EMBO/SourceData huggingface dataset:
Figure 5A. Exponentially growing S. cerevisiae WT, ste∆, tag∆ and yeh2∆ cells were treated with 100 µg/mL zeocin for 90 minutes. This treatment duration recurrently results in more cells accumulating in the G1 phase of the cell cycle After 90 min, cells were washed to remove zeocin and resuspended in fresh, zeocin-free medium, and samples were retrieved every hour, as indicated. All samples were processed for western blot analysis and cytometry. The de-activation of the DDR was monitored following the progressive de-phosphorylation of Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. Top: samples were migrated using commercial 3-8% gradient gels. Bottom: samples were migrated in home-made resolutive gels (see M&M).B. The percentage of Rad53-P was quantified at each time point as in Fig 2B. The plotted values represent the mean value of at least 3 independent experiments and the variability is represented by the SEM. Unpaired one-way ANOVA tests were conducted to compare the potential differences of the means of the four conditions at each time point. Only the p-value(s) for the comparisons being significantly different are indicated, where *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Figure 6B. Details as in (A) but for the indicated strains. A one-way ANOVA for multiple comparisons against the WT values (not plotted) was applied to evaluate whether there were significant differences between the means of the 210 time-point. p-values are indicated by asterisks. *, p < 0.05.C. Left: Representative images obtained after immunofluorescence to detect P-ATM (ATM phosphorylation at Serine 1981) signal in RPE-1 and Huh-7 fixed cells that had been treated (or not) with 0.005% MMS or 10 µg/mL zeocin for 2 hours; pre-treatments were done for 4 hours with 60 µM oleate. Size bars are 10 µm. Right: Quantification of the percentage of P-ATM-positive cells. The values obtained from 3 to 4 independent experiments are plotted as black spots, and the mean of them is shown as an orange bar. At least 200 cells were counted per condition and experiment. The p-values obtained after performing an unpaired t-test are shown.D. RPE-1 and Huh-7 cells were treated or not with MMS 0.005% for 2 hours. Thereafter, the ATM inhibitor AZD0156 was added at a final concentration of 1, 10 or 50 nM for 1 hour. Cells were collected and phosphorylation on Thr68 of CHK2 and tubulin levels were analyzed by western blot.E. Details as in (C) but to detect phosphorylated H2AX (yH2AX). The graph shows the mean fold-change in signal per nucleus out of at least 300 nuclei counted per condition and experiment. 3 to 4 independent experiments were performed. The bar height indicates the value of the mean of the means and, the error bars, the associated SEM.
Figure 7B. A hydrophobic membrane on which 100 pmol of the indicated lipid species were spotted was incubated with immunopurified FLAG-Tel1 and further developed using an anti-FLAG antibody. The bar lengths in the graph represent the mean value of 4 independent experiments and the error bars, the associated SEM. To assess whether the mean values differed significantly, a t-test was performed and p-values are shown. Additionally, an identical lipid-containing membrane was incubated with 3xFLAG peptide and subsequently developed using an anti-FLAG antibody to control that the observed signals were not due to FLAG peptide binding alone. PI; phosphatidylinositol; PI(4)P, phosphatidylinositol-4-phosphate; PI(4,5)P, phosphatidylinositol-4,5-bisphosphate; PI(3,4,5)P, phosphatidylinositol-3,4,5-triphosphate.D. Proximity ligation assay (PLA) to assess whether ATM and PI(4)P are in close proximity (< 40 nm). Top: representative pictures of RPE-1 cells, in which PLA signals are pictured in green. Size bar is 10 µm. Bottom left: the graph shows the mean PLA signal area occupied per cell when doing the experiment with both antibodies (experiment) or by omitting one primary antibody at a time (technical controls). In practice, each point is the value of having measured all the PLA signals present in one photo (20 to 40 cells) and divided this value by the number of nuclei. To account for reproducibility, we used SuperPlots to draw the graphs each independent experiment is plotted in a different colour, where the mean value of each independent experiment is highlighted by a more intense colour than the individual values of that experiment, for which the colour is more translucid. Last, the solid horizontal line marks the mean of the means. A one-way ANOVA for multiple comparisons was applied to evaluate whether there were significant differences between the means. Bottom right: the graph shows the mean PLA signal area occupied per cell when doing the experiment either in cells transfected with an siRNA Control (experiment) or with three different siRNAs targeted against ATM (biological controls). The meaning of each plotted point, of the horizontal line and the statistical analysis is as explained above.
