EMBO/sd-character-level-ner huggingface dataset:
Increased Autophagy in daf-2/insulin/IGF Receptor Mutants Is Independent of the daf-16/FOXO and pha-4/FOXA Transcription Factors.Figure 6(A) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan. (B) Average number of LGG-1::GFP-containing puncta in daf-16(mu86); daf-2(e1370) double mutants compared to daf-2(e1370) animals; p = 0.50, unpaired, two-tailed t-test. N2 wild-type animals (WT) are shown for comparison. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. The double mutant expressing the LGG-1 reporter had a mean lifespan similar to non-transgenic daf-16; daf-2 double mutants (data not shown, [65]). Please see Table S1 for quantification of all data.
Mycolactone synergizes with PIs for induction of MM cell apoptosis.Figure 2A MM.1S, JIM3 and KMS-11 cells were incubated with increasing concentrations of BZ for 24 h. The proportion of live (Annexin V- PI-) cells was then measured by flow cytometry as in Appendix Figure S1. Data are Mean % of live cells from technical duplicates, relative to vehicle controls (Ctrl). B MM.1S were treated for 24 h with mycolactone and/or BZ at the indicated concentrations. Data are Mean % of live, apoptotic and dead cells from technical duplicates, gated as in Appendix Figure S1, relative to total cells. C Synergy between mycolactone and BZ in the MM.1S, JIM3 and KMS-11 cell lines, when treated as in (B). Data are Mean Loewe scores ± SD shown as heatmaps. N = 6 (cumulative data of 3 independent experiments with technical duplicates) for MM.1S and KMS-11, and N = 4 (cumulative data of 2 independent experiments with technical duplicates) for JIM3. Statistical significance was assessed by Student's t-test: *p<0.05; **p<0.01; ***p<0.001. D,E MM.1S cell were treated as in (B) but using carfilzomib instead of BZ (D). Synergy scores (E) were calculated as in (C). Data are Mean Loewe scores of technical duplicates, shown as heatmaps.
Figure 10The details of this schematic model are described in the text. PM, plasma membrane; CT, cytoplasm; ER, endoplasmic reticulum; NC, nucleus.
Figure 1(A) Nissl-stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc/unc and Clcn3+/+ mice at P45 or after 20 months. In contrast, the hippocampus was absent (indicated by asterisk) in 3 months-old Clcn3-/- mice (scale bar: 200 µm). (B) Nissl-stained paraffin sections show intact retinal layers in 20 months old Clcn3unc/un mice. Neurodegeneration in Clcn3-/- mice however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (C) Immunoblots for ClC-3, -4 and -5 of membrane fractions of WT (+/+), Clcn3unc/unc (unc/unc), Clcn3unc/+ (unc/+) and Clcn3-/- (-/-) mice. β-actin, loading control. (D) Representative Western blot for ClC-4 of membrane fractions from brain and kidney of WT (+/+), Clcn3+/- (+/-) and Clcn3-/- (-/-) mice. β-actin, loading control. (E) Quantification of ClC-4 immunoblots including those shown in (C and D) (normalized to actin). Mean values ± s.e.m. Average from ≥5 animals per genotype and at ≥2 immunoblots per animal. *** p < 0.0005 , ** p < 0.005, * p < 0.05 (two-tailed unpaired t test).
Figure 8(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG) 100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.
Figure 5Trf1lox/lox p53-/- MEFs were transduced with eGFP-tagged Trf1 WT or mutant alleles to overexpressed TRF1 depicted variants. Endogenous TRF1 was deleted by transduction with Cre recombinase. Opera high-content screening (HCS) system was used to quantify the GFP spot intensity per cell. Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicates Growth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates Western blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicates Representative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.
Figure 4Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h, stained with CellROX Green, and signal analyzed via flow cytometry. Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected.
