EMBO/soda-nel huggingface dataset:
Rewiring Phospholipid Biosynthesis Reveals Resilience to Membrane Perturbations (2022)
Figure 3

Fig 3. Transposon mutagenesis screens to identify genes required for adaptation and lipid trafficking in the rewired strains.

A) Outline of the SATAY screening procedure. Cells expressing plasmids containing the PE and PC synthesizing enzymes and a plasmid harboring the galactose inducible transposase (TPase) and a transposon (TN) disrupting the TRP1 gene were grown for ~56h in galactose-containing medium to induce transposition (step 1). Cells were inoculated in synthetic medium lacking tryptophan in either KennedyON or OFF conditions, grown for several generations (step 2) and harvested for DNA extraction and sequencing of transposon insertion sites (TNs) (step 3). TNs are mapped to the genome to identify genes that become required under certain rewired conditions (step 4). An illustrative example of TN insertions of four libraries visualized in the UCSC genome browser highlights a genetic requirement for DRS2 (green bar) in KennedyOFF conditions.

B) GO term enrichments in the top 200 (red) and top 500 (blue) most variable genes (defined as genes with the highest standard deviation of TN insertions across libraries) identified using the YeastMine web server. Significance was determined using Holm-Bonferroni corrected p-values with a cut-off of 0.05, the size of the circles is inversely proportional to the p-values (i.e. bigger circles indicate higher significance).

C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel).

D) Schematic (top panel) illustrating the requirement for SAM transport across the mitochondrial inner membrane by Sam5 for PC production by Pmt in the mitochondrial matrix (MM). Lipids can be produced by the Kennedy pathway (red) independent of Sam5 activity. Transposon insertion maps generated in the UCSC genome browser for SAM5 for the library where PC is produced in the MM in either KennedyON or OFF conditions (bottom panel).
Rewiring Phospholipid Biosynthesis Reveals Resilience to Membrane Perturbations (2022)
Figure 5

Fig 5. Differential genetic requirements common to rewired libraries.

A) Volcano plot comparing the number of transposon insertions per gene of all 24 rewired libraries and two wild-type (wt) libraries. Genes that are significantly (p-value < 0.05) required (red, log2(fold change) < -1.5; blue, log2(fold change) < -1) or dispensable (green, log2(fold change > 2)) in the rewired libraries are highlighted. Genes depicted in grey are essential due to differential growth requirements in the preparation of the two wt libraries compared to the rewired strains, or correspond to genes deleted in the rewired strains.

B) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries. Left panel, schematic depicting PE and cardiolipin (CL) synthesis in mitochondria. The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow. Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM. Right panel, transposon numbers (normalized to wt libraries) for the indicated (set of) libraries for MDM31, PGS1, and CRD1.
Rewiring Phospholipid Biosynthesis Reveals Resilience to Membrane Perturbations (2022)
Figure 7

Fig 7: Csf1 is involved in phospholipid remodeling during cold stress.

A) Top: Co-localization of Csf1-GFP cells in log phase with genomically integrated Pex10-mCherry (peroxisome marker) and mtBFP plasmid (mito marker). Bottom: Co-localization of log growing Csf1-GFP cells with mCherry-Ubc6 (tail-anchor) (ER marker) and mtBFP plasmid (mito marker). Scale bar, 2 µm.

B) Five-fold serial dilutions of strains of the indicated genotypes grown on YPD plates at 30 °C or at 16 °C. Growth assays are representative images of biological replicates.

C) Five-fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures, in the presence or absence of 10 mM ethanolamine (etn).

D) Bubble plots depicting the changes in abundance for each phospholipid class, classified based on the fatty acid length and degree of unsaturation, in wild-type (WT) and csf1ΔC cells grown at 30°C (red-half) or at 16°C for 14.5 hours (blue-half). Significant changes for WT versus csf1ΔC at 16 °C or at 30 °C are indicated (* p<0.05, #p<0.005). p-values were calculated with the two-sample t-test.

E) Bar graphs depicting the fraction of more saturated species (defined as fatty acid chains with one double bond for ≤30 carbon chains or with two double bonds for ≥32), and less unsaturated species (defined as fatty acid chains with zero double bond for ≤30 carbon chains or with one double bond for ≥32) for each phospholipid class with different head groups. Bars represent the means of two or three biological replicates (individual measurements are shown as points and triangles). p-values are calculated with a two-sample t-test.

F) Ten-fold serial dilutions of indicated strains on YPD plates with or without 0.1% oleic acid at 20 °C or at 30 °C.
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 1

Figure 1. Stage-dependent upregulation of the expression of ADAR1 and interferon-stimulated genes during thymic T cell maturation.

A Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.

B, C The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).

D The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 2

Figure 2. Inhibition of T cell maturation by deletion of ADAR1 in the thymus.

A Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice. Values are displayed as the mean ± SEM (n = 7 for each group; Mann-Whitney U-test, N.S.; not significant).

B, C Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B). The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C). Values are displayed as the mean ± SEM (n = 7 for each group; Mann-Whitney U-test, **p < 0.01).

D, E Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D). The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E). Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05).

F Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel). Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05).

G Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel). Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05, N.S.; not significant).

H Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel). Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05).
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 3

Figure 3. Impaired thymic selection in CD4+ cell-specific Adar1 knockout mice.

A, B CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels). Values are displayed as the mean ± SEM (n = 4 for each group; Mann-Whitney U-test, *p < 0.05, N.S.; not significant).

C Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel). Values are displayed as the mean ± SEM (Mann-Whitney U-test, *p < 0.05, **p < 0.01, N.S.; not significant).

D The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.

E Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05) (right panel) by setting the mean value of stimulated WT samples at 60 sec as 1.
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 4

Figure 4. Spontaneous colitis observed in CD4+ cell-specific Adar1 knockout mice.

A Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group; Fisher's exact test, *p < 0.05). B Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm.

C Histological scores for colitis were compared between WT (n = 4) and Cd4cre A1fl/fl (n = 7) mice (Mann-Whitney U-test, **p < 0.01).

D Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel). Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05).

E Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel). Values are displayed as the mean ± SEM (n = 6 for each group; Mann-Whitney U-test, **p < 0.01).

F Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel). Values are displayed as the mean ± SEM (n = 4 for each group; Mann-Whitney U-test, *p < 0.05).

G Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 5

Figure 5. Upregulation of ADAR1-medited RNA editing during thymic T cell maturation.

A Distribution of the potential RNA editing sites found in 4SP thymocytes.

B The top 12 categories of biological processes, which are ranked according to the p-value, for the genes that contained potential RNA editing sites at the 4SP stage. Fisher's exact test was used for the statistical analysis. GO; gene ontology.

C, D RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05).

E The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one.

F Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.
ADAR1-mediated RNA editing is required for thymic self-tolerance and inhibition of autoimmunity (2018)
Figure 7

Figure 7. Disruption of the MDA5 signaling pathway ameliorates colitis in CD4+ cell-specific Adar1 knockout mice.

A Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice. Values are displayed as the mean ± SEM (Mann-Whitney U-test, *p < 0.05, N.S.; not significant).

B No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl mice, the data shown in Fig 4A was included.

C Representative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm.

D Histological scores for the colitis in Ifih1-/- (n = 3) and Cd4cre A1fl/fl Ifih1-/- (n = 4) mice. For side-by-side comparison with Cd4cre A1fl/fl mice, the data shown in Fig 4C was included.

Expanded View Figure Legends
The ion-coupling mechanism of human excitatory amino acid transporters (2021)
Figure 1

Figure 1 Na+ and transmitter binding

A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively.

B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9.

C. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.

D. Log-log plot of L-asp KD as a function of Na+ concentration. Empty circles are KD values (n=20) at pH=9.0 in the absence of UCPH101 (blue), and solid line is the fit of a straight line. For comparison, average L-asp KD at 1 mM Na+ in the presence pf of UPCH101 is also shown (n=3; red).

Data information: in (C) symbols represent average and s.e.m. values of at least three independent titrations. In (D), blue symbols represent individual KD values from n number of independent titrations, and red symbol represents average and s.e.m values from three experiments.
The ion-coupling mechanism of human excitatory amino acid transporters (2021)
Figure 3

Figure 3 H+ and transmitter binding

A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.

B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.

C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..

D. Potential H+-bond network between protonated E406386 (TM7b) and residues in HP2b in EAAT1CRYST transmitter-bound structure. HP2 is depicted in yellow and the 398NMDG motif in green. Residue numbering corresponds to EAAT1WT, and the interatomic distance (angstrom) is parenthesis.

Data information: in (A) bar plots depict averages and s.e.m. values of three independent experiments (empty circles) performed in triplicate. In (B,C) symbols represent individual KD values from n number of independent titrations.
The ion-coupling mechanism of human excitatory amino acid transporters (2021)
Figure 4

Figure 4 Counter-transported ion binding site

A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.

B. Superimposition of Na+/transmitter and Rb+ bound EAAT1CRYST structures, respectively, with tranD depicted in orange (Na+/transmitter) and pink (Rb+). TM4 and UCPH101 are omitted for clarity. Black mesh depicts anomalous-difference map contoured at 3.5σ around the tranD. Rb+ coordination is shown in the inset. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is parenthesis.

Data information: in (A) bar plots depict averages and s.e.m. values of three independent experiments (empty circles) performed in triplicate.
The ion-coupling mechanism of human excitatory amino acid transporters (2021)
Figure 5

Figure 5 Divalent cation binding

A. Superimposition of Rb+/Ba2+ and Ba2+ bound EAAT1CRYST structures with tranD depicted in pink and green, respectively, and TM4 removed for clarity. Black mesh depicts anomalous-difference map in the absence of Rb+ contoured at 4σ around the tranD, and inset shows details of Ba2+ coordination at Na3. Residue numbering corresponds to EAAT1WT, and the coordination distance (angstrom) is parenthesis.

B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.

C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).

Data information: in (B) symbols represent average and s.e.m. values of at least three independent titrations. In (C), blue symbols represent individual KD values from n number of independent titrations.
Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit (2016)
Figure 1

Figure 1. miR-499 is dynamically regulated during muscle fiber type transition and effects mitochondrial function.

(A) RT-qPCR analysis of Myh7b/miR-499 and Myh7/miR-208b levels during differentiation of myoblasts into mature myotubes (n = 3 independent experiments). *p = 0.0005 (Myh7b), *p < 0.0001 (miR-499), *p = 0.0002 (Myh7), *p < 0.0001 (miR-208b).

(B) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP).

(C) Mean expression levels (RT-qPCR) in white vastus (WV) and soleus (Sol) muscle from 8-week-old male wild type mice (n = 5 mice per group). *p < 0.0001 (Myh7b), *p < 0.0001 (miR-499), *p < 0.0001 (Myh7), *p < 0.0001 (miR-208b).

(D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP).

(E) (Left) Schematic depicts the increments of speed over time. (Right) Respiratory exchange ratio (RER) during a graded exercise regimen as described in Materials and Methods (n = 5 mice per group). Notably, MCK-miR-499 mice were able to exercise at a higher speed before exhaustion. *p < 0.05.

(F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month).

(G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).

All values represent the mean ± SEM. p-value was determined using two-tailed unpaired Student’s t-test.