Figure 8B. Left: RPE-1 cells untreated or exposed to 10 µg/mL zeocin for 2 h were used either for whole-cell protein extraction (W) or for fractionation into cytoplasmic (C) and nuclear (N) fractions in order to assess relative ATM distribution. Fractionation quality was assessed by checking for histone 3 (H3) (nucleus) and GOLPH3 (cytoplasm) signals. Right: To quantify the relative distribution of total ATM between nucleus and cytoplasm, total ATM signals were normalized to total Ponceau signals from the corresponding fraction. Each dot of a given color belongs to an independent experiment, the bar height shows the mean of those experiments, and the error bars represent the SEM. The p-value is derived from an unpaired t-test.D. Exponentially growing S. cerevisiae WT cells transformed with an empty vector ("control") or with a plasmid overexpressing OSH4 were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for inspection by fluorescence microscopy to evaluate the percentage of cells displaying GFP-Tel1 foci. Top: illustrative images of GFP channel (nuclear Tel1 signals) in the indicated conditions. Note that we systematically monitor mCherry-Pus1 to ascertain whether signals are nuclear or not. Scale bar is 6 µm. Bottom: graph displaying the mean value of the percentage of cells forming GFP-Tel1 foci and the SEM of 3 independent experiments for the indicated conditions. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.
Figure 9A. Western blot showing P-ATM (ATM phosphorylation at Serine 1981) detection. Calnexin is used as a loading control The experiment assesses the ability of the ATM inhibitor AZD0156 (ATMi) or the OSBP1 inhibitor schweinfurthin G (SWG) to extinguish P-ATM signals triggered by the previous exposure to different genotoxins. Thus, RPE-1 cells were treated either with 0.005% MMS, or 10 µg/mL zeocin, or 120 ng/mL neocarzinostatin (NCZ), or 200 µM camptothecin for 2 h, then 500 nM AZD156 (ATMi) or 10 nM SWG were added in the sustained presence of the genotoxins for 2 additional hours. The images are illustrative of three to four independent experiments whose quantifications are shown in the bottom. Each point illustrates the observed raw P-ATM signal for each condition in each experiment, and similarly coloured dots belong to a same experiment. All the signals belonging to a same experiment were obtained from simultaneously run and transferred gels and simultaneously hybridized and exposed membranes. Thick dashed black lines represent the median of the experiments, and their specific value is shown as a red number on top of each violin plot, while thin dashed black lines indicate first and third quartiles.B. Top: RPE-1 cells transfected after 3 days with siRNA control or against OSBP1 (3 different siOSBP1 sequences were assessed independently) were exposed to 0.005% MMS for the indicated times and the phosphorylation status of KAP-1 on Ser 824 was assessed by western blot. Total KAP-1 signals and the Ponceau staining are used as loading controls. Bottom: graph displaying the P-KAP-1 / KAP-1 ratio. Each plotted value is the mean out of three independent experiments, and the error bars account for the SEM. The displayed p-value highlights the significant difference of the concerned means after applying an unpaired t-test.D. RPE-1 cells were grown with 10 µM BrdU in the culture medium for the last 16 h prior to further processing. Cells were then either mock-treated, or pre-treated for 1 h with 500 nM of the ATM inhibitor (ATMi) AZD0156 or 10 nM of the specific OSBP1 inhibitor schweinfurthin G (SWG). Then, 200 µM CPT was added and cells were processed for BrdU detection at the indicated time points. Left: an illustrative image of BrdU-positive nuclei, indicated by yellow arrowheads. Scale bar is 15 µm. Right: the percentage of cells in the population whose nuclei were positive for BrdU was established by visual inspection of the acquired images. The graph shows the mean percentage out of three independent experiments and the error bars indicate their associated SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value for the one being significantly different is indicated.
