EMBO/soda-nel huggingface dataset:
Rab11-FIP1 mediates Epithelial-Mesenchymal Transition and invasion in esophageal cancer (2020)
Figure 2

Figure 2. Endocytic recycling genes are downregulated in mutant p53 cells

A. RNA-seq was performed on WT, mutant, and null p53 low passage cell lines. Principle component analysis (PCA) highlights distinct genetic profiles based on p53 status.

B. Relative fold change in expression level of endocytic recycling genes in invasive vs. non-invasive cells, and in mutant vs. WT p53 cells based on microarray and RNA-seq analysis, respectively.

C. qPCR analysis of recycling-related gene expression in mutant p53 relative to WT p53 cells (red and black bars, respectively). Graphs represent mean ± SEM (n=3 biological replicates for Rab11-FIP1, Rab25, Myo5b and Clic3; n=2 biological replicates for Rab5). Rab11-FIP1 *p=0.0109, Rab25 **p=0.0021, Myo5b ***p=0.0001, Clic3 **p=0.0051, Rab5 no significant differences (n.s.). (Unpaired t-test).
Rab11-FIP1 mediates Epithelial-Mesenchymal Transition and invasion in esophageal cancer (2020)
Figure 3

Figure 3. Knockdown of Rab11-FIP1 promotes epithelial disorganization and increases 3D organoid size

A. Western blot confirming knockdown of Rab11-FIP1 by shRNAs. Relative intensity by densitometry of Rab11-FIP1 results are shown at the bottom.

B-F 3D organoids were generated from WT and mutant p53 cells expressing non-targeting control (NT) or Rab11-FIP1 shRNAs (sh1 and sh2). B. Diameter of organoids (n= 80-130 organoids). Bar graph represents mean diameter ± SEM. ****p<0.0001 WT or mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0006 WT non-targeting control vs Rab11-FIP1 shRNA2, ****p<0.0001 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). ****p<0.0001 WT vs mutant p53 non-targeting control (Unpaired t-test). C. Percent of WT p53 (top panel) and mutant (bottom panel) organoids exhibiting a solid, intermediate, or stratified morphology (n>50 organoids generated from three different cell passages quantified). D. Percent of WT p53 and mutant organoids exhibiting a basaloid-like morphology (n>50 organoids generated from three different cell passages quantified). Bar graph represents mean diameter ± SEM. n.s. WT p53 non-targeting control vs Rab11-FIP1 shRNAs, *p=0.046 mutant p53 non-targeting control vs Rab11-FIP1 shRNA1, ***p=0.0002 mutant p53 non-targeting control vs Rab11-FIP1 shRNA2 (One-way Anova). E. WT p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm. F. Mutant p53 organoids stained for H&E (left), Rab11-FIP1 (middle), and the basal cell marker p63 (right) by IHC. 40X. Scale bar = 100 μm.
Rab11-FIP1 mediates Epithelial-Mesenchymal Transition and invasion in esophageal cancer (2020)
Figure 4

Figure 4. Loss of Rab11-FIP1 promotes mutant p53 driven invasion

A. EPC2-hTERT-EGFR-p53R175H cells grown in OTC stained for RCP. Arrow indicates invasive region. (scale bar = 100 μm).

B. Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non-targeting control (NT) and Rab11-FIP1 shRNAs (sh1 and sh2) (n=9 technical replicates from 3 different passages). Error bars represent ±S.E.M. n.s. WT p53 NT vs sh1, ***p=0.0005 WT p53 NT vs sh2, n.s. Mutant p53 NT vs sh1,** p=0.0026 Mutant p53 NT vs sh2, (One-way Anova) *p=0.0173 WT p53 NT vs Mutant p53 NT, (unpaired t-test).

C-D. WT and mutant p53 cells expressing non-target control and Rab11-FIP1 shRNAs were grown in OTC. C. H&E staining of OTC with WT p53 (top) and mutant p53 (bottom) with invasive regions. Scale bar = 50 μm. D. Graph shows relative invasive area from OTC of WT p53 non-target control and Rab11-FIP1 shRNAs. n=5 technical replicates in WT p53 NT, n=6 technical replicates in WT p53 sh1, n=6 technical replicates in WT p53 sh2. Error bars represent ±S.E.M. **** p<0.0001 WT p53 NT vs sh1, n.s. WT p53 NT vs sh2(One-way Anova).
Rab11-FIP1 mediates Epithelial-Mesenchymal Transition and invasion in esophageal cancer (2020)
Figure 5

Figure 5. Loss of Rab11-FIP1 induces epithelial-mesenchymal transition in ESCC

A. Immunofluorescent staining for Rab11-FIP1 in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm

B. Immunofluorescent staining for f-actin in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm

C. Immunofluorescent staining for E-cadherin, in TE1 and TE2 ESCC cell lines expressing a non-targeting control shRNA or shRNA against Rab11-FIP1. Scale bar= 50 μm

D. qPCR analysis of epithelial and mesenchymal gene expression in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). n.s. TE2 SNAI2, *p<0.05,(Unpaired t-test).

