EMBO/soda-nel huggingface dataset:
Nucleome programming is required for the foundation of totipotency 
in mammalian germline development (2022)
Figure 4

Figure 4. Generation of minimal LADs.

(A) Correlation between compartment score and ChIP-seq enrichment at 50 kb resolution.

(B) Correlation between differential compartment score and differential ChIP-seq enrichment between mESCs and GSCs at 50kb resolution.

(C) Representative chromosome-wide distributions of compartment score and lamin B1 enrichment for mESCs and GSCs.

(D) LAD occupancies in different cell types (Peric-Hupkes et al., 2010; Poleshko et al., 2017; Robson et al., 2016; Yattah et al., 2020).

(E) Venn diagram of LADs called in GSCs, union of LADs called in all other cell types in this study, and union of LADs identified from all other studies (Peric-Hupkes et al., 2010; Poleshko et al., 2017; Robson et al., 2016; Yattah et al., 2020).

(F) UpSet plot for the union set of LADs in different studies (Peric-Hupkes et al., 2010; Poleshko et al., 2017; Robson et al., 2016; Yattah et al., 2020). A majority of regions correspond to constitutive LADs.

(G) IF analysis for lamin B1 in (left) EpiLCs and d4c7 mPGCLCs, as well as (right) EpiLCs and GSCs. Symbols for each cell type are as indicated. Scale bars, 10 μm.

(H) Western blot for lamin B1 in different cell types (bottom) and quantification normalized by β-actin (top).

(I) Average distributions of lamin B1 enrichment across all chromosomes (1-19, X). Ribbons correspond to 95% confidence intervals of fitted GAMs.

(J) Lamin B1 ChIP-seq enrichment in the first (left/p-ter) and the last (right/q-ter) 300 Kb of each chromosome. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of chromosomes = 20 (autosomes and chromosome X).

(K) Representative chromosome-wide distributions of ChIP-seq enrichment for lamin B1 and H3K9me3/me2.

(L) (top) Representative images of FISH against major satellite repeats in EpiLCs and GSCs. Scale bars, 10 μm; (bottom) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 18/22 for EpiLC/GSC.
Nucleome programming is required for the foundation of totipotency 
in mammalian germline development (2022)
Figure 5

Figure 5. Heterochromatin re-organization.

(A) (left) H3K9me3 ChIP-seq tracks, with TEs in different classes shown below; (right) Distribution of domain widths for H3K9me3-enriched regions based on cross-correlation, as implemented in MCORE.

(B) Spatial-temporal dynamics of H3K9me3 domains (>10 Kb) analyzed using ChromTime.

(C) (Top) Enrichment of interaction between (upper) and within (lower) broad H3K9me3 domains (>50 Kb; identified in GSCs and overlap peaks in all other cell types).

(D) Correlation between H3K9me2/3 and lamin B1 ChIP-seq enrichment.

(E) IF analysis for H3K9me3 (left) and H3K9me2 (right) in EpiLCs and GSCs. Arrowheads: GFP+ GSCs; arrows: EpiLCs. Scale bars, 10 μm.

(F) Odds ratio and significance of overlap between H3K9me3 domains conserved across all cell types and different repeat families. Error bars denote 95% confidence intervals.

(G) Scatter plot of lamin B1 enrichment in GSCs vs the aggregated density of select TEs (L1, ERV1 and ERVK) in 1mb bins, with points colored by H3K9me3 enrichment in GSCs.

(H) Expression of H3K9 methyltransferases as measured by RNA-seq (Ishikura et al., 2016; Ohta et al., 2021; Sasaki et al, 2015). Two biological replicates in each cell type were analyzed.

(I) (left) Western blot for G9a, GLP, Setdb1 and α-tubulin; (right) quantification normalized by α-Tubulin.

(J) (left) Scatter plot of H3K9me3 enrichment across all promoters in d2 mPGCLCs and d4c7 mPGCLCs, with 728 genes (red) showing substantially higher H3K9me3 levels in d2 mPGCLCs than d4c7 mPGCLCs; (right) pathway enrichment of the 728 genes using g:Profiler.

(K) Aggregate plot of H3K9me3 around the TSSs of Setdb1-repressed germline genes (Karimi et al, 2011). The thick line marks the mean while the upper and lower limits indicate standard errors.

(L) Normalized H3K9me3 tracks around the TSSs of Dazl and Ddx4.