Figure 1D, E Detection of FLAIL and asFLAIL gene expression in Col-0 and flail mutants by strand-specific RT-qPCR. Transcript levels were normalized to UBQ expression levels. Y-axis showed relative values compared to the expression level of Col-0.G Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0. Data represented the mean of six independent experiments.
Figure 2B, C Detection of sense FLAIL and asFLAIL transcript expression in Col-0, flail3 mutant and complementation lines by strand-specific RT-qPCR. pFLAIL:gFLAIL18/88 and pasFLAIL:gasFLAIL18/39 represented sense and antisense FLAIL complementation lines, respectively with flail3 35S:GUS transgenic line as a control. Positions of primers used for strand-specific RT-qPCR analyses were the same as those in Figure 1A. Transcript levels were normalized to UBQ expression levels. Y-axis showed relative values compared to the expression level of Col-0.D Representative morphological phenotypes of 4-week-old plants of Col-0, flail3 mutant, transgenic lines. Scale bar: 2 cm. E Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0.
Figure 3A Heatmap of 13 flowering genes and sense FLAIL was cropped with systematic gene names illustrating that the expression of dysregulated flowering genes in flail3 was fully (AGL24, CO, COOLAIR, CIR1, FBH4, LAC8, PLL25, PRE1) or partially (AGL20, DDF1, MAF4, NMT3, PHYA) rescued in complementation line. DEGs analyzed by DESeq2 with |log2 fold change| > 1 and adjusted p value < 0.05 were considered significant differential expression. Three biological replicates for Col-0, flail3, and two for flail3 pFLAIL:gFLAIL18 (complement). Flowering genes were highlighted in red. The color scale reflected the log 2-fold change in gene expression, ranging from down-regulated (blue) to up-regulated (red).Genome browser screenshots illustrating the expression of sense FLAIL and dysregulated flowering genes in flail3 were rescued in complementation line (top panel). Normalized read counts (TPM from RNA-seq) for differentially expressed flowering genes in Col-0, flail3, and flail3 pFLAIL:gFLAIL18 plants (bottom panel).
Figure 4E, F Genome browser screenshots illustrating two of FLAIL bound targets CIR1 (E) and LAC8 (F) in ChIRP-seq.
Figure 5FLAIL affects AS of LAC8. Light blue arrows indicate probes used for gel electrophoresis in C; LAC8 isoforms were detected by RT-PCR with black arrow indicating LAC8.1, grey arrow indicating LAC8.2, and brown arrow indicating LAC8.3. -RT, negative control without adding reverse transcriptase. Isoform bands were quantified using Image Lab software relative to the respective UBQ band of each sample.F-H ChIRP-qPCR using probe pools FLAIL-asDNA (Odd and Even) identified FLAIL binding snRNAs U1, U2 and U5. Luc-asDNA probes retrieved RNAs as a negative control. Retrieved FLAIL transcript was served as a positive control; UBQ and a non-coding, unspliced, conserved snoRNA lncCOBRA1 (AT1G05913) as negative controlsI Morphological phenotypes of 4-week-old plants of Col-0, flail3, lac8-52-34, lac8 flail3-55-1 mutants. Scale bar: 2 cm. J Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0. Data represented the mean of at least seven independent experiments in Col-0, flail3, lac8-52-34, lac8 flail3-55-1.
Figure 1B, CPP score during the pre-test and CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Test of ASE-sdC mice, t = 1.306, p = 0.3586 versus Pre-test of ASE-sdC mice; Test of ACE-sdC mice, t = 10.91, p < 0.0001 versus Pre-test of ACE-sdC mice. ASE-sdC group, n = 18 mice; ACE-sdC group, n = 24 mice. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA with Sidak's multiple comparisons test or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, **, p < 0.01 versus ASE-sdC mice.
