EMBO/soda-nel huggingface dataset:
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 1

Rab25 expression alters cells sensitivity to nutrient stress

A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780 pcDNA control and (b) p < 0.05 A2780 Rab25 versus A2780 pcDNA control.

B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25.

C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal.

D. Electron microscopic analysis of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments.

E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.

E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal.

F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel).

G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose.

H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780 pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 2

Expression of Rab25 regulates cellular ATP and glycogen levels

Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29ht pcDNA cells, 6.21−10 moles/mg for IOSE29ht Rab25 cells, 8.14−11 moles/mg for IOSE80ht pcDNA cells, 1.05−10 moles/mg for IOSE80ht Rab25 cells, 2.85−10 moles/mg for A2780 pcDNA cells, 3.81−10 moles/mg for A2780 Rab25 cells, 7.23−10 moles/mg for HEY pcDNA cells and 9.82−10 moles/mg for HEY Rab25 cells.

Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.

Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.

Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.

Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.

Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780 pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence of absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.

Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.

Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 3

Rab25 regulates bioenergetics in ovarian cancer cell through the AKT pathway

A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS).

B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells.

C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells.

D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 h glucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 h SF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 h SF, b, p < 0.01 15 min SF versus 5% FBS.

D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF).

E. Utilization of glycogen to produce ATP.

F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.

G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 4

Rab25 increases glucose uptake

A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.

B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.

C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.

D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescent staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM.

E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.

E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEY pcDNA cells.

F. Down-regulation of AKT by siRNA specific to AKT.

G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 5

Detection of physical association between AKT and Rab25 by protein complementation assay (PCA)

Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow.

Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody.

Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.

Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.
Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress. (2012)
Figure 6

Prediction in clinical samples

A. Rab25 expression correlates with glycogen levels in patient tumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using QPCR and glycogen assay, respectively.

B–C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curves for patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).

B. Tothill et al, 2008.

C. Dressman et al, 2007.

D. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction.

E. Proposed model for the mechanism by which Rab25 regulates cellular bioenergetics.
Root endophyte induces plant thermotolerance by constitutive chromatin modification at heat stress memory gene loci (2020)
Figure 3

Fig. 3: SA187-induced thermotolerance is associated with higher expression of heat-responsive genes and chromatin modification

(a,b) Dynamics of HSFA2 and HSP101 transcript levels in control (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed and SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C. SA187-colonized (HS+187) and thermoprimed (LAT) plants showed higher transcript levels in comparison to plants exposed at 44°C HS for 30 min (HS) after 1 h of recovery at 22°C.

(c,d). Transcript levels of heat stress memory genes HSP18.1 and APX2 in control plants (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C . Transcript levels were normalized to tubulin as reference gene and the respective 22°C NHS plants were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance.

(e) Schematic representation of the experimental set-up and sampling times for ChIP-PCR. 9 day old plants were thermoprimed at 37°C for 3 h before incubation at 22oC for 24 h or 72 h.

(f) APX2 and HSP18.2 gene models drawn to scale (grey boxes, 5' untranslated region; orange boxes, exons; angled arrow, transcription start site). The underneath numbers with grey bar indicates the positioning of regions analyzed for ChIP-PCR, three regions of APX2 and 2 regions of HSP18.2.

(g) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2. Amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants.

Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments).
Root endophyte induces plant thermotolerance by constitutive chromatin modification at heat stress memory gene loci (2020)
Figure 4

Fig. 4: Ethylene and HSFA2 signaling mediates SA187-induced thermotolerance in Arabidopsis.

(a) Phenotypes of SA187-colonized or non-colonized wild type Col-0, hsfa2, hsfa1q and ein3-1 mutant plants upon long-term aquired thermotolerance treatment (LAT): 9 d old plants without and with SA187 (LAT, LAT+187) were treated at 37°C for 3 h, then returned to 22°C for 2 d. At d 11, plants were heat stressed at 44°C for 30 min and incubated for recovery at 22°C; or direct heat stress treatment (HS): 11 d old plants without and with SA187 (HS, HS+187) were treated at 44°C for 30 min and incubated for recovery at 22°C.