(M) Distribution of differences in promoter H3K9me3 between d2 and d4c7 mPGCLCs for germline genes (Kurimoto et al., 2015), Setdb1-repressed germline genes (Karimi et al., 2011) and other genes. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. From top to bottom, Number of genes = 19559, 21, 99. Significances are computed using Wilcoxon rank-sum tests, p-values from top to bottom: 2.36e-3, 1.14e-9, 4.43e-1.
Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death (2013)
figf1

(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool. (b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments). (c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.
Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death (2013)
figf2

(a) Chemical structure of the NMS-859 covalent inhibitor. (b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom. (c) Relative positions of ADP and Walker A residues Cys522 and Lys524 (the latter two are within hydrogen-bonding range), as found in the C chain D2 ADP-binding pocket of VCP (PDB code 3CF1).
Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death (2013)
figf4

(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments. (b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP. (c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches. (d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.
Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death (2013)
figf5

(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM). (b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP–Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP–Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP–Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm. (c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units. (d) VCP and proteasome inhibitors induce cell killing in distinct cell targets. IC50 data are plotted as log10 ratio, calculated according to the following equation: log10 ratio = log10 (IC50 cell line/mean IC50 of all cell lines). Data shown are representative results of one of three independent experiments.
A non-death function of the mitochondrial apoptosis apparatus in immunity (2019)
Figure 1

Figure 1 Treatment with Bcl-2-family inhibitors causes the secretion of cytokines from human cells

A-E HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 (A-D) or the Mcl-1-inhibitor S63845 (E) for 72 h. Cytokines were measured by ELISA. CTRL, non-targeting control gRNA; Bax/Bak, both Bax and Bak had been deleted by CRISPR/Cas9; Bcl-XL, over-expressing Bcl-XL; STING, deletion of STING.

(A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).

(B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).

(C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).

(D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).

(E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).

F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).

G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).

H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µm

I HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).

Data information: *, p<0.05 for treated vs. untreated (solid lines) or for modified cell lines vs. CTRL (unpaired two tailed t-test for different cell lines and paired two tailed t-test for treated against untreated conditions in the same cell line).
A non-death function of the mitochondrial apoptosis apparatus in immunity (2019)
Figure 2

Figure 2 Pathogen infection induces DNA-damage and a DNA-damage-response through the mitochondrial apoptosis pathway

HeLa cell derivatives were infected with viral, bacterial or protozoan pathogens. CTRL, cells carrying a non-targeting gRNA; CAD and Bax/Bak, cell lines deficient in CAD or Bax and Bak (CRISPR/Cas9-mutants); Bcl-XL, cells over-expressing Bcl-XL.

A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)

B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.

D, E Cells were infected with influenza A virus (IAV) (MOI=1; analysis was 16 h post-infection). D, detection of viral M2 protein was used as infection control.

F, G Cells were infected with Herpes simplex virus 1 (HSV-1) (MOI=1; analysis was 16 h post-infection).

H Cells were infected with Chlamydia trachomatis (Ctr) (MOI=1; analysis was 30 h post-infection).

I Cells were infected with Salmonella Typhimurium (Sty) (MOI=50; analysis was 24 h post-infection).

J Cells were infected with the protozoan parasite Toxoplasma gondii (MOI=5; analysis was 16 h post-infection).

Data information: (B, D, F, H-J) Cells were tested for the DNA-damage-response marker γH2AX by Western blotting. Western blots are representative of at least three independent experiments. (C, E, G) Cells were subjected to single-cell gel electrophoresis (Comet assay). Each dot depicts one cell. Three experiments per condition were performed and are shown (150 cells per condition). Bars show means. The experiments in (D and F) were done in parallel, and the uninfected controls shown are the same.
A non-death function of the mitochondrial apoptosis apparatus in immunity (2019)
Figure 3

Figure 3 The mitochondrial apoptosis pathway contributes to cytokine secretion during infection

HeLa cell lines with an intact (CTRL) or a deficient (Bax/Bak-double deficient or Bcl-XL-over-expressing) mitochondrial apoptosis apparatus were infected with pathogens; supernatants were analysed for cytokines.

A Infection with MVA (MOI=10 for 16 h; IL-6).

B infection with HSV-1 (MOI=1 for 16 h; IL-6).

C, D Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6, IL-8).

E, F Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6, CXCL-1).

G-J Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6, IL-8, CXCL-1, FGF-2).

Data information: Data are means/SEM of three independent experiments. *, p<0.05 between control and mutant cells (unpaired two-tailed t-test).
A non-death function of the mitochondrial apoptosis apparatus in immunity (2019)
Figure 4

Figure 4 The mitochondrial apoptosis apparatus contributes to contain intracellular bacterial growth

A, B (A) HeLa cell lines or (B) AGS cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six (A) or 4-5 (B) experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).

(B) Growth of C. trachomatis was measured in AGS cells as increase in GFP-fluorescence. The data show means/SEM of 4 to 5 independent experiments (genotype effect: p<0.0001; time-genotype interaction: p<0.0001) (12 h and 38 h, n=4; 18 h, n=5).

C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)

D CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).

E CTRL, Bax/Bak-deficient or Apaf-1-deficient HeLa cells were infected with Salmonella Typhimurium (MOI=50). CFU/well were calculated as in (D). n=3.

Data information: For (A, B and D), the significance was tested as described in methods. For (C and E), significance was calculated by unpaired two tailed t-test. *, p<0.05 between control and mutant cells.