Figure 2A, The number of c-Fos-positive neurons after CPP test in mPFC and its subregions of Cg1, PrL, IL. The c-Fos (red) and NeuN (green) are used as markers for evaluating cell activity and labeling neuron, respectively. Two-tailed unpaired t test. mPFC, t = 2.89, p = 0.0161; Cg1, t = 2.912, p = 0.0155; PrL, t = 2.311, p = 0.0435; IL, t = 0.8092, p = 0.4372. n = 6 mice per group. Scale bar, 400 μm/100 μm. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex; ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood.B, The correlation of number of mPFC c-Fos-positive neurons with the corresponding ΔCPP score in ACE-sdC mice. mPFC, r = 0.9048, p = 0.0132; Cg1, r = 0.9206, p = 0.0092; PrL, r = 0.8646, p = 0.0262; IL, r = 0.4181, p = 0.4094. n = 6 mice. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex ; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.E, Quantification of ΔF/F. ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.
Figure 3A, Volcano plot illustration showed the upregulated, downregulated and unchanged genes in response to cocaine administration in mPFC. Each dot represents one gene. Red, blue and gray dots represent the upregulated, downregulated and not changed genes, respectively.C, Dusp1 mRNA levels on P74. Two-tailed unpaired t test. t = 0.3691, p = 0.7307. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE miceD, DUSP1 protein levels on P74. Two-tailed unpaired t test. t = 1.251, p = 0.2792. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood; The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE miceI, Mean gray value of DUSP1 in CaMKII-positive neurons. Two-tailed unpaired t test. t = 5.751, p = 0.0045. n = 3 mice per group. Scale bar, 400 μm/100 μm. Data information ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK: t = 3.338, p = 0.0289; p-ERK: t = 3.143, p = 0.0348; ERK1/2: t = 1.030, p = 0.3611. n = 3 biological replicates per group. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.
Figure 4B, Dusp1 mRNA levels on P78. Two-tailed unpaired t test. t = 4.413, p = 0.0116. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.C, DUSP1 protein levels on P78. Two-tailed unpaired t test. t = 4.071, p = 0.0152. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.G, The total distance traveled by mice during CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Ctrl virus-treated ACE-sdC mice, t = 0.4813, p = 0.9975 versus Ctrl virus-treated ASE-sdC mice; OE virus-treated ASE-sdC mice, t = 1.068, p = 0.8734 versus Ctrl virus-treated ASE-sdC mice; OE virus-treated ACE-sdC mice, t = 1.425, p = 0.6527 versus Ctrl virus-treated ACE-sdC mice. ASE-sdC group, n = 10 mice per group; ACE-sdC group, n = 13 mice per group. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.I, The percentage of c-Fos-positive neurons in virus-transfected neurons. Two-tailed unpaired t test. t = 3.624, p = 0.0047. n = 6 mice per group. Scale bar, 200 μm/100 μm. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK, t = 3.503, p = 0.0248; p-ERK, t = 9.063, p = 0.0008; ERK, t = 0.1728, p = 0.8712. n = 3 biological replicates. Data information: Ctrl, control virus-treated mice; OE, overexpression virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, #, p < 0.05, versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.
Figure 5B, Dusp1 mRNA levels on P78. Two-tailed unpaired t test. t = 9.250, p < 0.0001. n = 4 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.C, DUSP1 protein levels on P78. Two-tailed unpaired t test. t = 11.00, p = 0.0004. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.G, Average distance traveled during CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Ctrl virus-treated ACE-sdC mice, t =0.6755, p = 0.9851 versus Ctrl virus-treated ASE-sdC mice; KD virus-treated ASE-sdC mice, t = 0.02773, p > 0.9999 versus Ctrl virus-treated ASE-sdC mice; KD virus-treated ACE-sdC mice, t = 1.670, p = 0.4832 versus Ctrl virus-treated ACE-sdC mice. ASE-sdC group, n = 10 mice per group; ACE-sdC group, Ctrl, n = 7 mice; KD, n = 11 mice. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.I, The percentage of c-Fos-positive neurons in virus-transfected neurons. Two-tailed unpaired t test. t = 3.976, p = 0.0073. n = 4 mice per group. Scale bar, 200 μm/100 μm. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK, t = 3.293, p = 0.0301; p-ERK, t = 3.903, p = 0.0175; ERK, t = 0.1856, p = 0.8618. n = 3 biological replicates per group. Data information: Ctrl, control virus-treated mice; KD, knocking-down virus-treated mice. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus Ctrl virus-treated mice.