EMBO/SourceData huggingface dataset:
Figure 5(A) Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9-WT (x-axis) and EV to SOX9-T236/T240A (y-axis). Each dot represents one transcript, with colored dots indicating transcripts upregulated (log2 FC>0.585; p<0.05; red dots) or downregulated (log2 FC<-0.585; p<0.05; blue dots) in both comparisons. The transcript corresponding to the SOX9 gene is highlighted as a red filled dot. Indicated r-value indicates Spearman's rank correlation coefficient between the two FC profiles.(B) Box-and-Whisker plot displaying the sample specific expression of three FBW7 isoforms; FBW7α (NM_033632), FBW7β (NM_018315) and FBW7γ(NM_001013415'). Each box represents the expression distribution over three replicates.(D) Heat map of the row-wise z-scores of 16 EMT hallmark genes in SOX9-WT and -T236/T240A samples. Heat map was generated using the GenePattern software (Reich et al., 2006).(E) Quantitative PCR (qPCR) of SNAI2 (SLUG), VIM and POU3F1 (OCT6) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9-WT and SOX9-T236/240A. Cells were induced for 8 or 24 hours with doxycycline.(F) Western blot of SOX9, HA-SOX9, pSOX9-T236, SNAI2 (SLUG), VIM and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9-WT and SOX9-T236/240A. Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control.(G) Heat map of the row-wise z-scores of 14 pro-metastasis genes differentially expressed between SOX9-WT and -T236/T240A samples and previously identified as SOX9 target genes (Kadaja et al., 2014, Larsimont et al., 2015, Oh et al., 2014). Heat map was generated using the GenePattern software (Reich et al., 2006).
Figure 6(A) Cisplatin dose response curves of (i) MB002, (ii) Daoy, and (iii) MIC in the absence (EV) or presence of SOX9 by Alamar blue. Cells were pre-conditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from 3 independent repeats, each containing 5 technical replicates.(B) Cisplatin dose response curves of SOX9-expressing (i) Daoy and (ii) MIC in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A)(C) Overall survival analysis of mice bearing Daoy or Daoy-expressing dox-inducible SOX9 treated with cisplatin. The dox-preconditioned cells (105 cells) were orthotopically xenografted to Nude-Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin (2mg/kg) intraperitoneally for every other day for a total of 6 doses.(D) Heat map of the row-wise z-scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9-WT or Sox9-T236/T240A. Heat map was generated using the GenePattern software.(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni's post-test.(F) Time course western blotting of HA-SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204 and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9-WT or SOX9-T236/240A. GAPDH was used as a loading control.
Figure 7(A) Western blotting of pT236- and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363, 8186, or 2014. The set of blots shown is a representative from 3 independent repeats.(B) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014. The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment. Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.(C) Quantification of resazurin-based Daoy and MB002 cell viability following treatment with Cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy were treated for 72 hours, while MB002 were subjected to 5 day course of treatment. The cellviability were calculated relative to the vehicle-treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software. Error bars indicate standard deviation.(D) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin, AZD2014, or their combination. β-actin were used as a loading control. The set of blots shown is representative from 3 independent experiments.(E) Western blotting profiling of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 48 hours treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative from 3 independent experiments.(F) Quantification of cell viability of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 5 days treatment with either of 10 μM cisplatin (black bars), 0.5 µM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from 3 independent experiments, from which statistical significance were analysed by 2-way ANOVA multiple comparisons with Bonferroni's post-test.(G) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy-SOX9/T-FBW7α. The cells were either untreated or incubated with 5 µg/mL doxycycline for 72 hours prior to being harvested for gel electrophoresis. The set of blots shown is representative from 2 independent experiments.
Figure 1(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e.(B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.
Figure 2(A) HeLa cells were transfected with siRNAs for autophagy-related gene ATG5 (siATG5) or with a non-silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5-ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR).(B) Control or ATG5‐KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e.(C) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH).(D) Control or ATG7‐KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.
Figure 3(A) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed.(B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green).(C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.
Figure 4(B) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy.(C) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.
Figure 5(A) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3).(B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution.(C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy.(D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test).(E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments.(F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test).(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.
Figure 2(I) Representative high-resolution confocal images of C. elegans expressing mitochondria-targeted GFP in the body wall muscle cells (scale bar= 10 μm). Quantification of mitochondrial morphology (elongation) is reported in the right panel (one-way ANOVA with Holm-Sidak's correction, n=37-47 animals from 3 independent experiments). Data information: , middle line represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(J) Representative western blot analysis and densitometry of zcIs13[hsp-6p::GFP] expression in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates). Data information: , bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(M) Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
Figure 4(A) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with Oil Red O (ORO) (scale bar= full image-100 μm; hindgut magnification-20 μm). (B) Quantification of hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes stained with ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(E) Representative images of wt, cest-2.2 O/E and cest-2.2 (lf) animals stained with ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of ORO staining in hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(I-J) Representative images of nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining. Data information: bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(K) Representative thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.(N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.