Figure 6(A) Immunofluorescence microscopy staining of Fst1 (green) and α-SMA (red) in NRCFbs at 24 hours after recombinant TGF-β1 (10ng/ml) stimulation. Nuclei were stained by DAPI. Scale bar indicates 50μm. (B) Time course changes of Fstl1 protein expression in NRCFbs after stimulation of recombinant TGF-β1 (10ng/ml). Protein expression of Fstl1 was detected by immunoblotting. Tubulin was used as a loading control (n=3 for each time point). Two independent experiments were performed. (C) Transcript level of Fstl1, S100a4 and Acta2 mRNA in NRCFbs was determined by qPCR analysis. The samples were harvested at 24 hours after stimulation with recombinant TGF-β1 (10ng/ml) or control vehicle. Error bars represent mean ± SEM (Fstl1 and S100a4: n=6 for each group, Acta2: n=3 for each group). Statistical analysis was performed by unpaired t-test (2-tailed) for Fstl1 and S100a4, and non-parametric unpaired t-test (2-tailed) for Acta2. Two independent experiments were performed.
A comprehensive model of spongiogenesis.Figure 7Prion infection result a cascade of events affecting a number of enzymes in various cellular compartments, and eventually resulting in depletion of PI(3,5) P2 as the proximal cause of spongiosis. Red bars indicate therapeutic interventions.
Figure 2(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice). (B) Vector copy number (VCN) in peripheral blood eight weeks post-transplantation (mean ± SD, each dot represents an individual mouse). (C) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA). (D) MFI of MHC class II positive macrophages identified by F4/80 expression in the BM (mean ± SD, each dot represents an individual mouse; WT IFNγ vs WT CTRL: * p = 0.0221, Mann-Whitney test). (E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse). (F) Maturation state (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) of CD8+ lymphocytes (mean ± SD, each dot represents an individual mouse; WT IFNγ vs WT CTRL: **** p ≤ 0.0001, ordinary One-Way ANOVA).
Positioned nucleosomes, chromatin states and transcriptional activity.Figure 2A Bar chart showing the number of nucleosomes that map to selected chromatin states [20]. Active (State 1); Insulator (State 8); Repressed, Rep, (State 12) and heterochromatin, Hetero (State 13), and to open chromatin (based on ATAC-seq). Shown are the observed number of nucleosomes per chromatin state (black) and the expected number calculated assuming a random distribution across the genome (grey). B Bar chart showing the number of CTCF sites that map to different chromatin states and open chromatin, using the same methodology as A.
Figure 6A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green). Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CNX as ER membranes marker. Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CLIMP63 as ER membranes marker. G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001. Data information: Scale bars 10 μm. H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001). Data information: Scale bars 10 μm.
Acetylation of FXR at K217 inhibits its SUMO2 modification, partly by blocking the interaction with PIASy A, B In‐cell SUMO assay: COS‐1 cells were transfected with the plasmids as indicated, and levels of SUMO2‐FXR were detected by IP/IB.Figure 5A, B In‐cell SUMO assay: COS‐1 cells were transfected with the plasmids as indicated, and levels of SUMO2‐FXR were detected by IP/IB. At the bottom, levels of SUMO2‐FXR relative to total FXR are shown. C Mice were infected with Ad‐FXR‐WT or the K217Q mutant and treated with GW4064, and SUMO2‐FXR levels were detected by IP/IB (n = 3 mice). D Experimental outline for in vitro acetylation and SUMOylation. E, F In vitro assay: Purified flag‐FXR‐WT was incubated with purified p300 and acetyl‐CoA as indicated, and acetylated FXR was detected by IB (E). Effects of acetylation of FXR on its SUMOylation (F). G, H Effects of the Ac‐mimic K217Q mutation on the interaction of FXR with PIASy in COS‐1 cells (G) or in liver extracts pooled from three mice (H) as detected by CoIP.
Distinct roles of LLO and PlcA/B in the modulation of host AA starvation pathways.Figure 3(A) qPCR analysis of ATF3 induction following infection of HeLa cells with WT, TKO, PlcA/B− or LLO− Listeria strains for 1-4 h. Values are means±s.e.m. n=3. (B) HeLa cells were infected with WT, PlcA/B− or LLO− Listeria strains for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies. (C) HeLa cells left unstimulated (CTR) or infected with WT, PlcA/B− or LLO− Listeria strains for 1 h, analysed by IF using antibodies against mTOR and LAMP2. The arrow shows a Listeria vacuole positive for mTOR and LAMP2, and arrowheads indicate mTOR‐ and LAMP2‐positive late endosomes or lysosomes. (D, E) Percentage of cells infected with WT, PlcA/B− or LLO− Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. **P0.01. (F) Percentage of cells infected with WT Listeria, in the presence (black bars) or absence (white bars) of rapamycin, displaying one or several GFP‐LC3+ Listeria autophagosomes. Values are means±s.e.m. n=2. Scale bars: 5 μm.Source data for this figure is available on the online supplementary information page.
