as mean ± SD , of grown as in ( A ) ( R8 , grey bars ) or at 30 µmol . m - 2 . s - 1 660 - nm red ( R30 , pink bars ) showing partial insensitivity of pap8 - 1 to PBG overexpression . Measurements are given in source data ; in order of graph n equals ( 50 , 133 , 58 , 43 , 36 , 112 , 25 , 60 ) δ - ( comparison of mean ) R8 : δPBG / PBGp8 - 1 = 20 . 38 corresponding to p - value < 10 - 31 δwt / p8 - 1 = 0 . 5 < 1 . 96 not significant at α set to 0 . 05 ) .

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B Hypocotyl length, given as the mean ± SD, of plants grown as in (A) (R8, grey bars) or at 30 µmol.m-2.s-1 660-nm red light (R30, pink bars) showing partial insensitivity of pap8-1 to the PBG overexpression. Measurements are given in source data; in the order of the graph n equals (50, 133, 58, 43, 36, 112, 25, 60) δ-test (comparison of the mean) R8: δPBG/PBGp8-1= 20.38 corresponding to p-value < 10-31 δwt/p8-1= 0.5 < 1.96 not significant at α set to 0.05).


accumulation of PBG observed under GFP excitation in given genotypes . ( C , D ) Epi - microscopy . ( E , F ) Confocal microscopy showing of bodies . Scale bars equal 5 µm .

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Nuclear accumulation of PBG observed under GFP excitation in the given genotypes. (C,D) Epi-fluorescence microscopy. (E,F) Confocal microscopy showing the size of the nuclear bodies. Scale bars equal 5 µm.


( G ) Box plot ( Min , 1st quartile , median as central band , 3rd quartile , max ) on diameter of bodies ( NBs ) ; n equals number of records .

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(G) Box plot (Min, 1st quartile, median as the central band, 3rd quartile, max) on the diameter of the nuclear bodies (NBs); n equals the number of records.


H Immuno - blots using a GFP antibody or a PAP8 antibody showing respectively of PHYB - GFP PAP8 in given genotypes grown in for 3 days or in ; n . a . , not applicable as pap8 - 1 mutant can only be visually distinguished from wild type after exposure ; 2 lines ( L # 06 L # 07 ) for PBG / pap8 - 1 were tested . Coomassie blue presented as loading ; signals were quantified using ImageJ .

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H Immuno-blots using a GFP antibody or a PAP8 antibody showing respectively the levels of PHYB-GFP and PAP8 in the given genotypes grown in the dark for 3 days or in light; n.a., not applicable as the pap8-1 mutant can only be visually distinguished from wild type after light exposure; 2 lines (L#06 and L#07) for PBG/pap8-1 were tested. Coomassie blue staining presented as loading; signals were quantified using ImageJ.


I RT - qPCR on wild type , pap8 - 1 , PBG PBG pap8 - 1 . Seedlings were grown in ( D ) or under ( L , 30 µmol . m - 2 . s - 1 ) ; of transcripts are given relative to EF1α ; error bars correspond to standard errors on technical triplicates sample is wild - type PBG . δ - ( comparison of mean ) * * * , P < 10 - 72 .

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I RT-qPCR analysis on wild type, pap8-1, PBG and PBG pap8-1. Seedlings were grown in the dark (D) or under white light (L, 30 µmol.m-2.s-1); levels of transcripts are given relative to EF1α; error bars correspond to standard errors on technical triplicates and the dark sample is the wild-type PBG line. δ-test (comparison of the mean) ***, P < 10-72.


J Immuno - blots showing of PIF1 , PIF3 , HY5 in given genotypes grown in or condition as noted : p5 / + , mix of an heterozygous pap5 - 2 siblings progeny undistinguishable from wild type ; p5 - 2 , pap5 - 2 pifq , quadruple pif1 - 1 pif3 - 3 pif4 - 2 pif5 - 3 mutant ; Histone H3 ( H3 ) , RbcL PAP8 were used as controls ; n . a . , not applicable .