Figure 1Representative Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042.Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005.Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039.Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.
Figure 2Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm.Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005.Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028.ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008.Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005.Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.
Figure 3A Representative Western blots showing separation of lysosome- and mitochondria-enriched fractions by a density gradient. Total extracts from different HD models were fractionated by the density gradient. Eight fractions were collected from top (#1) to the bottom (#8). The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient. n = 3.B, C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild-type (WT) and HD transgenic R6/2 mice showing mitochondrial (as indicated by yellow arrows) association with lysosomal vacuoles (shown by red arrows). The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted. Analysis (of six independent images) was carried out by an observer blinded to the experimental conditions. *P < 0.023; **P < 0.035. Scale bar: 1 μm.D Western blot showing association of mitochondrial membrane with lysosomal membrane. The presence of membranes of each organelle was assessed using anti-MFN1, VDAC (as mitochondria markers), and LAMP1 (as a lysosomal marker). n = 3.E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03.F Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of normal fibroblast cells and HD patient-derived fibroblasts. Mitochondrial protein levels were normalized to the cytosolic protein, enolase. n = 3. *P = 0.008; **P = 0.002; #P = 0.0008.G Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of mice striatal cells with Q7 and Q111. n = 3. *P = 0.0008; **P = 0.0007; #P = 0.001.H Western blot showing mitochondrial contents (Tom20 and aconitase) in the total lysates of brain tissues from wild-type (WT) and HD transgenic mice. n = 2. *P = 0.02; **P = 0.03 *§P = 0.01; **§P < 0.04.
Figure 4Western blots showing co-immunoprecipitation of GAPDH from the total lysates and isolated mitochondria of PC12 cells with anti-GAPDH antibody. Anti-EGFP antibody detects the presence of polyglutamine repeats. Bottom panel shows co-immunoprecipitation of GAPDH in the total lysate of PC12 cells treated with formaldehyde to cross-link proteins. Ctr* = control. n = 3.Western blots showing co-immunoprecipitation of GAPDH from the total lysate (left) and isolated mitochondria (right) of brain tissues of wild-type (WT) and transgenic R6/2 (HD) mice with anti-GAPDH antibody. The presence of huntingtin was examined with anti-huntingtin antibody and anti-polyglutamine antibody, respectively. Co-immunoprecipitated huntingtin or polyglutamine (polyQ) was normalized to GAPDH. Left: n = 2. *P = 0.03; **P = 0.02. Right: One representative experiment from two repeats.Sub-mitochondrial localization of GAPDH and expanded polyglutamine repeats. Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent (Triton X-100). n = 3.
Figure 5Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 before (represented as NT: no treatment) and after overexpression of inactive GAPDH (represented as G). n = 3. *P < 0.035; **P = 0.001; #P = 0.001.Representative Western blot showing levels of mitochondrial proteins in the total lysates of mice striatal cells with Q7 or Q111 before (NT) and after overexpression of inactive GAPDH (G). n = 2. *P = 0.006; **P = 0.007; #P = 0.001.Determination of mitochondrial ROS production in different HD models. Results are shown as percent of control. n = 3. *P = 0.008; #P = 0.054; **P = 0.03.Determination of mitochondrial membrane potential using JC-1 in different HD models and control cells (MMP, ΔΨm). The difference in membrane potential between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in cells with expanded polyglutamine repeats (PC12 cells with Q74 and HD patient-derived fibroblasts) was not statistically significant. n = 3.ATP levels in normal and HD patient-derived fibroblasts. The difference in ATP levels between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in HD patient-derived fibroblasts was not statistically significant. n = 2.Cell viability determined by colorimetric assays in different HD models and control cells. n = 3. *P = 0.02; **P = 0.02; ##P = 0.01.Immunofluorescence of normal and HD patient-derived fibroblasts, with and without overexpression of inactive GAPDH, stained for late endosome with anti-Rab7 antibody and mitochondria with anti-Tom20 antibody. Images were acquired at 63× magnification, and brightness and contrast of all images were adjusted by 30%. Correlation coefficient (juvenile): **P = 0.007; *#P = 0.005. Mitochondrial interconnectivity (juvenile): ##P = 0.001; ##*P = 0.02. Correlation coefficient (adult): §§P = 0.001; *§P = 0.005. Mitochondrial interconnectivity (adult): ≠≠P = 0.043; ≠≠*P = 0.015.