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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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).


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 .

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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 the central band , 3rd quartile , max ) on the diameter of the nuclear bodies ( NBs ) ; n equals the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 complex with PAP5 ( red ) . Grey areas depict changes of signals in the 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 and black respectively . The units on the y - axis are normalized values of the integrals of the signal measured in the 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 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 .

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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 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 .

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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 assay of designed TAD constructs . The spot assay was performed at 30ºC ( left ) and 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 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 + ) .

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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 ) and ATP / ADP ( E ) in latently infected and reactivated cells . Data were normalized using the 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 cells infected with Tat - deficient HIV - 1pNL4 - 3 as compared to cells 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 the limiting rate enzyme of the pentose phosphate pathway , i . e . G6PD ( B ) in HIV - 1 infected as compared to mock infected cells .

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

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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 ) 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 ´

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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´


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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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).


E MEFs were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were treated with DMSO or T + S + Z for 24 h . Cell viability 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 .

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E MEFs were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for 24 h. Cell viability 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.


F HEK293T cells were transfected with NC or miR - 324 - 5p , together with pmirGLO - mouse MLKL - 3 ' UTR or the mutated form of mouse 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 ) .

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F HEK293T cells were transfected with NC or miR-324-5p, together with pmirGLO-mouse MLKL-3'UTR or the mutated form of mouse 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).


A U937 cells were treated with 100 ng / ml IFN - α , 100 ng / ml IFN - β , 100 ng / ml IFN - γ , 100 ng / ml LPS , or 25 μg / ml poly ( I : C ) for 24 h . Identical concentrations were used in later experiments unless otherwise stated . qPCR analysis for the expression of MLKL . C U937 cells were treated with PBS , IFN - α , IFN - β , IFN - γ , LPS , or poly ( I : C ) for 24 h . qPCR analysis for the expression of miR - 324 - 5p . 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 .

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A U937 cells were treated with 100 ng/ml IFN-α, 100 ng/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR analysis for the expression of MLKL. C U937 cells were treated with PBS, IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. qPCR analysis for the expression of miR-324-5p. 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.


B U937 cells were treated with IFN - α , IFN - β , IFN - γ , LPS , or poly ( I : C ) for 24 h . and western blotting analysis of MLKL and β - actin ( left ) . Quantification of MLKL normalized to β - actin levels ( right ) . 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 .

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B U937 cells were treated with IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. and western blotting analysis of MLKL and β-actin (left). Quantification of MLKL normalized to β-actin levels (right). 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 U937 cells were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were treated with IFN - β ( D ) or IFN - γ ( E ) for 24 h . Western blotting analysis of MLKL and β - actin .

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D, E U937 cells were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with IFN-β (D) or IFN-γ (E) for 24 h. Western blotting analysis of MLKL and β-actin.


F , G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN - β ( F ) or IFN - γ ( G ) treatment . After 24 h , qPCR analysis for the expression of miR - 324 - 5p . After 48 h , western blotting analysis of p - STAT1 , STAT1 , 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 .

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F, G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN-β (F) or IFN-γ (G) treatment. After 24 h, qPCR analysis for the expression of miR-324-5p. After 48 h, western blotting analysis of p-STAT1, STAT1, 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.


H , I U937 cells were transfected with NC , or STAT1 siRNA oliogs ( siSTAT1 ) . After 48 h , cells were treated with IFN - β ( Η ) or IFN - γ ( Ι ) . qPCR analysis for the expression of miR - 324 - 5p . Western blotting analysis of STAT1 , 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 .

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

H, I U937 cells were transfected with NC, or STAT1 siRNA oliogs (siSTAT1). After 48 h, cells were treated with IFN-β (Η) or IFN-γ (Ι). qPCR analysis for the expression of miR-324-5p. Western blotting analysis of STAT1, 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.


J HT - 29 cells were treated with IFN - γ for 24 h . The cell lysate was collected for ChIP analysis . STAT1 binding to miR - 324 - 5p promoter DNA region was determined by Chip - qPCR . The amount of precipitated DNA was calculated as percent input . 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 .

