EMBO/SourceData huggingface dataset:
Figure 4(A) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (A) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.(B) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (B) small hairpin RNA-mediated LRH-1 knockdown (shLRH-1). Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(C) Cell cycle distribution of T-ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(D) Simultaneous flow cytometric analysis of cell cycle distribution by EdU/DAPI staining and cell death by AnnexinV staining in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase distribution as % of viable, AnnexinV negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(E) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated 3d2 concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(F) Flow cytometric analysis of 3d2-induced cell death by AnnexinV staining in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(G) Immunoblots of full-length and cleaved (cl.) PARP, P-p38, c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.
Figure 6(A) Immunoblots of full-length and cleaved (cl.) PARP, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated for 24 h with 10 µM dexamethasone (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.(B) Flow cytometric analysis of cell death by AnnexinV staining in MOLT-4 cells treated with 3d2, 30 µM Dexa and/or 50 µM pan caspase inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.(C) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout (Bim KO) CEM-C7 cells treated with 3d2 and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.(D) Immunoblots of BimEL, BimL and BimS, Bcl-2 and Tubulin from MOLT-4 expressing a non- (shcontrol) or LRH-1- (shLRH-1) targeting small hairpin RNA construct.(E) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout (GR KO) MOLT-4 cells treated with 3d2 and 30 µM Dexa. Mean values of n=3 biological replicates ± SD are shown. Data information: Two-way ANOVA E); ns = not significant, ** p < 0.01, *** p < 0.001.(F/G) mRNA expression levels of human GR (F) and Bim (G) in shcontrol and shLRH-1 CEM-C1 cells were determined by real-time quantitative PCR after treatment with 1 µM Dexa for indicated times. Fold induction relative to the untreated shcontrol sample (0h) was calculated after normalization to beta-actin. Individual values of technical triplicates of one representative of two biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.(H) Immunoblots of full-length and cleaved (cl.) PARP, GR and phospho-AMPK (P-AMPK), c-Myc, full-length and cl. Caspase 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 and/or 10 µM Dexa. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø) was used as a solvent control.
Figure 7(A) Ex vivo response of human T cell acute lymphoblastic leukemia (T-ALL) patient-derived xenografts (PDX), co-cultured on hTERT-immortalized primary bone marrow MSCs, to treatment with indicated concentrations of 3d2 and/or dexamethasone (Sample 1, 3 and 4: 1 nM; Sample 2: 0.1 nM). Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage (%) of the dimethyl sulfoxide treated control. Data information: All displayed experiments display one biological replicate.(B) Synergy scores (Z-scores) between 3d2 and dexamethasone of human T-ALL patient-derived xenografts (PDX) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and/or dexamethasone using SynergyFinder tool. Z-scores ≥ 0 indicate additivity or synergism and < 0 antagonism. Data information: All displayed experiments display one biological replicate.(C) Expression levels of human GRα, LRH-1 and Bim mRNA in human PDX were determined by real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Data information: All displayed experiments display one biological replicate.(E) In vivo leukemia progression of human T-ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle (PBS control), 40 mg/kg 3d2 and or 10 mg/kg Dexamethasone starting 3 days post transplantation. Engraftment was calculated as % of human vs total (human+mouse) CD45 positive (CD45+) cells as assessed by flow cytometric analysis of peripheral blood. Data information: All displayed experiments display one biological replicate.
Figure 1(a) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.
Figure 2(c) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7). Data information: Scale bars, 50 µm.(d) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001. Data information: Scale bars, 50 µm.
Figure 3(a) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.
Figure 4Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µmImmortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm
Figure 5(c) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border. (d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001. Data information: Scale bars, 100 µm(e) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.
Figure 152-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test)., I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.
Figure 2Immunoblots showing CIB1 (A) protein levels in WT and co-1 backgrounds (A) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).B. Immunoblots showing CO (B) protein levels in WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).The relative levels of CIB1 in WT and co-1 backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.D. The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.
Figure 3A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm. Data information: For each experiment, two or more biological replicates were performed.C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC. Data information: For each experiment, two or more biological replicates were performed.D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs. Data information: For each experiment, two or more biological replicates were performed.F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN). Data information: For each experiment, two or more biological replicates were performed.
Figure 4A. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0. Data information: data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.
Figure 5A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μB. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.
Figure 1MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1.
Figure 2MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3).MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3).
Figure 3HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).
Figure 4MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).