EMBO/soda-nel huggingface dataset:
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 2

Figure 2. LUBEL catalyzes the synthesis of linear Ub chains.

A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band.

B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody.

C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.

D The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody.

E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 3

Figure 3. Endogenous linear Ub chains are present in Drosophila.

A Endogenous linear Ub chains enriched in total protein extracts (input, lane 1) from adult w1118 (w-) flies by GST-Linear-TUBE (lane 3). The enriched samples were further treated with recombinant DUBs, vOTU (lane 4) or OTULIN (lane 5). Ub chains were visualized by immunoblotting using anti-linear Ub antibody or anti-Ub antibody. Loading of GST proteins was analyzed by Ponceau S staining.

B Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.

C Mass spectrometry analysis using an identical sample as Figure 3B lane 6. MS/MS spectra of the prototypic linear Ub chain peptide (GGMQIFVK) is shown.
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 4

Figure 4. dCYLD hydrolyzes linear and K 63-linked Ub chains.

A, B Schematic diagram of Drosophila (D. melanogaster) CYLD (dCYLD) and human (H. sapiens) CYLD (A), and a multiple amino acid sequence alignment of a catalytic region of the ubiquitin carboxyl-terminal hydrolase (UCH) domain in different species (B). dCYLD is 639 amino acids in length, and contains one cytoskeletal-associated protein-glycine-conserved (CAP-Gly) domain (Red) and the UCH domain (Green) (A). Human CYLD is 965 amino acids in length and contains three CAP-Gly domains, an unstructured phosphorylation region (Phos-site (Pink); specific for CYLD chordata species), and a UCH domain (Green). A conserved active Cys (C284 in Drosophila) is indicated by * (B).

C In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band.

D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL.

E Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 5

Figure 5. Endogenous linear Ub chains are diminished in catalytically dead LUBEL mutant flies.

A Amino acid sequences of the mutations introduced in LUBEL by CRISPR/Cas9. Two LUBEL mutant lines were created for in vivo studies; w- parental line, C2690S/C2693S (CC/SS), and delR2, which has an indel mutation with an early stop codon at amino acid 2719 due to a frame shift. Mutations are indicated in red.

B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band.

C The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant.

D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 µm.
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response (2016)
Figure 6

Figure 6. Catalytically dead LUBEL flies are susceptible to high temperature.

A The level of poly-Ub chains in w- flies after heat shock. w- male flies were incubated in a 36 °C water bath for indicated times and the levels of poly-Ub chains in total protein extracts were compared by immunoblotting using antibodies against linear, K 48-, and K 63-linked Ub chains. Ponceau S staining was used to visualize the protein loading.

B Survival of catalytically dead LUBEL mutant flies upon heat shock. Young adult flies (15 males and 15 females) were incubated in a 36 °C water bath and immobilized flies were counted over the time indicated. Median survival time (hours): w- = 9.25, CC/SS #1 = 5.5, CC/SS #2 = 5, delR2 = 5.75, CC/SS #1/+ = 8.5, CC/SS #2/+ = 10, delR2/+ = 8. p-values calculated by Gehan-Breslow-Wilcoxon test between fly lines: w- and CC/SS #1, CC/SS#2, or delR2 > 0.0001, w- and CC/SS #1/+ = 0.0134, w- and CC/SS #2/+ = ns, w- and delR2/+ = 0.0002, CC/SS #1 and CC/SS #1/+ > 0.0001, CC/SS #2 and CC/SS #2/+ > 0.0001, delR2 and delR2/+ > 0.0001. Representative data are shown from six independent experiments.

C Survival of whole body or muscle-specific LUBEL knockdown flies upon heat shock. shRNA-based knockdown (KD) of LUBEL were driven by Tub-Gal4 or Mef2-Gal4 flies. Control fly lines (Tub-Gal4/+ and Mef2-Gal4/+) were used to compare with the KD flies. 15 males and 15 females per each line were used in this assay. Median survival time (hours): Tub-Gal4/+ = 7, Tub-Gal4>LUBEL = 3.5, Mef2-Gal4/+ = 7.5, Mef2-Gal4>LUBEL= 7. p-values calculated by Gehan-Breslow-Wilcoxon test: Tub-Gal4 <0.0001 (****), Mef2-Gal4 = 0.0029 (**). Representative data are shown from three independent experiments.