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

J HT-29 cells were treated with IFN-γ for 24 h. The cell lysate was collected for ChIP analysis. STAT1 binding to miR-324-5p promoter DNA region was determined by Chip-qPCR. The amount of precipitated DNA was calculated as percent input. 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.


A U937 cells were transfected with NC , siMLKL - 3 ' UTR , or miR - 324 - 5p . After 48 h , cells were exposed to 100 ng / ml IFN - γ plus 20 μM z - VAD for 24 h . Identical concentrations were used in later experiments unless otherwise stated . Cell viability 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 .

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

A U937 cells were transfected with NC, siMLKL-3'UTR, or miR-324-5p. After 48 h, cells were exposed to 100 ng/ml IFN-γ plus 20 μM z-VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability 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.


B qPCR analysis for the expression of MLKL ( left ) and miR - 324 - 5p ( middle ) in PBMC - derived macrophages that were treated with PBS or IFN - γ for 24 h . The corresponding western blotting analysis of MLKL and β - actin ( right ) . 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 .

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

B qPCR analysis for the expression of MLKL (left) and miR-324-5p (middle) in PBMC-derived macrophages that were treated with PBS or IFN-γ for 24 h. The corresponding western blotting analysis of MLKL and β-actin (right). 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.


C , D PBMC - derived macrophages ( C ) and HT - 29 cells ( D ) were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were exposed to S + Z or IFN - γ + S + Z for 24 h . Cell viability was determined by measuring ATP levels ( left ) . The corresponding western blotting analysis of MLKL and β - actin ( right ) . 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 .

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

C, D PBMC-derived macrophages (C) and HT-29 cells (D) were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were exposed to S+Z or IFN-γ+S+Z for 24 h. Cell viability was determined by measuring ATP levels (left). The corresponding western blotting analysis of MLKL and β-actin (right). 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.


E qPCR analysis for the expression of miR - 324 - 5p in miR - 324 - 5p knockout HT - 29 cells ( miR - 324 - 5p - / - cells ) . Altered miR - 324 - 5p DNA sequences were shown in miR - 324 - 5p - / - clone 19 # and 36 # . 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 .

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

E qPCR analysis for the expression of miR-324-5p in miR-324-5p knockout HT-29 cells (miR-324-5p-/- cells) . Altered miR-324-5p DNA sequences were shown in miR-324-5p-/- clone 19# and 36#. 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 MiR - 324 - 5p + / + HT - 29 , miR - 324 - 5p - / - - 19 # , and miR - 324 - 5p - / - - 36 # cells were treated with T + S + Z for the indicated times . Cell viability 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 .

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

F MiR-324-5p+/+ HT-29, miR-324-5p-/- -19# , and miR-324-5p-/- -36# cells were treated with T+S+Z for the indicated times. Cell viability 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 , H MiR - 324 - 5p + / + HT - 29 , and miR - 324 - 5p - / - HT - 29 cells were treated with IFN - β ( G ) or IFN - γ ( H ) plus 20 μM z - VAD for 24 h . Cell viability 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 .

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

G, H MiR-324-5p+/+ HT-29, and miR-324-5p-/- HT-29 cells were treated with IFN-β (G) or IFN-γ (H) plus 20 μM z-VAD for 24 h. Cell viability 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.


A qPCR analysis for the expression of IFN - β ( left ) and IFN - γ ( right ) in PBMC - derived macrophages that were infected with IAV ( H1N1 strain PR8 ) at an multiplicity of infection ( MOI ) of 0 . 2 for 24 h . B qPCR analysis for the expression of MLKL ( left ) and miR - 324 - 5p ( right ) in PBMC - derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV ( MOI = 0 . 2 ) for 24 h . 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 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR analysis for the expression of IFN-β (left) and IFN-γ (right) in PBMC-derived macrophages that were infected with IAV (H1N1 strain PR8) at an multiplicity of infection (MOI) of 0.2 for 24 h. B qPCR analysis for the expression of MLKL (left) and miR-324-5p (right) in PBMC-derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV (MOI=0.2) for 24 h. 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.


