EMBO/SourceData huggingface dataset:
Figure 6A. 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 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 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 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.
Figure 7A. 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. 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, 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, 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, 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.
f1a, 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 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 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.
f2a, 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 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 and the release of active caspase-1 and IL-1b was determined.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 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.
f3a-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 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 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 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 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.
f4a, 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 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.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.
Figure 1B 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 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 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 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.
Figure 2B 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 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 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) 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.
Figure 3B 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.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.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.
Figure 4(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.
Figure 5A 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).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.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.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.
Figure 6A 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.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.
Figure 7A 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.
Figure 1scRNA-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+).
Figure 2Relative ratios of NADH/NAD+ (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.
Figure 4Relative 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.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.
Figure 5Reactivation 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´s post-test (B). Solid lines represent the means.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.
Figure 2A 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.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.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.
Figure 3B 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).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.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.