Extended view
Targeted genome editing in vivo corrects a Dmd duplication restoring wild-type dystrophin expression (2021)
Figure 2

Figure 2. The Dup18-30 mouse model recapitulates DMD disease manifestations.

A) 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm.

B) The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei.

C) Percentage of dystrophin-positive fibers in the TA and triceps of WT and Dup18-30 mice. WT, n = 4; Dup18-30 n = 6-7.

D) Percentage of myofibers with centrally located nuclei in the TA and triceps of WT and Dup18-30 mice. WT, n = 4; Dup18-30 n = 6.

E) Forelimb grip strength was measured in 15-week-old WT and Dup18-30 mice. WT, n = 9; Dup18-30 n = 12.

F) Specific tetanic force was measured in 15-week-old mice using an in vivo muscle-function analyzer. WT, n = 11; Dup18-30 n = 15 mice.

G, H) Mice were tested in an open-field chamber in which total distance travelled (G) and vertical activity (H) were assessed. WT, n = 9; Dup18-30 n = 12.

Data information: Data is represented as means ± S.D. Statistical analyses were performed with Student's t-test. *** P < 0.001, **** P< 0.0001.
Targeted genome editing in vivo corrects a Dmd duplication restoring wild-type dystrophin expression (2021)
Figure 3

Figure 3. A single-sgRNA/Cas9 treatment removes the Dmd duplication and restores full-length dystrophin expression in the Dup18-30 mouse model.

A) Schematic of the single sgRNA CRISPR/Cas9 strategy to remove the duplication of the exons from 18 to 30 in the Dup18-30 mouse model. A single sgRNA targeting intron 21 (i21) was utilized to cut both copies of the duplicated region. Cas9 and the sgRNAs are represented as scissors.

B) Two-day-old neonatal Dup18-30 pups were injected with AAV9 carrying Cas9 and i21 gRNA (n=11; 3x1012 viral genomes) via temporal vein and sacrificed 7 weeks later.

C) The efficiency of the removal of the duplicated region at the DNA level was assessed via qPCR analysis by normalizing the signal obtained from the duplication junction to that of the total Dmd signal. Dup18-30 untreated, n = 3-4; Dup18-30 treated, n = 5-9.

D) The RNA editing efficiency was quantified in the same tissues analyzed in panel B via qPCR utilizing the expression ratio between the duplication junction and the WT Dmd transcript. Dup18-30 untreated, n = 4; Dup18-30 treated, n = 4-5.

E) RT-PCR analyzing the removal of the duplication from the heart, TA and triceps muscle of WT, Dup18-30 untreated, and Dup18-30 treated mice. Arrows correspond to primers in exon 17 and exon 33.

F, G ) Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.

H) Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm.

I) Quantification of dystrophin western blot in panels F and G. Dup18-30 untreated, n = 2; Dup18-30 treated, n = 4.

J) Percentage of dystrophin positive fibers from the immunostaining in the heart and TA of and Dup18-30 untreated and Dup18-30 treated mice. Dup18-30 untreated, n = 5; Dup18-30 treated, n = 10-11.

Data information: All data is represented as the mean ± S.D. Statistical analyses were performed with Student's t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Targeted genome editing in vivo corrects a Dmd duplication restoring wild-type dystrophin expression (2021)
Figure 4

Figure 4. The single-sgRNA/Cas9 treatment restores DGC expression and improves muscle pathology in Dup18-30 mice.

A) Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm.

B) TA, triceps and diaphragm muscle architecture was analyzed by H&E staining. Scale bars, 50 μm.

C) Central nuclei were quantified in the TA, triceps and diaphragm muscles. Dup18-30 untreated, n = 4-5; Dup18-30 treated n = 6-9. All data is represented as means ± S.D. Statistical analyses were performed with Student's t-test. *** P< 0.001, **** P< 0.0001.
The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity (2020)
Figure 4

Figure 4: AltFUS is necessary for FUS-associated inhibition of autophagy and accumulation of FUS/TDP-43 cytoplasmic aggregates

A, Images by confocal microscopy of mCherry-GFP-LC3 signal in HeLa cells across biological conditions: untreated mock, bafilomycin treated mock, altFUS, FUS, FUS(Ø), FUS-R495x and FUS(Ø)-R495x (representative images of n=3). The altFUS signal (white) is shown as an inset in the top or bottom left of the left panels. The white scale bar corresponds to 10 μm and the zoomed in region (right panels) is delimited by a white box.

B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance (see Appendix Fig S4B for a representative image). Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).

C, Images by confocal microscopy of altFUS (Flag tagged - white), FUS (GFP tagged - green) and TDP-43 (red) signals in HeLa cells transfected with the bicistronic GFP-FUS(Flag)-R495x or the monocistronic GFP-FUS(Ø-Flag)-R4955x constructs, or co-transfected with the monocistronic GFP-FUS(Ø-Flag)-R495x and altFUS-Flag constructs (representative images from n=3). The white scale bar corresponds to 10 μm.

D-E, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).