EMBO/soda-nel huggingface dataset:
H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis. (2015)
Figure 3

H19 stimulates proliferation of endometrial stromal cells

A–F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.

G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT–qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.

H A proposed model of the H19/let-7/IGF1R-mediated regulation of endometrial stromal cell proliferation. During the proliferative phase of the endometrium, E2 stimulates the expression of H19. As its level rises, H19 acts as a sponge to sequester let-7 and prevent it from inhibiting its target gene Igf1r. Increased IGF1R protein level leads to increased IGF1 signaling with a biological endpoint of increased proliferation of endometrial stromal cells.

Data information: Results are presented as mean ± SD, n = 6. P-values are indicated on the top of each plot.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 1

Figure 1 Telomere dysfunction increases with age in mouse cardiomyocytes.

A) Representative images of γH2AX immuno-FISH in troponin-C/α-actinin (top left and bottom left panels, respectively) positive mouse CMs (white - troponin-C/α-actinin; red - telo-FISH; green - γH2AX). Images are Z-projections of 0.1μm stacks taken with a 100x oil objective. Scale bar: 20μm. Right panels represent a single Z-plane where co-localisation between a γH2AX foci and telomere was observed, Scale bars 1μm. (Below) 3D reconstruction of Immuno-FISH using STED microscopy for γH2A.X and telomeres in a CM from 30 month old mice. Scale bars as indicated and scale the same for both individual TAF examples using STED.

B, C) Mean number of TAF (B) and % of TAF positive (C) α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.

D, E) Mean number of total γH2A.X foci (D) and % of γH2A.X foci -positive nuclei (E) in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.

F) Fold enrichment of γH2AX at telomere repeats by real-time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control, 3 and 30 month old whole mouse hearts. Data are mean ± SEM of n=3 mice per age group.

G) Quantitative PCR-ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL/6 mice whole heart lysates. Data are mean ± SEM of n=4 mice per age group.

H) Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q-FISH in young (4 months) and old (30 months) wild-type mice. Data are from n=3 mice. > 100 CMs were analysed per mouse.

I) % of telomere FISH signal loss in 30 months old wild-type mice and late generation Terc-/- mice (6 months old) in comparison to 4 months old wild-type mice. Data are from n=3 mice. >100 CMs were analysed per mouse.

Data information: Statistical analysis was performed using one-way ANOVA (Holm-Sidak method) for multiple comparisons and Mann-Whitney test and two-tailed t-test for single comparisons. ***P<0.001; **P<0.01.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 2

Figure 2 Stress-induced telomere-associated DNA damage is persistent in mouse embryonic cardiomyocytes, rat neonatal cardiomyocytes and H9C2 myoblasts

A) Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10Gy X-irradiation. Left panels represent troponin C-positive embryonic cardiomyocytes (troponin C - magenta; DAPI - light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z projections of 0.1µm slices, with white arrows indicating co-localisation. Co-localising foci are amplified in the right-hand panels (amplified images represent a single Z-plane where co-localisation was observed). Scale bars represent 10μm. Scale bars in single plane images 500nm.

B) (Left) Mean number of both TAF and non-TAF in troponin I-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3 independent experiments; 30-50 troponin positive cardiomyocytes were analysed per experiment. (Right) Mean percentage of γH2AX foci co-localising with telomeres (% TAF) in troponin C-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); * P<0.05. Significant differences were found for mean number of non-TAF, but not for mean number of TAF.

C) Mean number of both TAF and non-TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24h with H2O2. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); **P<0.01.

D) (Left) Representative images of γH2AX immuno-FISH in H9C2 myoblasts 3 days 10Gy X-irradiation (red - telo-FISH; green - γH2AX). White arrows identify areas shown in higher magnification panels. (Right) Mean number of both TAF and non-TAF in H9C2 myoblasts at days 3 and 5 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); ***P<0.001. Scale bars represent 1μm. Scale bars in single plane images 500nm.

E) Representative time-lapse images of H9C2 rat cardiomyoblasts expressing AcGFP-53BP1 from 3 days after 10Gy irradiation at the indicated times (mins). Images are maximum intensity projections with a 6.7µm focal depth. Scale bar represents 1μm.

F) Kaplan-Meyer survival curves for AcGFP-53BP1c DDR foci in H9C2 cells 3 days after 10Gy irradiation at 10 minute intervals for 24 hours. >500 foci from 10 cells were tracked per condition. Gehan-Breslow Test was used, P<0.001.

G) Schematic illustration showing 1 month old C57BL/6 mice treated with 2Gy whole body X-irradiation, followed by a recovery period of 11 months before culling at 12 months of age. Mean number of TAF in α-actinin positive cardiomyocytes. Data are mean ± SEM of n=3 mice per group. 90 α-actinin positive cardiomyocytes were quantified per condition. Statistical analysis performed using two-tailed t test; *P<0.05.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 3

Figure 3 TRF1-FokI fusion protein induces telomere-specific double strand breaks, senescence, and hypertrophy in rat neonatal cardiomyocytes.