C PBMC - derived macrophages were transfected with NC , siMLKL , or miR - 324 - 5p . After 48h , cells were treated with S + Z in the presence or absence of IAV ( MOI = 0 . 2 ) for 24 h . Cell viability 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 .

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

C PBMC-derived macrophages were transfected with NC, siMLKL, or miR-324-5p. After 48h, cells were treated with S+Z in the presence or absence of IAV (MOI=0.2) for 24 h. Cell viability 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.


D qPCR analysis for the expression of M gene of IAV in PBMC - derived macrophages that were transfected with NC , siMLKL or miR - 324 - 5p for 48h , followed by the infection with IAV ( MOI = 0 . 2 ) for 24 h . 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 .

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

D qPCR analysis for the expression of M gene of IAV in PBMC-derived macrophages that were transfected with NC, siMLKL or miR-324-5p for 48h, followed by the infection with IAV (MOI=0.2) for 24 h. 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.


A Freeze - fracture electron micrograph of wild - type yeast treated with DTT for 3 h . The left panel shows two spherical structures , one in a vacuole membrane invagination and one inside the vacuole . The middle panel shows the boxed area of the left panel at higher magnification . Arrows indicate membrane layers that are free of intramembrane particles . The right panel shows the protoplasmic fracture face ( P face ) of cortical ER with a high density of intramembrane particles . Cy , cytosol ; N , nucleus ; V , vacuole .

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A Freeze-fracture electron micrograph of wild-type yeast treated with DTT for 3 h. The left panel shows two spherical structures, one in a vacuole membrane invagination and one inside the vacuole. The middle panel shows the boxed area of the left panel at higher magnification. Arrows indicate membrane layers that are free of intramembrane particles. The right panel shows the protoplasmic fracture face (P face) of cortical ER with a high density of intramembrane particles. Cy, cytosol; N, nucleus; V, vacuole.


B Fluorescence images of cells expressing GPD promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . The upper and lower panel show vacuole - associated and perinuclear GFP - Pho8 stretches and an intravacuolar GFP - Pho8 ring , respectively . Scale bars : 2 µm .

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B Fluorescence images of cells expressing GPD promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. The upper and lower panel show vacuole-associated and perinuclear GFP-Pho8 stretches and an intravacuolar GFP-Pho8 ring, respectively. Scale bars: 2 µm.


C Correlative light and electron microscopy of cells expressing GPD promoter - driven GFP - Pho8 and Sec63 - mCherry . The lower panel shows the boxed area in the electron micrograph at higher magnification . The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack .

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C Correlative light and electron microscopy of cells expressing GPD promoter-driven GFP-Pho8 and Sec63-mCherry. The lower panel shows the boxed area in the electron micrograph at higher magnification. The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack.


D Fluorescence images of cells expressing Hmg2 - GFP and either additionally expressing Sec63 - mCherry or stained with CMAC . The upper and lower panel show cytosolic and vacuolar Hmg2 - GFP rings , respectively . Scale bars : 2 µm .

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D Fluorescence images of cells expressing Hmg2-GFP and either additionally expressing Sec63-mCherry or stained with CMAC. The upper and lower panel show cytosolic and vacuolar Hmg2-GFP rings, respectively. Scale bars: 2 µm.


A Individual frames from time - lapse imaging of a cell expressing GAL promoter - driven GFP - Pho8 and stained with the vacuole membrane dye FM4 - 64 . Numbers indicate the time in minutes after the start of the image sequence . Scale bar : 2 µm .

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A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm.


B Electron micrograph of a cell expressing GPD promoter - driven GFP - Pho8 . Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination . Black arrowheads point to vacuole membrane that encases the bent stack and the whorl .

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B Electron micrograph of a cell expressing GPD promoter-driven GFP-Pho8. Arrows indicate the edges of ER cisternae that are part of a bent stack in a vacuole membrane invagination. Black arrowheads point to vacuole membrane that encases the bent stack and the whorl.


C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks . n = 43 .

C Fraction of uptake intermediates that were converted into vacuolar whorls or stacks. n = 43.