D Heat-induced mRNA expression of HSP70. w-, CC/SS #2 and delR2 flies were heat treated for 60 min and recovered for 1 hour, and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w- untreated sample was used as calibrator to calculate the expression ratio. Data are presented as meanSD (*<0.01, ***<0.001, ****<0.0001). Representative data are shown from three independent experiments.
Loss of autophagy in the central nervous system causes neurodegeneration in mice (2006)
figf1

a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in the Atg7-deficient brain. Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was used as a loading control. Data shown are representative of three separate experiments. b, Kaplan–Meier survival curves of Atg7flox/+; nestin-Cre (n = 41) and Atg7flox/flox; nestin-Cre (n = 26) mice over 28 weeks. c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28. When lifted by the tail, Atg7flox/+; nestin-Cre mice behave normally, extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs. d, Movement ataxia in Atg7flox/flox; nestin-Cre mice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/+; nestin-Cre (n = 5) and Atg7flox/flox; nestin-Cre (n = 5) mice were placed on a rod rotating at 20 r.p.m., and the time spent on the rod was counted. Data show mean ± s.d. *, P 0.01 (Students t-test). There was no significant sex difference in survival rate and onset-stage of abnormal limb-clasping and tremor in Atg7flox/flox; nestin-Cre mice.
Loss of autophagy in the central nervous system causes neurodegeneration in mice (2006)
figf2

a–f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a–d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex. g–j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells. k–p, TUNEL staining of the cerebral cortex (k, l) and cerebellum (n, o) at P56 in Atg7flox/+; nestin-Cre (k, n) and Atg7flox/flox; nestin-Cre (l, o) sections. TUNEL-positive cells are indicated with arrows and shown as higher magnification images beneath panels l, n and o. Histograms show the average number (± s.d.) of TUNEL-positive cells in ten sections for three animals of each genotype (m, p). *, P 0.05 (t-test). Scale bars, 1 mm (a, b), 100 µm (c–j), 250 µm (k, l, n, o). We observed no sex difference in brain morphology or neuronal loss in Atg7flox/flox; nestin-Cre mice.
Loss of autophagy in the central nervous system causes neurodegeneration in mice (2006)
figf3

a–l, The presence of ubiquitin-positive dots was examined immunohistochemically in several regions including cerebral cortex (a, b), cerebellum (c, d), hippocampus (e, f), hypothalamus (g, h), amygdala (i, j) and pontine nuclei (k, l) of Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants. Scale bars, 50 µm. m, n, Electron micrographs of the brain of Atg7flox/flox; nestin-Cre mice. Inclusion bodies (arrows) were often observed in Atg7flox/flox; nestin-Cre hypothalamus. The boxed region in m is shown in n. Inclusion bodies were not detected in Atg7flox/+; nestin-Cre brain (data not shown). Scale bars, 5 µm (m), 1 µm (n). o, Immunoelectron micrograph of ubiquitin in a representative Atg7flox/flox; nestin-Cre hypothalamus. N, nucleus. Scale bar, 1 µm. p, q, Immunohistochemical detection of ubiquitin-positive inclusions in the cerebral cortex (p) and hypothalamus (q) at P9, P18 and P56. Brain sections of each genotype at the indicated ages were immunostained with an anti-ubiquitin antibody. Ubiquitin-positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox/flox; nestin-Cre mice. Scale bars, 50 µm.
Loss of autophagy in the central nervous system causes neurodegeneration in mice (2006)
figf4

a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice. b, Peptide hydrolysis activity of 20S and 26S proteasomes. Homogenates from P28 Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glycerol density gradient centrifugation (10–40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26S proteasomes are indicated with arrowheads. Note that whereas 26S proteasomes exist in active forms in tissues, 20S proteasomes are latent and are activated artificially by a low concentration of SDS. c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.
Src‐dependent autophagic degradation of Ret in FAK‐signalling‐defective cancer cells (2012)
Figure 1