A) Representative images of rat neonatal CMs 4 days following transfection with a FLAG-tagged TRF1-FokI-D450A (top row) or TRF1-FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in Figure) (red - telo-FISH; green - γH2A.X). Images are z-projections of 0.1µm stacks taken with 100X objective. White arrows indicate co-localisation between telomeres and γH2A.X, with co-localising foci amplified in the right panels (taken from single z-planes where co-localisation was found). Scale bar represents 3.5μm. Scale bar in magnified images showing individual co-localisation 500nm.

B, C) % of γH2A.X foci co-localising with telomeres (B) and mean number of Telomere-associated Foci (TAF) (C) in FLAG-tagged TRF1-FokI-D450A- and TRF1-FokI-expressing CMs. Data are mean ± SEM of n=4 independent experiments. >50 cells were analysed per condition. Statistical analysis was performed by two-tailed t-test ***P<0.001.

D) Histograms displaying telomere intensity for telomeres co-localising or not co-localising with γH2AX foci. Red dotted lines represent median. Mann-Whitney test show no significant difference in telomere intensity between TAF and non-TAF.

E) Mean % of FLAG-labelled CMs positive for SA-β-Gal activity. Data are mean ± SEM of n=3 independent experiments. >100 cells were quantified per condition. Statistical analysis performed using two tailed t test; **P<0.01.

F) Expression of p21 mRNA (as a function of β-actin and Gapdh) by real-time PCR in TRF1-FokI-D450A and TRF1-FokI expressing CMs. Data are mean ± SEM of n=6 independent experiments. Statistical analysis performed using two tailed t test; ***P<0.001.

G) Mean± SEM cell surface area (μm2) of FLAG-labelled CMs expressing TRF1-FokI-D450A and TRF1-FokI. Statistical analysis performed using two tailed t test; ***P<0.001. Scale bar represents 50μm.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 4

Figure 4 Aged cardiomyocytes activate senescence pathways but not a typical SASP.

A) Schematic illustrating CM isolation procedure.

B) Real-time PCR gene expression analysis in isolated mouse CMs from C57BL/6 mice. Data are mean ± SEM of n=3-4 per age group. Statistical analysis performed by one-Way ANOVA (Holm-Sidak method); *P<0.05.

C) Mean % of p21-positive CM nuclei from 3, 15, and 30 month old C57BL/6 mice by immunohistochemistry (IHC). Data are mean ± SEM of n=4 per age group. >100 CMs were quantified per age group. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); *P<0.05.

D) Mean % of 3 and 24 month old mouse CMs staining positive for SA-β-Gal in vivo with representative images above (blue - SA-β-Gal; green - troponin C; red - WGA). Black arrows indicate SA-β-Gal expression in a troponin C expressing CM. Statistical analysis performed using two-tailed t-test; *P<0.05. Data are mean ± SEM from the analysis of >500 CMs per mouse, 4 mice per age group. Scale bar 20µm.

E) Mean % of SADS-positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo, as detected by centromere-FISH. Data are mean ± SEM of n=4 per age group. >200 CMs per mouse were quantified. Statistical analysis performed using two-tailed t-test; *P<0.05.

F) SASP heatmap: Pearson correlation clustered heatmap showing a curated list of known SASP genes (top panel) or a selection of secreted SASP proteins (bottom panel) in young (3 months) and old (20 months) mouse CMs (n=5 per age group). The colour intensity represents column Z-score, where red indicates highly and blue lowly expressed.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 5

Figure 5 Cardiomyocyte SASP induces fibrosis and reduces proliferation in fibroblasts and induces hypertrophy in cardiomyocytes.

A) (Above) RNA-sequencing of purified CMs from 4 mice per age group reveal age-dependent increased expression of 3 secreted proteins Edn3, Tgfb2, and Gdf15; the colour intensity represents column Z-score, where red indicates highly and blue lowly expressed. (Below) mRNA expression of Edn3, Tgfb2, and Gdf15 was independently validated by RT-PCR in young and old isolated adult CMs. Data are mean ±S.E.M of n=8 mice per group.

B) CMs were isolated by Langendorff heart perfusion, purified and cultured for 48h. Conditioned medium (CM) was collected and added to cultures of neonatal fibroblasts in the presence of 10μM EdU.

C) Representative images of immunofluorescent staining against α-SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs.

D, E) Quantification of % of EdU incorporation (D) and % of a-SMA positive cells (E) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition.

F) Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3, and Gdf15 for 48h and immunostained against α-SMA and EdU (fibroblasts) and α-actinin (CMs).