The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl . Scale bar : 2 µm .

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The image sequence shows formation of a vacuolar whorl that becomes a cytosolic whorl. Scale bar: 2 µm.


Fluorescence images of ∆ atg7 and ∆ atg8 cells ( A ) expressing GAL promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . Scale bar : 2 µm .

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Fluorescence images of ∆atg7 and ∆atg8 cells (A) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.


Fluorescence images of ∆ snf7 and ∆ vps4 cells ( B ) expressing GAL promoter - driven GFP - Pho8 and the ER marker Sec63 - mCherry and stained with CMAC to label the vacuole . Scale bar : 2 µm .

Fluorescence images of ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.


C Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 in tunicamycin - treated cells . The dotted line indicates the activity in ∆ atg7 cells . Mean + SEM , n ≥ 3 .

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C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3.


D , E Relative activity of endogenous Pho8 in untreated cells ( D ) or after tunicamycin treatment ( E ) . Background activity determined in ∆ pho8 mutants was subtracted from the activity in all other strains . Mean + SEM , n = 3 . WT , wild - type .

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D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type.


F Western blot of GFP from cells expressing Sec63 - GFP and treated with tunicamycin ( Tm ) where indicated . The two Atg7 - independent cleavage products of around 55 kDa were used as readout for micro - ER - phagy . Pgk1 served as a loading control . G Quantification of Sec63 - GFP cleavage normalized to the WT from western blots as shown in panel ( F ) . Mean + SEM , n = 4 .

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F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control. G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4.


A Electron micrograph of wild - type yeast treated with DTT for 3 h . Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies ( white arrows ) . Cy , cytosol ; V , vacuole .

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A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.


B Relative activity of cyto - Pho8 ∆ 60 after nitrogen starvation , which reports on non - selective macroautophagy of cytosol . Mean + SEM , n ≥ 3 . WT , wild - type .

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B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after nitrogen starvation ( C ) Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3.


Relative activity of the ER - phagy reporter Yop1 - Pho8 ∆ 60 after tunicamycin treatment ( D , E ) . Mean + SEM , n ≥ 3 .

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Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.


F , G Growth assays of untreated and tunicamycin - treated WT , ∆ atg39 / 40 , ∆ snf7 and ∆ snf7 ∆ atg39 / 40 cells . Numbers indicate growth relative to WT cells based on the areas under the curves . Mean ± SEM , n = 3 .

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F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.


A Fraction of wild - type ( WT ) , ∆ vps23 , ∆ snf7 and ∆ vps4 cells with GFP - Pho8 structures . Mean + SEM , n ≥ 3 .

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A Fraction of wild-type (WT), ∆vps23, ∆snf7 and ∆vps4 cells with GFP-Pho8 structures. Mean + SEM, n ≥ 3.


B Fraction of GFP - Pho8 structures classified as stacks , cytosolic whorls , uptake intermediates or vacuolar whorls . The number of GFP - Pho8 structures in WT , ∆ vps23 , ∆ snf7 and ∆ vps4 cells was 562 , 345 , 367 and 449 , respectively . The table shows the results of Chi - Square tests for independence for all pairs of strains . * * * , highly significant ( P < 0 . 00001 ) ; ns , not significant ( P > 0 . 05 ) .

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B Fraction of GFP-Pho8 structures classified as stacks, cytosolic whorls, uptake intermediates or vacuolar whorls. The number of GFP-Pho8 structures in WT, ∆vps23, ∆snf7 and ∆vps4 cells was 562, 345, 367 and 449, respectively. The table shows the results of Chi-Square tests for independence for all pairs of strains. ***, highly significant (P < 0.00001); ns, not significant (P > 0.05).


C Fraction of stacks classified as peripheral , perinuclear or vacuole - associated . The number of stacks in WT and ∆ snf7 cells was 252 and 351 , respectively . The observed distributions in the two strains were analyzed with a Chi - Square test for independence . * * * , highly significant ( P < 0 . 00001 ) .