(b,c) Fresh weight and percent survival of Col-0, hsfa2, hsfa1q and ein3-1 plants in HS, HS+187, LAT and LAT+187 treatments. Due to the dwarf size of hsfa1q mutants, LAT treatment was performed on d 18 and HS at d 20. All treatments are compared with plants upon 44°C HS.

(d) Fresh weight of 1µM 1-aminocyclopropane-1-carboxylic acid (ACC) treated wild type plants. 5 d old plants were transferred to 1µM ACC containing plates and 11 d old plants were HS treated at 44°C for 30 min before recovery at 22°C for 3 d (HS, HS+ACC). LAT: 9 d old plants without and with ACC treatment (LAT, LAT+ACC) were primed for 3 h at 37°C before incubation for 2 days at 22°C and further heat stress of 44°C was performed at day 11 for 30 min

(e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions.

(f) Fresh weight of hsfa2 and control Col-0 plants treated with and without ACC under non-heat stress condition (NHS, NHS+ACC) of 22°C and heat stress (HS, HS+ACC) condition of 44°C for 30 min. Percent survival was scored at day 4 of recovery from 44°C heat stress. All treatments were compared with plants upon HS.

Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).
Root endophyte induces plant thermotolerance by constitutive chromatin modification at heat stress memory gene loci (2020)
Figure 5

Fig. 5: SA187-induced sustained H3K4me3 levels at APX2 and HSP18.2 memory genes depend on ethylene and HSFA2 signaling

(a,b) Transcript levels of HS memory genes HSP18.2 and APX2 in ein3-1 non-heat stressed (NHS, NHS+187), 44°C heat stressed SA187-colonized (HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C .

(c) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2 (Fig 3F).

(d,e) Transcript levels of HS memory genes HSP18.2 and APX2 in hsfa2 mutant.

(f) Relative enrichment of H3K4me3 levels at APX2 and HSP18.2 memory genes in hsfa2 mutant.

Data information: For transcript level analysis, the data were normalized to tubulin as reference gene and the respective 22°C NHS, NHS+187 control plants, that were harvested at the same time points. For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.
MALT1 targeting suppresses CARD14-induced psoriatic dermatitis in mice (2020)
Figure 1

Figure 1.
Perinatal lethality of CARD14E138A transgenic mice is rescued by MALT1 deficiency

(A) Schematic representation of the Rosa26LSL-CARD14-E138A transgene construct (LSL = LoxP-stop-LoxP).

(B) Ratio of genotypes obtained of 1 week-old pups after crossing K5creTg/+ CARD14E138A+/+ male mice with K5cre+/+ CARD14E138ATg/+ female mice; CARD14E138A+/+ = Rosa26+/+, CARD14E138Atg/+ = Rosa26LSL-CARD14-E138A/+

(C) Representative Western blot showing CARD14 protein levels in skin and liver lysates of E18.5 pups. Actin is shown as a loading control.

(D) Representative images of K5creTg/+ CARD14E138A+/+ and K5creTg/+ CARD14E138ATg/+ pups at E18.5. (E) Ratio of genotypes obtained of 1 week-old pups from crossing Malt1fl/fl K5creTg/+ CARD1414E138A+/+ male mice with Malt1fl/fl K5cre+/+ CARD14E138Atg/+ female mice.

(F) Representative image of 6 month-old Malt1fl/fl K5cretg/+ CARD14E138A+/+ and Malt1fl/fl K5cretg/+ CARD14E138Atg/+ mice.