E. qPCR analysis of epithelial and mesenchymal gene expression following ZEB1 depletion in TE1 (left) and TE2 (right) cells expressing Rab11-FIP1 shRNA relative to control cells (dotted line). Graph error bars represent mean SEM (n=3 technical replicates from three different cell passages). **p<0.01, * p<0.05, n.s. TE1 CDH1, VIM. SNAI1, SNAI2, TE2 VIM, SNAI1 (Unpaired t-test).

Expanded View Figure Legends
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients (2020)
Figure 1

Figure 1. Anti-cytokine activity of baricitinib.

(A) IC50s for baricitinib (orange) in cytokine-stimulated human CD4+ T cells, CD8+ T cells, NK cells, and monocytes are shown for IL-2/pSTAT5, IL-4/pSTAT6, IL-3/pSTAT5, IL-6/pSTAT3, IL-10/pSTAT3, IL-15/pSTAT5, IL-21/pSTAT3, G-CSF/pSTAT3, GM-CSF/pSTAT5, and IFNγ/pSTAT1. Average daily percent STAT inhibition was calculated using in vitro concentration response curves and published human exposure data. Cytokine treatments that did not result in sufficient pSTAT stimulation are denoted as "NS" in the radar plots.

(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.

Data information: IC50, half maximum inhibitory concentration; NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription.
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients (2020)
Figure 2

Figure 2. NAK binding affinities of baricitinib and other comparators.

(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)

(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.

(D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).

(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).

(G) Schematic showing the timeline for the experiment shown in panel H.

(H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients (2020)
Figure 3

Figure 3. Viral detection and immunological features of four COVID-19 patients treated with baricitinib.

(A) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.

(B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter).

(C) Total serum IgG, IgA, and IgM levels are shown for all patients.

Data information: Ct, cycle threshold; E, envelope membrane; ED, emergency department; N, nucleocapsid protein; NK, natural killer; RdRp, RNA-dependent RNA polymerase.
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients (2020)
Figure 4

Figure 4: Seroconversion of patients during baricitinib treatment. Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients (2020)
Figure 5

Figure 5. Standard laboratory and radiologic features of the four COVID-19 cases treated with baricitinib. (A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.

(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients.

(C) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).

Data information: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; CT, computed tomography; WBC, white blood cells.
Fate and propagation of endogenously formed Tau aggregates in neuronal cells (2020)
Figure 1

Figure 1: Spreading of K18-ATTO 594 fibrils in CAD cells.

(A) Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, below are pictures after 24 hours of co-culture of the two populations (left), and of acceptor cells treated with conditioned medium from donor cells for 24 hours (SN, right). In white is the cell membrane labelling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm.

(B) Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after co-culturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 hours (secretion). The total transfer is arbitrarily set at 100%, and cell-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+SD) of 4 independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08).

(C) Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the co-culture. Left, data represent the means (132%) +SD, normalized to non-treated co-culture arbitrarily set at 100%, of 3 independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean= 47.7%, 3 independent experiments, *P = 0.020).

(D) Representative confocal images (40x) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24h after fibrils addition, fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture, (z10), not attached to the substrate). On the right and below z10 pictures, are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm.
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 1

Figure 1. Nedd4-2 cKO mice exhibit alterations in hippocampal synaptic functions and L-LTP.

(A) Representative western blots of Nedd4-2 and GAPDH from Nedd4-2 WT and Nedd4-2 cKO forebrains at postnatal day 14.

(B) Patch-clamp recording from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 14-17. Representative mEPSC traces (left) and quantification of mEPSC amplitude and frequency are shown (right) (n = 17 and 18 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively).

(C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm.

(D) Synaptic input-output relationship was obtained by initial slope of fEPSPs against the stimulus intensity at Schaffer collateral synapses of hippocampal slices from Nedd4-2 WT (n = 14 slices from 11 mice) and Nedd4-2 cKO (n = 17 slices from 12 mice) mice at 6-8 weeks of age.