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J Immuno-blots showing the levels of PIF1, PIF3, HY5 in given genotypes grown in the Dark or Light condition as noted: p5/+, mix of an heterozygous pap5-2 siblings progeny undistinguishable from wild type; p5-2, pap5-2 and pifq, quadruple pif1-1 pif3-3 pif4-2 pif5-3 mutant; Histone H3 (H3), RbcL and PAP8 were used as controls; n.a., not applicable.


A Bimolecular complementation using in combination PAP8ΔcTP - NY ( P8Δc - NY ) with PAP5ΔcTP - YC ( P5Δc - YC ) or PAP8ΔcTP - YC ( P8Δc - YC ) with PAP5ΔcTP - NY ( P5Δc - NY ) ; PAP10 - RFP ( P10 - RFP ) was used as internal positive control for transfection ; in grey depicts number of green - over red - . Arrowheads indicate nuclei . See Appendix Fig . S6 for control experiments ; transgenes expressed under CaMV35S promoter .

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A Bimolecular fluorescence complementation tests using in combination PAP8ΔcTP-NY (P8Δc-NY) with PAP5ΔcTP-YC (P5Δc-YC) or PAP8ΔcTP-YC (P8Δc-YC) with PAP5ΔcTP-NY (P5Δc-NY); PAP10-RFP (P10-RFP) was used as internal positive control for transfection; the ratio in grey depicts the number of green-fluorescent cells over red-fluorescent cells. Arrowheads indicate nuclei. See Appendix Fig. S6 for control experiments; transgenes expressed under CaMV35S promoter.


B Overlay of 1H - 15N correlation 2D NMR spectra of free 15N - labelled PAP8 alone ( blue ) or in with PAP5 ( red ) . Grey areas depict changes of signals in PAP8 spectrum .

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B Overlay of 1H-15N correlation 2D NMR spectra of free 15N-labelled PAP8 alone (blue) or in complex with PAP5 (red). Grey areas depict changes of signals in the PAP8 spectrum.


C 15N - Filtered Diffusion Ordered Spectroscopy - NMR measurements to PAP8 . Exponential decay curves of PAP8 in absence or in presence of MBP - PAP5 are shown in red black respectively . units on y - axis are normalized values of integrals of signal measured in amide proton region .

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C 15N-Filtered Diffusion Ordered Spectroscopy-NMR measurements to PAP8. Exponential decay curves of PAP8 in absence or in presence of MBP-PAP5 are shown in red and black respectively. The units on the y-axis are normalized values of the integrals of the signal measured in the amide proton region.


A Immuno - blots showing of PAP8 , PAP5 , Histone H3 ( H3 ) , Rubisco ( RbcL ) , HY5 ; D , ; Ph , photomorphogenic growth conditions from germination on . * , HY5 is detected as a modified form ( + 6 kDa ) ; arrows indicate two different post - translational modified forms of HY5 for which accumulation occurs upon exposure .

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A Immuno-blots showing the levels of PAP8, PAP5, Histone H3 (H3), Rubisco (RbcL), and HY5 ; D, Dark; Ph, photomorphogenic growth conditions from germination on. *, HY5 is detected as a modified form (+ 6 kDa); the arrows indicate the two different post-translational modified forms of HY5 for which accumulation occurs upon light exposure.


B Relative contents normalized to histone H3 during transition from skotomorphogenesis to photomorphogenesis , we propose a phase of photo - initiation corresponding to 5 minutes of allowing PAP8 PAP5 to rise to nearly 100 % of their maximum . first macroscopic signs of greening are indicated around 3 hours while photosynthetic apparatus bursts at 8 hours to be fully accumulated at 48 hours .