(G) Body weight and ear thickness of untreated WT and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ male and female mice (between 4 and 7 months old). Each symbol represents one mouse; the line represents the mean value (n≥4 mice per group). WT = Malt1fl/fl K5cre+/+ CARD14E138A+/+, Malt1fl/fl K5cre+/+ CARD14E138ATg/+ and Malt1fl/fl K5creTg/+ CARD14E138A+/+ mice. Statistical difference between two groups was determined using a Mann-Whitney U-test (ns: p-value>0.05)

(H) Representative H&E-stained histological sections of ear and skin tissue of Malt1fl/fl K5cre+/+ CARD14E138A+/+ and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ mice (scale bar represents 100 μm).
MALT1 targeting suppresses CARD14-induced psoriatic dermatitis in mice (2020)
Figure 2

Figure 2.
Keratinocyte-specific deletion of MALT1 reduces CARD14E138A-induced psoriasiform inflammation

(A) Schematic representation of the experimental induction of CARD14E138A transgene using tamoxifen. WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+

(B) Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen. Malt1EKO = Malt1fl/fl. The combined results of five independent experiments are shown (Malt1+/+ WT n=17, Malt1fl/fl WT n=8, Malt1+/+ ieCARD14E138A n=18, Malt1fl/fl ieCARD14E138A n=12).

(C) Representative H&E-stained histological sections of ear tissue of tamoxifen-treated mice (Scale bar represents 200 μm). Arrowheads indicate infiltrates of immune cells.

(D) Epidermal thickness measured on ear sections. Each symbol represents the mean of at least ten measurements for each ear; the line represents the mean value. The combined results of three independent experiments are shown.

(E) Analysis of infiltrating immune cells in the ears of tamoxifen-treated mice using flow cytometry. Cell count of neutrophils (CD45+ CD3/CD19- CD64- CD11b+ Ly6G+), eosinophils (CD45+ CD3/CD19- CD64- Ly6G- SiglecF+), T cells (CD45+ CD3/CD19+, MHCII-), DCs (CD45+ CD3/CD19- CD64- MHCII+CD11c+) in single cell suspensions of the ear (n≥4).

(F) mRNA expression levels of the indicated genes in ears relative to reference genes (Hprt1, Rpl13a and Tbp1). Each symbol represents one mouse; the line represents the mean value (n≥3).

Data information: Statistics: Error bars reflect SEM. Differences between Malt1+/+ ieCARD14E138A and Malt1EKO ieCARD14E138A groups were determined using REML analysis (B) or a Mann-Whitney U-test (D-E-F) (*p-value<0.05, **p-value <0.01, ****p-value<0.0001).
MALT1 targeting suppresses CARD14-induced psoriatic dermatitis in mice (2020)
Figure 3

Figure 3.
Inhibition of MALT1 proteolytic activity attenuates CARD14E138A-induced psoriasis

(A) Schematic representation of the experimental set-up of CARD14E138A induction and MALT1 inhibitor treatment.

(B) Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle. Actin is shown as a loading control. Each lane represents one mouse.

(C) Changes in relative ear thickness and body weight in vehicle- and MALT1 inhibitor-treated mice upon CARD14E138A induction. M1 inh= MALT1 inhibitor, WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+. Combined results of two independent experiments are shown (WT + vehicle: n=9, WT + M1 inh: n=8, ieCARD14E138A + vehicle: n=9, ieCARD14E138A + M1 inh: n=10).

D, E (D) Representative histological sections of ear tissue stained with hematoxylin and eosin and (E) measurements of epidermal thickness of tamoxifen-treated mice. Combined results of two independent experiments are shown. Each symbol represents the mean of at least ten epidermal thickness measurements for each ear; the line represents the mean value.

(F) IL-6 and IL-17a production by ear draining lymphocytes upon ex vivo restimulation with anti-CD3/anti-CD28 for 3 days (n≥3 biological replicates).

(G) Heatmap showing relative mRNA expression levels of the indicated genes in ear tissue normalized to reference genes. Values represent median of each group (n≥3).

Data information: Statistics: Data are representative for 2 independent experiments. Error bars reflect SEM. P-values were determined using REML analysis (C), two-way ANOVA with Sidak correction for multiple comparisons (D) or Mann-Whitney U-test (E) (*p-value<0.05, ***p-value<0.001, ****p-value<0.0001).

Expanded View Figure Legends