G-I) Quantification of α-SMA (G) and EdU positive neonatal fibroblasts (H) and surface area (μm2) (I) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.

Data information: Asterisks denote statistical significance with one-way ANOVA (multiple comparisons) (G, H, I) or two-tailed t-test (A, D, E). ***P<0.001; **P<0.01; *P<0.05. For all panels scale bars represent 50μm.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 6

Figure 6 Mitochondrial dysfunction is a feature of cardiomyocyte senescence and drives TAF in mouse cardiomyocytes in vivo.

A) Mito and ETC genes with GSEA analysis: Clustered heatmap showing all genes associated with the "Mitochondrion" GO term in young and old, mouse CMs as observed by the GSEA pre-ranked list enrichment analysis (normalised enrichment score: -1.70; FDR q-value < 0.05). Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain (ETC) GO ontology. In both instances, genes are by column and samples by row with the colour intensity representing column Z-score, where red indicates highly and blue lowly expressed.

B) Real-time PCR gene expression analysis of MAO-A, MnSOD and catalase in isolated mouse CMs from young (3 months) and old (20 months) mice. Data are mean ± SEM of n=4-5 per age group. Asterisks denote a statistical significance at P<0.05 using two-tailed t-test.

C) Mean % of 4-HNE- (top) or 8oxodG- (bottom) positive CMs from 3 month (young) or 30 month (old) aged mice. Data are mean ± SEM of n=4 per age group. 100 CMs were quantified per age group. Asterisks denote a statistical significance at P<0.05 using two-tailed t-test.

D-G) Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in wild-type (control) compared to MAO-A transgenic mice with or without drinking water supplemented with 1.5g/kg/day NAC from the age of 4 to 24 weeks, MnSOD+/+ vs MnSOD-/+, Catalase+/+ vs. Catalase-/-, WT vs. POLG double mutant mice. Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group. Statistical analysis was performed using two-tailed t test (E-G) or one-way ANOVA (for multiple comparisons) (D); *P<0.05.

H) Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control (pre-treated for 30 minutes before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF-positive nuclei (bottom graph). Data are mean ± SEM from 4 separate CM cultures isolated from 3 month-old mice. 50 CMs were quantified per condition. Asterisks denote a statistical significance at P<0.05 using one-way ANOVA (Holm-Sidak method).
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 7

Figure 7 Genetic clearance of p16 positive cells in aged mice reduces cardiomyocyte senescence, cardiac hypertrophy and fibrosis.

A) Examples of individual short axis cine-MR images of 3 or 20 month old mouse hearts. Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders. % change in wall thickening calculation based on wall thickness at the 4 points indicated. Measurements were made in all cine slices at end diastole and end systole. Graphs representing data obtained from MRI analysis of >7 animals per age group. Data are mean ± S.E.M. Asterisks denote a statistical significance at P<0.05 using Mann Whitney U-test. Scale bars represent 5mm.

B) Comparison between mean number of TAF per CM and CM area in 22 month old animals. Data are mean ± S.E.M. of n=4. >100 CMs were quantified per mouse. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); *P<0.05.

C) Schematic depicting experimental design for Figures d-f: 27 month old INK-ATTAC mice were treated 4 times with AP20187 (or Vehicle), 3 days in a row, every 2 weeks (2-month-long treatment in total) and were sacrificed afterwards for analysis.

D) Comparison between the % of p16- or eGFP-positive CMs by RNA-in situ hybridization per plane in INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n=5 per age group. 100 CMs were analysed per mouse. Asterisks denote a statistical significance at *P<0.05 or ***P<0.001 using two-tailed t-test.

E) Mean number of TAF (left graph) and mean % of TAF-positive nuclei (right graph) in CMs. Data are mean ± S.E.M. of n=6 per age group. 100 CMs were analysed per mouse. ***P<0.001; **P<0.01; *P<0.05.

F) Mean CM area μm2. Data are mean ±S.E.M. of n=6 per age group, >150 CMs analysed per mouse. Asterisks denote a statistical significance at *P<0.05 using two-tailed t-test.

G) % of fibrotic area evaluated by Sirius Red staining. Data are mean ±S.E.M. of n=6 per age group. Asterisks denote a statistical significance using Mann Whitney test. ***P<0.001.
Length-independent telomere damage drives cardiomyocyte senescence (2019)
Figure 8

Figure 8 Pharmacological clearance of senescent cells with Navitoclax (ABT 263) reduces cardiomyocyte senescence and stimulates cardiomyocyte regeneration.

A) Schematic depicting experimental design. Mice at 100 weeks (23-months) of age were treated with vehicle (Veh) or navitoclax (Nav) intermittently for 2 weeks. At 104 weeks mice were injected every day with EdU for 1 week.