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C Fraction of stacks classified as peripheral, perinuclear or vacuole-associated. The number of stacks in WT and ∆snf7 cells was 252 and 351, respectively. The observed distributions in the two strains were analyzed with a Chi-Square test for independence. ***, highly significant (P < 0.00001).


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


Fluorescence images of a cell expressing non - fluorescent GFP ( Y66F ) - Pho8 , Snf7 - mNeonGreen and the ER marker mCherry - Ubc6 , and stained with the vacuole dye CMAC . Numbers indicate the time in minutes after the start of the image sequence . White arrows indicate a Snf7 - mNeonGreen punctum adjacent to an ER whorl . Black arrow point to a vacuole membrane invagination . Panel ( E ) illustrates transient appearance of Snf7 at an ER whorl . Panel ( F ) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination . In this series , mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time . Scale bar : 2 µm .

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Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.


A Electron micrographs of wild - type and ∆ snf7 cells treated with DTT for 3 h . Cy , cytosol ; V , vacuole . Scale bars : 300 nm .

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A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.


B Quantification of vacuolar whorls for which the uptake process was complete or incomplete . The observed distributions in the two strains were analyzed with a Fisher ' s exact test .

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B Quantification of vacuolar whorls for which the uptake process was complete or incomplete. The observed distributions in the two strains were analyzed with a Fisher's exact test.


C , D Electron micrographs of serial thin sections of a ∆ snf7 cell ( C ) or a wild - type cell ( D ) treated with DTT for 3 h . Note that the whorl appears separated from the cytosol in some sections ( e . g . section 300 nm ) but is still in contact with the cytosol ( see sections 600 nm and 700 nm ) . In the wild - type cell ( D ) , the vacuolar whorl is completely separated from the cytosol . Scale bar : 300 nm .

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C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.


Callus , de novo shoot , and root formation in wild - type ( WT ; Col ) , hag1 - 6 , hag1 - 7 , and 35S : : FLAG : HAG1 hag1 - 7 root explants . Root explants were transferred onto CIM for 1 week ( w ) and then transferred onto fresh CIM , SIM , or RIM for callus , de novo shoot , or root induction , respectively . Scale bar : 1 cm . Graph on the right shows the percentage of explants with shoots formed as scored at 18 days ( d ) on SIM . 36 explants of each genotype were used for scoring .

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Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.


Shoot regeneration assay using Col and hag1 - 6 root explants on media with different cytokinin ( 2IP ) to auxin ( IAA ) ratios . Col and hag1 - 6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg / ml IAA - containing media supplemented with 0 , 500 , or 5 , 000 mg / ml 2IP . Pictures were taken at 27 d after transfer onto each media using representative explants . Scale bar : 2 mm .

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Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.


Root explant - derived calli of Col and hag1 - 6 at 4 w on CIM ( left ) . Scale bar : 1 mm . 4 - w - old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight ( right ) .

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Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).


Histochemical GUS staining of CYCB1 ; 1 : GUS in Col ( left ) and CYCB1 ; 1 : GUS in hag1 - 6 . Shown are root explants at 4 d on CIM . Scale bar : 1 mm .

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Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm.


Statistic chart of differentially expressed genes ( DEGs ) in Col and hag1 - 6 roots ( R ) , 4 d CIM - incubated root explants ( C ) , 4 d CIM - incubated + 2 d SIM - incubated root explants ( S ) , and 4 d CIM - incubated + 2 w SIM - incubated root explants ( S2W ) . Green : up - regulated genes , log2 ratio ≥ 1 ; red : down - regulated genes , log2 ratio ≤ - 1 ; FDR < 0 . 01 .

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Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.


Immunoblot analysis of HAG1 : HA protein . Samples are as described in ( E ) . 1 w CIM - incubated Col root explants ( Col ) were used as negative control . Ponceau staining was performed as loading control .

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Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.


pWOX5 : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with propidium iodide ( PI ) staining ( red ) .

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pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

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pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 root tips at various time points on CIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

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pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


WOX5 transcript levels in Col and hag1 - 6 hypocotyl ( D ) and cotyledon ( E ) explants at indicated days on CIM .

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WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.