(E) Paired-pulse ratio of fEPSPs at Schaffer collateral synapses over an interpulse interval range of 25-400 ms from Nedd4-2 WT (n = 13 slices from 9 mice) and Nedd4-2 cKO (n = 12 slices from 8 mice) slices.

(F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right.

(G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.

Data information: Student's t-test was used. Data are presented as mean ± SEM with *p < 0.05, **p < 0.01 and NS: non-significant.
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 2

Figure 2. Male Nedd4-2 cKO mice exhibit deficits in hippocampus-dependent learning and memory.

(A) Fear conditioning test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of fear conditioning test (left). Mice were placed in the fear conditioning chamber for 180 s with a metal grid floor and received two electrical shocks at 120 and 150 s (0.5 mV, 2s). Twenty-four hrs later, mice were placed in the same chamber for 180 s in the absence of the shock. Freezing behavior during contextual fear conditioning test at 24 hrs after training is shown (right). Freezing rate from Nedd4-2 WT (n = 3) and Nedd4-2 cKO (n = 3) mice without shocks during the training (unshock) was also shown (right).

(B) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant).

(C) Novel object recognition test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of the test (left). Mice explored the chamber with two identical objects for 10 min during the training day. Twenty-four hrs later, mice returned to the chamber with one of the objects replaced by a novel object. Exploration time of familiar and novel objects on testing session (middle) and the preference index for the novel object (right) were measured. The dash line represents the 50% chance of exploring either of the objects.

(D) Open field test from Nedd4-2 WT (n = 7) and Nedd4-2 cKO (n = 8) mice. Representative image showing the explorative behavior of Nedd4-2 WT and Nedd4-2 cKO mice during 5 min open field test period (left). Quantification of locomotor activity and anxiety-like behavior are shown by average velocity, total distance traveled, immobile time, time spend in center zone, number of entries in center zone, and defecation number (right).

Data information: Student's t-test was used. Data are presented as mean ± SEM with *p < 0.05, **p < 0.01, ***p < 0.001 and NS: non-significant.
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 3

Figure 3. The actin dynamics is impaired in Nedd4-2 cKO mice during the basal state and upon induction of cLTP.

(A) Western blots of F-actin to G-actin ratio from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification are shown (n = 10 mice for Nedd4-2 WT and Nedd4-2 cKO).

(B) Western blots of F-actin to G-actin ratio with or without cLTP induction from Nedd4-2 WT or Nedd4-2 cKO cultured neurons. Schematic representation of the experimental design for cLTP inductions (top left). Representative western blots (bottom left) and quantification of F-actin to G-actin from Nedd4-2 WT or Nedd4-2 cKO (right) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis (n = 11 and 8 for Nedd4-2 WT and Nedd4-2 cKO cultures, respectively). For quantification, data from cLTP induction were normalized to data from control (CTL) condition.

Data information: Student's t-test (A) and one-way ANOVA with Tukey test (B) were used. Data are presented as mean ± SEM with *p < 0.05, **p < 0.01 and NS: non-significant.
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 4

Figure 4. Cofilin phosphorylation is impaired in Nedd4-2 cKO mice.

(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown.

(B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm.

(C) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.

Data information: Student's t-test (A, B) and one-way ANOVA with Tukey test (C) were used. Data are presented as mean ± SEM with *p < 0.05, ***p < 0.001 and NS: non-significant.
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 5

Figure 5. Inhibition of cofilin restores hippocampal synaptic functions and L-LTP in Nedd4-2 cKO mice.

(A) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16 treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM). Representative secondary dendrites of Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) neurons are shown. Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons is on the right (n = 32-36 neurons). Scale bar: 5 µm.

(B) Patch-clamp recordings from Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM) at DIV 14-17. Representative mEPSC traces from Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) are shown. Quantification of mEPSC amplitude and frequency from Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons are shown (n = 22-24 neurons).

(C) fEPSP recording from Nedd4-2 WT (left; n = 7 slices from 6 mice for TAT and n = 6 slices from 6 mice for TAT-pS3) and Nedd4-2 cKO (right; n = 9 slices from 9 mice for both TAT and TAT-pS3) hippocampal slices of mice at age 6-8 weeks. The slices were pretreated with TAT or TAT-pS3 peptide (20 μM) for 1 hr before the induction of L-LTP at Schaffer collateral synapses. Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showed the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses.

Data information: One-way ANOVA with Tukey test (A, B) and Student's t-test (C) were used. Data are presented as mean ± SEM with *p < 0.05, **p < 0.01, ***p < 0.001 and NS: non-significant.