Intracellular localization of Ret is altered in the absence of FAK in SCC cells. (A) FAK+/+ and FAK−/− murine SCC cells were lysed and FAK, Ret or Src immunoprecipitated and immunoblotting carried out using anti‐FAK, anti‐Ret, anti‐Src and anti‐actin. (B) Cells were fixed and stained with either anti‐Ret (green in the upper left panels) or anti‐Ret PY1062 (green in the lower left panels) and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate localization at adhesions while broken arrows show localization in puncta. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). Lysates were immunoblotted with anti‐Ret PY1062 and anti‐actin (lower right panels). (C) Cells were fixed and stained with anti‐Ret (green) and anti‐FAK (red). Merged images are also shown. Solid arrows indicate co‐localization between Ret and FAK at adhesions, while broken arrows indicate its absence in puncta. Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma.
Src‐dependent autophagic degradation of Ret in FAK‐signalling‐defective cancer cells (2012)
Figure 2

Ret localizes to Src‐positive autophagosomes in FAK‐deficient SCC cells. Cells were fixed and stained with either (A) anti‐Ret (green) and anti‐PY416 Src (red) or (B) anti‐PY416 Src (red) and anti‐LC3B (green). Merged and zoomed images are shown. Solid arrows indicate co‐localization at adhesions, while broken arrows show co‐localization in intracellular puncta. (C) Cells were transfected with scrambled, Atg5 or Atg7 siRNA, trypsinized and allowed to readhere before immunoblotting with anti‐Atg5, anti‐Atg7 and anti‐actin (right panels). Cells were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Merged images are shown. Solid arrows indicate co‐localization between Ret and paxillin at adhesions, while broken arrows indicate its absence in puncta. (D) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (E) Cells, either untreated or treated with chloroquine for 24 h, were fixed and stained with anti‐Ret (green) and DAPI (blue). Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma; siRNA, short interfering RNA.
Src‐dependent autophagic degradation of Ret in FAK‐signalling‐defective cancer cells (2012)
Figure 3

Src‐dependent phosphorylation of FAK is required for Ret/FAK binding. (A) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret. (B) Cells were fixed and stained with anti‐Ret (green) and either anti‐FAK (red in the upper panels) or anti‐PY416 Src (red in the lower panels). Merged images are shown. Solid arrows indicate Ret in adhesions, while broken arrows show Ret in puncta. Scale bars, 20 μM. (C) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). FAK, focal adhesion kinase; Ret, rearranged during transfection.
Src‐dependent autophagic degradation of Ret in FAK‐signalling‐defective cancer cells (2012)
Figure 4

Src activity is required for targeting of Ret to puncta in both the presence and absence of FAK. (A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret. (B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.
Mutant Lef1 controls Gata6 in sebaceous gland development and cancer (2019)
Figure 1

Figure 1: ΔNLef1 drives Gata6 expression and SG transcriptional signature.

(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts.

(B) Tail epidermal whole-mounts from WT and K14ΔNLef1 mice labelled with Fabp5, Krt15 and counterstained with Dapi.

(C) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody (red) as compared to input alone (grey) and negative control (Neg. Ctl., black). DNA motif analysis revealed Lef1 classical consensus sequence. Representative plots of ChIP-Seq reads aligned to the Vgll4, Runx1 and Gata6 loci.

(D) Heat-map (Pearson correlation) of differentially expressed genes (DEG) between different micro-dissected regions (IFE, HF and SG) from WT mice (left panel) (Donati et al, 2017). ΔNLef1 Chip-seq data show ΔNLef1 direct transcriptional targets in black (right panel).

(E) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature.

(F) RT-qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence (scr.). Data are means ± SEM of three independent wells.

(G) RT-qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1- or ΔN34Lef1-expressing plasmid. ∆N34Lef1 is the human ortholog of murine ∆N32Lef1, which is expressed in K14∆NLef1 transgenic mice (Takeda et al, 2006). Data are means ± SEM of three independent experiments.

(H) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6.

(I) Heat-maps of DEG from WT- or K14ΔNLef1-unfractionated (all) or sorted (Itga6+Cd34-, basal; Itga6+Cd34+, HFSC) keratinocytes in comparison with IFE, SG, and HF gene signatures ranked from high to low expression.

(J) Heat-map depicting similarities, as Pearson's correlation coefficient, between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis (left panel). Additional correlation with ΔNLef1 Chip-seq data is depicted in the right heat-map.

(K) GO enrichment analysis of ΔNLef1 direct target genes among DEG between WT and K14ΔNLef1 mice.

Data information: (A, B and H) Scale bars: 50 μm. (F, G) Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.