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B Relative protein contents normalized to histone H3 during the transition from skotomorphogenesis to photomorphogenesis, we propose a phase of photo-initiation corresponding to the 5 minutes of light allowing PAP8 and PAP5 to rise to nearly 100% of their maximum. The first macroscopic signs of greening are indicated around 3 hours while the photosynthetic apparatus bursts at 8 hours to be fully accumulated at 48 hours.


Spot of designed TAD constructs . spot was performed at 30ºC ( left ) during heat shock at 37ºC ( right panel )

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Spot assay of designed TAD constructs . The spot assay was performed at 30ºC (left) and during heat shock at 37ºC (right panel)


scRNA - Seq of expression of entire glycolytic pathway or of glucose phosphate isomerase only ( GPI ) in CD4 + T - infected in vitro ( D , E ) or CD4 + T - of PLWH ( F ) . In panels ( D , E ) , were infected with VSVG - HIV - 1 - GFP sorted for viral expression as detailed in ( Golumbeanu et al , 2018 ) . Following latency establishment , were left untreated or HIV - 1 expression was reactivated through suberoyl anilide hydroxamic acid ( SAHA ) or α - CD3 - CD28 engagement . Clusters 1 2 were identified by principal component as described in ( Golumbeanu et al , 2018 ) . In panel ( F ) , CD4 + T - were isolated from total blood of PLWH under ART as described in ( Cohn et al , 2018 ) . Viral expression was reactivated by treatment with phytohemagglutinin ( PHA ) were sorted using antibodies against Env Gag . Sorted were then subjected to scRNA - Seq . expression level of HUMAN - GLYCOLYSIS pathway in ( D ) was calculated as average expression of genes comprising gene list ; expression in cluster 1 2 were compared using Wilcoxon rank sum . For panels ( E , F ) , significance of GPI differential expression level between clusters ( E ) or between control Env + Gag + conditions ( F ) was assessed by Wilcoxon rank sum encoded in FindMarkers Seurat R function . * * p < 0 . 01 , * * * p < 0 . 001 ; * * * p < 0 . 0001 . For panels ( D , E ) n = 1 donor , 43 ( untreated ) , 90 ( SAHA ) , 91 ( TCR ) , for panel ( F ) n = 3 donors , 109 ( control ) 85 ( Gag + Env + ) .

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scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only (GPI) in primary CD4+ T-cells infected in vitro (D,E) or CD4+ T-cells of PLWH (F). In panels (D, E), cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in (Golumbeanu et al, 2018). In panel (F), CD4+ T-cells were isolated from total blood of PLWH under ART as described in (Cohn et al, 2018). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env+ Gag+ conditions (F) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function. ** p< 0.01, *** p< 0.001; *** p< 0.0001. For panels (D,E) n = 1 donor, 43 cells (untreated), 90 cells (SAHA ), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag+ Env+).


Relative ratios of NADH / NAD + ( D ) ATP / ADP ( E ) in latently infected reactivated . Data were normalized using matching uninfected control .

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Relative ratios of NADH/NAD+ (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.


Relative expression of HIV - 1 gag in infected with Tat - deficient HIV - 1pNL4 - 3 as compared to infected with wild type HIV - 1 pNL4 - 3 .

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Relative expression of HIV-1 gag in cells infected with Tat-deficient HIV-1pNL4-3 as compared to cells infected with wild type HIV-1 pNL4-3.


relative expression of limiting rate enzyme of pentose phosphate pathway , i . e . G6PD ( B ) in HIV - 1 infected as compared to mock infected .

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relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B) in HIV-1 infected as compared to mock infected cells.


relative expression of genes regulating thioredoxin glutathione antioxidant pathways , i . e . TrxR1 ( C ) GCLC ( D ) , in HIV - 1 infected as compared to mock infected .

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relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.