SCR transcript levels in Col and hag1 - 6 hypocotyl ( F ) and cotyledon ( G ) explants at indicated days on CIM .

SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.


H3Ac enrichment at the WOX5 ( A ) , SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) loci in Col ( upper black diagram ) and hag1 - 6 ( lower red diagram ) calli ( 1 w CIM ) as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 5e - 2 for WOX5 ( A ) and 1e - 5 for SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) . ChIP - seq data were visualized using Integrated Genome Browser ( IGB ; http : / / bioviz . org / igb / index . html ) . The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position , respectively . Schematic representation of each locus is depicted at the bottom . Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines . 5 ' and 3 ' UTRs are represented as gray boxes . Transcription start sites are indicated with arrows .

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H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.


ChIP - qPCR analysis of H3Ac levels at the WOX5 ( E ) , SCR ( F ) , PLT1 ( G ) , and PLT2 ( H ) loci in Col and hag1 - 6 . Root ( R ) , 1 w CIM ( C ) , and 1 w CIM + 2 w SIM ( S ) samples were used for assays .

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ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays.


Direct targeting of HAG1 : HA protein to WOX5 ( I ) , SCR ( J ) , PLT1 ( K ) , and PLT2 ( L ) chromatin as determined by ChIP - qPCR .

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Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR.


Transcript levels of WOX7 ( A ) and WOX14 ( B ) in Col and hag1 - 6 roots ( R ) , 4 d CIM explants ( C ) , 4 d CIM + 2 d SIM explants ( S ) , and 4 d CIM + 2 w SIM explants ( S2W ) . Levels of Col R were set to 1 after normalization with UBQ10 .

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Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.


De novo shoot regeneration of Col , Nossen ( No ) , wox5 - 3 , wox7 - 1 , wox14 - 1 , and wox5 - 3 wox7 - 1 wox14 - 1 root explants . Col is the WT control for wox5 - 3 and wox7 - 1 , whereas No is the WT control for wox14 - 1 . The picture was taken at 3 w on SIM using representative explants of each genotype . Scale bar : 1 cm . Percentage of explants with shoots was scored at indicated weeks on SIM ( D ) .

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De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).


H3Ac enrichment at the WOX14 locus in Col ( upper black diagram ) or hag1 - 6 ( lower red diagram ) calli as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 1e - 5 . See Figs 3A - D for schematic .

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H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.


ChIP - qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1 - 6 . Regions tested are indicated in the schematic in ( E ) . Sample preparation and data presentation were performed as described in Figs 3E - H .

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ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.


Direct targeting of HAG1 : HA protein to WOX14 chromatin as determined by ChIP - qPCR .

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Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.


De novo shoot regeneration using Col and scr mutants ; scr - 3 ( A and B ) and scr - 6 ( A and C ) . Explants with shoots were scored at indicated days on SIM using Col and scr - 3 root explants that had been pre - incubated on CIM for 2 w ( B ) or Col and scr - 6 hypocotyl explants that had been pre - incubated on CIM for 1 w ( C ) . Scale bar : 1 mm .

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De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.


De novo shoot regeneration of Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 . The percentage of root explants with shoots ( E ) and the number of shoots per root explant ( F ) were scored at 15 d on SIM . The explants had been pre - incubated on CIM for 2 w before transfer onto SIM . Scale bar : 1 cm .

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De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.


Transcript levels of PLT1 ( G ) and PLT2 ( H ) in Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 roots ( R ) , 1 w CIM explants ( C ) , and 1 w CIM + 2 w SIM explants ( S2W ) as determined by RT - qPCR . Levels of Col R were set to 1 after normalization with UBQ10 .

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Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.


Activation of WOX5 by DEX treatment of 35S : : GVG - WOX5 in Col and hag1 - 6 . Root explants of 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX . DEX was treated for 3 - d - pulse period on CIM or / and SIM ( see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure ) . Morphology of shoots regenerated from 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 root explants ( A ) . Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX ( B ) .

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Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).


Activation of SCR by DEX treatment of UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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

Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.


Activation of WOX5 and SCR by DEX treatment of 35S : : GVG - WOX5 UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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

Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.