Genetic architecture of recA influences pulsing in individual M.Figure 2Schematics of the recA regulatory region and of genetically modified variants. The -10 and -35 regions of the two promoters are underlined. The transcriptional start sites are indicated by arrows. The consensus recognized by LexA (red) and ClgR (green) are shown in gray (wt) and orange (mutated) boxes. Gray arrowheads represent the insertion of fluorescent markers. The RecA translation initiation codon is bolded. Genomic loci (left and bottom) and representative time-lapse images (right) of the four fluorescent reporters in exponential phase are color-coded. Phase-contrast and fluorescence images are merged. Scale bar, 10 µm. Color-coded single-cell time traces of the four reporters expressed as percent of the generation time (B). Population coefficients of variation of fluorescence (%) are in color-coded boxes. Single-cell VMR of fluorescence (C) and fluorescence averaged over the lifetime of the cell (D) in exponential phase. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ***p = 0.0002; ****p < 0.0001. The data shown are from 2 independent experiments (n = 105 cells per strain). Single-cell fluorescence decreases but variation does not change in strains devoid of positive regulation. Single-cell fluorescence increases and variation decreases in the strain devoid of negative regulation. Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments. Single-cell analysis of independent lineages separated by black vertical lines. Color-coded traces represent single-cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours (n = 20). White and gray vertical bands represents cell divisions. The data shown are from 2 independent experiments. Aligned pulse profiles averaged for each reporter strain (color-coded as in A). Error bars are SD (4 < n < 37). The data shown are from 2 independent experiments. Pulses are abrogated in the strain devoid of negative regulation. Analysis of pulse characteristics. Black lines indicate means and SD (52 < n < 88). Significance by Kruskal-Wallis nd Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments. Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time (52 < n < 88). Different strains are color-coded. Statistical analysis by one-way ANOVA and Holm-Sidak's multiple comparisons test. Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time.
GaMTs efficiently deliver post-Golgi cargos to the cell leading edge.Figure 6Projections of 120 frame movies present leading edge delivery of two different types of cargos. Left: Ecad cargo; Right: Rab5 cargo. Yellow line mark the cell boundary. Scale bar: 20 μm. Examples of cargo delivery towards the leading edge, time series present at 40 s, 80 s and 120 s, respectively. B: control cell; C: CAMSAP2 KD cell; D: CLASPs&GCC185 KD cell. Red arrow heads indicate cargo current position. Size of cropped regions: 30 μm x 5 μm. Box-whisker plot presents the Ecad cargo number on GaMTs and non-GaMTs. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test. Box-whisker plot presents the number of Ecad cargos finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 24 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, two-tailed Mann-Whitney. Box-whisker plot shows the number of Rab5 cargos that finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 23 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, two-tailed Mann-Whitney.
Fig.Figure 4(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.) We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM. We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM. We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM. We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM. We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.
Fibronectin and LDLR depend on LMF1 for secretion.Figure 7Bottom, center and top slices from a z-stack of a cld/wt or cld/cld MEF cell. YPet-tagged fibronectin is in green and mCherry-Sec61β, an ER marker, is in red. Note fibronectin assembly into the extracellular matrix in cld/wt cells. cld/wt cells had significantly higher amounts of fibronectin outside of the ER than cld/cld cells by a two-tailed student's t-test. Bottom, center and top slices show that LDLR is retained in the ER in cells HEK293∆LMF1 cells. LDLR-GFP is in green and mCherry-Sec61β is in red. Note the clumping of LDLR in the ER in HEK293∆LMF1 cells. The ratio of LDLR within the ER/total LDLR was significantly higher for HEK293∆LMF1 cells than HEK293 or HEK293+LMF1 cells by a 2-tailed student's t-test.
Figure 3(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate).  (B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.  (C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.  (D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.  (E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.  (F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.  (G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).  H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.  I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).