Reactivation from HIV - 1 latency ( A ) relative viability ( B ) in Th17 following 24 h treatment with Trx inhibitor auranofin ( AF ; 500 nM ) , GSH inhibitor buthionine sulfoximine ( BSO ; 250 μM ) , or a combination of two . GFP - HIV - 1 expression was determined by FACS . Data are expressed as mean ± SD of three replicates were analyzed by One - Way ANOVA followed by Tukey ' s post - ( A ) or Two - Way ANOVA followed by Sidak ´

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Reactivation from HIV-1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP-HIV-1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by One-Way ANOVA followed by Tukey's post-test (A) or Two-Way ANOVA followed by Sidak´


of integrated HIV - 1 following treatment for 48 h with AF / or BSO in CD4 + T - derived from PLWH under suppressive antiretroviral therapy . Live were sorted after treatment , integrated was measured by Alu - PCR . latency reactivating agent SAHA was used as a reference compound ( Archin et al , 2012 ) . Data were analyzed by non - parametric Friedman ´ s followed by Dunn ' s post - .

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Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4+ T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al, 2012). Data were analyzed by non-parametric Friedman´s test followed by Dunn's post-test.


A HT - 29 were transfected with NC , siRIPK1 or miR - 324 - 5p . After 48 h , were treated with TNF - α ( T ) or T + S + Z for an additional 6 h . Western blotting of p - RIPK1 , RIPK1 , p - RIPK3 , RIPK3 , p - MLKL , MLKL β - actin .

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A HT-29 cells were transfected with NC, siRIPK1 or miR-324-5p. After 48 h, cells were treated with TNF-α (T) or T+S+Z for an additional 6 h. Western blotting analysis of p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL and β-actin.


B Western blotting of RIPK1 , RIPK3 , MLKL β - actin in HT - 29 that were transfected with NC , siRIPK1 , siRIPK3 , MLKL siRNA oliogs ( siMLKL ) , miR - 324 - 5p .

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B Western blotting analysis of RIPK1, RIPK3, MLKL and β-actin levels in HT-29 cells that were transfected with NC, siRIPK1, siRIPK3, MLKL siRNA oliogs ( siMLKL), and miR-324-5p.


C HT - 29 were harvested 48 h after transfection with NC , siMLKL , miR - 324 - 5p . MLKL expression was analyzed by qPCR ( upper ) or RT - PCR ( lower ) . Data information : Data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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C HT-29 cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. MLKL expression was analyzed by qPCR (upper) or RT-PCR (lower). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


D , E MKN45 ( D ) 174T ( E ) were harvested 48 h after transfection with NC , siMLKL , miR - 324 - 5p . qPCR for expression of MLKL ( upper ) western blotting of MLKL β - actin ( lower ) . Data information : Data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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D, E MKN45 (D) and 174T (E) cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. qPCR analysis for the expression of MLKL (upper) and western blotting analysis of MLKL and β-actin (lower). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


F HT - 29 were transfected with NC , siMLKL , miR - 324 - 5p , or anti - miR - 324 - 5p . After 48 h , were treated with DMSO or T + S + Z for an additional 24 h . survival was determined by measuring ATP . Data information : Data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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F HT-29 cells were transfected with NC, siMLKL, miR-324-5p, or anti-miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for an additional 24 h. Cell survival was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


G Western blotting of MLKL β - actin in HT - 29 that were harvested 48 h after transfection with NC , siMLKL , miR - 324 - 5p , or an anti - miR - 324 - 5p .

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G Western blotting analysis of MLKL and β-actin in HT-29 cells that were harvested 48 h after transfection with NC, siMLKL, miR-324-5p, or an anti-miR-324-5p.