B) Quantification of mean number of TAF and % of TAF-positive CMs in 24 months old wild-type mice treated with vehicle or navitoclax (50mg/kg/day). Data are mean± SEM of n=5-8 mice per group. More than 100 CMs were quantified per animal.

C) CM cross-sectional area in 24 months old wild-type mice treated with vehicle or navitoclax. Data are mean± SEM of n=8 mice per group.

D) (Left) Representative images of Sirius red staining and (right) % of fibrosis area in 24 month-old mice treated or not with navitoclax. Data are mean mean±S.E.M of n=3 per group. Scale bar represents 50µm.

(E-G) MRI analysis of ejection fraction (EF%), left ventricle mass (LVmass) index and % of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.

H) Examples of confocal microscopy images of CMs positive for CM marker troponin C (TropC), EdU, Ki67 and Aurora B from navitoclax treated animals. (Upper Right panel) white arrows identify two nuclei in the same CM that have incorporated EdU. (Lower Right panel) white arrow identify EdU expressing Aurora B symmetrically between two nuclei. Scale bars represent 20µm.

I) Table summarising numbers of CMs quantified in (J).

J) Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group.

K) % of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.

Data information: Asterisks denote a statistical significance using two-tailed t test (B, C, K) or Mann Whitney test (D, J). ***P<0.001; **P<0.01; *P<0.05.

Expanded View Figure legends
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 4

Figure 4. Expression of autophagy and inflammatory genes in wt and mdx mice treated with NaB or DFZ.

A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.

J-K Representative blotting and bar chart with individual points showing the expression and/or phosphorylation of pAKT/AKT and COX2 in the gastrocnemius of the indicated six groups of mice.

Data Information: Each bar is the mean ± S.E.M. from 5 independent biological replicates. **** = p≤0.0001; *** = p≤0.0003; ** = p≤0.005; * = p≤0.05 versus the indicated experimental group calculated using ANOVA.
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 5

Figure 5. Measurement of endocannabinoid system activity in the plasma and skeletal muscle of wt and mdx mice treated with NaB and DFZ.

A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.

C-D Bar charts with individual points showing the mRNA expression levels of CB1 and CB2 measured in the gastrocnemius of the indicated groups of mice.

E Representative blots showing the expression levels of CB1 and CB2 proteins in the gastrocnemius of the indicated groups of mice.

F Quantification of CB1 and CB2 proteins to the housekeeping protein GAPDH.

Data Information: Each bar is the mean ± S.E.M. of 4-5 independent biological samples. *** = p≤0.0003; ** = p≤0.003; * = p≤0.05 versus the indicated experimental group calculated using ANOVA.
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 6

Figure 6. Effect of rimonabant on autophagy in mdx mice.

A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).

D Representative blots showing the expression levels of LC3I and LC3II proteins in the gastrocnemius of the indicated groups of mice.

E, F Quantification of LC3I and LC3II proteins to the housekeeping protein GAPDH.

Data Information: Each bar is the mean ± S.E.M. from 3 independent biological samples. ** = p≤0.005 * = p≤0.05 versus the indicated experimental group calculated using ANOVA.
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 7

Figure 7. Effect of NaB on LPS-stimulated C2C12 cells.

A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).

C Time-dependent effect of NaB (3 mM), MK1903 (1 μM) and rosiglitazone (1 μM) on autophagosome formation measured in C2C12 myoblasts. Data are expressed as fluorescence intensity normalized to controls (%).

Data Information: Each bar is the mean ± S.E.M. of at least 3 independent replicates. * = p≤0.05 versus the veh group. ± = p≤0.05 versus the LPS group; # = p≤0.05 versus the other experimental groups (A) or the veh group (B) calculated using ANOVA.
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 8

Figure 8. Effect of LPS on the endocannabinoid system activity in C2C12 cells.

A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).

G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h.

I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells.

Data Information: Each bar is the mean ± S.E.M. from 3 independent biological replicates. ** p≤ 0.005; * = p≤0.05 versus the veh group; ± = p≤0.05 versus the LPS group calculated using ANOVA.
Targeting gut dysbiosis against inflammation and impaired autophagy in Duchenne muscular dystrophy (2022)
Figure 9

Figure 9. Effect of LPS on the expression of miRNAs targeting the Cnr1 gene.

A Schematic representation of miRNAs targeting the 3′-UTR region of both murine and human CB1 gene.

B Heatmap representation of the expression of selected miRNAs in the indicated biological replicates. Red, upregulated; green, downregulated.

C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).

Data Information: Each bar is the mean ± S.E.M. from 3 independent biological replicates. ± = p≤0.05 vs veh group; ** = p≤0.03 vs LPS group; # = p≤0.05 versus the other experimental groups calculated using ANOVA.