B HEK293T were transfected with NC , MLKL siRNA oligos targeting 3 ' UTR of MLKL ( siMLKL - 3 ' UTR ) , or miR - 324 - 5p , together with pmirGLO - MLKL - 3 ' UTR or mutated form of MLKL - 3 ' UTR shown in A . Relative luciferase activity of MLKL - 3 ' UTR . Data are presented as firefly luciferase activity / renilla luciferase activity ± SD . In B , data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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B HEK293T cells were transfected with NC, MLKL siRNA oligos targeting the 3'UTR of MLKL (siMLKL-3'UTR), or miR-324-5p, together with pmirGLO-MLKL-3'UTR or the mutated form of MLKL-3'UTR shown in A. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD. In B, data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


C HEK293T were transfected with NC , miR - 324 - 5p , or mutated form of miR - 324 - 5p together with pmirGLO - MLKL - 3 ' UTR . Relative luciferase activity of MLKL - 3 ' UTR . Data are presented as firefly luciferase activity / renilla luciferase activity ± SD ( n = 5 , biological replicates ) .

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C HEK293T cells were transfected with NC, miR-324-5p, or the mutated form of miR-324-5p together with pmirGLO-MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n=5, biological replicates).


D Western blotting of MLKL β - actin in HeLa - endogenous MLKL ( HeLa stably expressing human RIPK3 ) HeLa - exogenous MLKL ( Mlkl - / - HeLa stably expressing human RIPK3 coding ( CDS ) of MLKL ) that were harvested 48 h after transfection with NC , miR - 324 - 5p , or MLKL siRNA oligos targeting CDS region of MLKL ( siMLKL - CDS ) .

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D Western blotting analysis of MLKL and β-actin in HeLa-endogenous MLKL cells (HeLa cells stably expressing human RIPK3) and HeLa-exogenous MLKL cells (Mlkl-/- HeLa cells stably expressing human RIPK3 and the coding sequence (CDS) of MLKL) that were harvested 48 h after transfection with NC, miR-324-5p, or MLKL siRNA oligos targeting the CDS region of MLKL (siMLKL-CDS).


E qPCR for expression of MLKL in HeLa - exogenous MLKL that were harvested 48 h after transfection with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . Data information : data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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E qPCR analysis for the expression of MLKL in HeLa-exogenous MLKL cells that were harvested 48 h after transfection with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


F , G HeLa - endogenous MLKL ( F ) HeLa - exogenous MLKL ( G ) were transfected with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . After 48 h , were treated with T + S + Z for 24 h . survival was determined by measuring ATP . Data information : number of surviving was normalized to number of surviving control , which were treated with DMSO . data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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F, G HeLa-endogenous MLKL cells (F) and HeLa-exogenous MLKL (G) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with T+S+Z for 24 h. Cell survival was determined by measuring ATP levels. Data information: The number of surviving cells was normalized to the number of surviving control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


H HeLa expressing MLKL ( 1 - 190 aa ) fused to DmrB ( HeLa - MLKL ( 1 - 190 ) - Dmir ) were transfected with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . After 48 h , were treated with DMSO or dimerization agent AP20187 ( 60 nM ) for 24 h . survival was determined by measuring ATP . Data information : number of surviving was normalized to number of surviving control , which were treated with DMSO . data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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H HeLa cells expressing MLKL(1-190 aa) fused to DmrB (HeLa-MLKL(1-190)-Dmir cells) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with DMSO or the dimerization agent AP20187 (60 nM) for 24 h. Cell survival was determined by measuring ATP levels. Data information: The number of surviving cells was normalized to the number of surviving control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


C African green monkey kidney epithelial ( Vero ) were transfected with NC , siMLKL , or miR - 324 - 5p for 48 h . qPCR for expression of MLKL . Data information : data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown .

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C African green monkey kidney epithelial (Vero) cells were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


D MEFs were transfected with NC , siMLKL , or miR - 324 - 5p for 48 h . qPCR for expression of MLKL western blotting of MLKL β - actin . Data information : data are represented as means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - . experiments were performed at least three times , representative data are shown . β - actin serves as an internal control ( D ) .

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D MEFs were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL and western blotting analysis of MLKL and β-actin. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown. β-actin serves as an internal control (D).