EMBO/soda-nel huggingface dataset:
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 7

Figure 7. Role of BAG6 in the localization of the Golgi apparatus and glycoprotein transport to the plasma membrane.

(A, B) BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).

(C) Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated.

(D-F) Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Representative image from a cell surface glycoprotein quantification assay with Alexa FluorTM488-conjugated Lectin GS-II as a probe (D). The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E). Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment (see also Appendix Fig S6). *p < 0.05 compared with control knockdown (Student's t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).

EXPANDED VEIW Figure legends
MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 2

Figure 2. FGF signaling inhibits the Insulin/IGF signaling via JNK-mediated IRS1 degradation.

A. MS-phospho-proteomics analysis of RCS with indicated genotypes (ctrl=wild type) treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours, showing biological processes regulated by FGF signaling (in blue: up-regulated, in green: down-regulated). N=4 biological replicates were analyzed. FDR <0.05.

B. Proteomic/phospo-proteomic signaling network modulated by FGF18 in chondrocytes. Red= activating phosphorylation; blue= inhibitory phosphorylation. Cubes dimensions and colors are relative to level of protein regulations.

C,D. Representative western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-AKT (T308), AKT, p-cJUN (s73) and cJUN in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 12h. MG132 (10 μM for 6h) and JNK inhibitor (50 μM for 12h) to inhibit proteasome (C) and JNK (D) activity, respectively. β-actin was used as loading control. N=3 independent experiments. Bar graphs showed quantification of indicated proteins normalized to their totals. IRS1 was normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test **p<0.005; *p<0.05; NS not significant.

E. Co-immunofluorescence of p-IRS1 S307 mouse (green) and p-S6 S240/S242 (red) ribosomal protein in IRS1-overexpressing RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. Nuclei (N) were stained with DAPI (blue). Scale bar 15 μm. Quantification analysis of p-S6 fluorescence intensity in IRS1-overexpressing vs non expressing RCS chondrocytes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=35 cells analyzed. Student's paired T-test *p<0.05.
MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 6

Figure 6. TFEB overexpression induces ER-phagy in U2OS and MEFs cells via FAM134B.

A. qRT-PCR analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells. Values were normalized to Hprt gene and expressed as fold change values relative to mock. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Student's un-paired T-test *p<0.05; NS not significant.

B. Western blot analysis of Fam134b isoforms in mock, TFEB S142A:S211A and TFE3 S246A:S312A over-expressing U2OS cells showing induction of FAM134B-2, but not of FAM134B-1 isoform by TFEB/TFE3. Filamin A was used as loading control. Blot is representative of N=3 independent experiments.

C,D. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (TMEM192-HA, red) in control and Sh-FAM134B U2OS cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (C). In (D), Quantification of CLIMP63 fluorescence in TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 21 (vehicle ctrl), N=33 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle shFAM134B), N= 30 (TFEB S142A:S211A-GFP shFAM134B) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.

E,F. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (Lamp1, red) in WT and Fam134bKO MEF cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (E). In (F), Quantification of CLIMP63 fluorescence in Lamp1 decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 20 (vehicle ctrl), N=22 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle Fam134bKO), N= 20 (TFEB S142A:S211A-GFP Fam134bKO) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.
MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B (2020)
Figure 7

Figure 7. FGF signaling controls ER-phagy in vivo.

A,B. Representative images of alcian blue (cartilage) and Alizarin red (bone) skeletal staining showing growth retardation in Fgfr3/4 dKO mice compared to age/sex wild type littermate at post-natal day 30. B. Femur, tibia and tail details.

C. Haematoxylin/Eosin staining of femoral growth plate sections from wild type and Fgfr3/4 dKO mice. Higher magnification insets showed a disorganized hypertrophic chondrocyte layer, in Fgfr3/4 dKO mice. Scale bar 60 μm.

D. Western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-JNK (Y185), JNK1/2 proteins in growth plate lysates of mice with indicated genotypes. N=3 mice/genotype. β-actin was used as loading control. Bar graph shows quantification. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.

E. Western blot analysis of LC3 and Lamp1 proteins from growth plate lysates of mice with indicated genotypes. β-actin was used as loading control. N=3 mice/genotype were analyzed. Bar graph shows quantification. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.

F. qRT-PCR analysis of Fam134b-2 expression from growth plate of mice with indicated genotypes. N=8 (wt mice) and N=9 (Fgfr3/4 dKO mice) were analyzed. Values were normalized to Hprt gene and expressed as fold change relative to control. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.

G. Western blot analysis of Fam134b-2 protein from growth plate lysates of mice with indicated genotypes. β-actin was used as loading control. N=6 (wt mice) and N=4 (Fgfr3/4 dKO mice) were analyzed. Bar graph showed quantification of Fam134b-2 normalized to β-actin. Mean +/- standard error of the mean (sem). Student's un-paired T-test *p<0.05.

H. Representative immunofluorescence staining of CLIMP-63 of femur growth plate sections from mice with indicated genotypes. Scale bar 10μm. Insets showed increased CLIMP-63 staining in Fgfr3/4 dKO mice. Scale bar 5 μm. Hypert= hypertrophic chondrocytes; pre-hypert= pre-hypertrophic chondrocytes; prolif = proliferating chondrocytes.

I. Representative TEM images of growth plate chondrocytes from mice with indicated genotypes. Arrows indicated the ER. N= nucleus. Scale bar 1μm.
Transient Dux expression facilitates nuclear transfer and induced pluripotent stem cell reprogramming (2020)
Figure 1

Figure 1. Dux promotes SCNT-mediated somatic cell reprogramming.

(A) Schematic illustration of the screening strategy.

(B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group.

(C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm.

(D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's  t-test.

(E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm.

(F) Schematic transient induction of Dux expression. All timings reported in this paper is hour post-activation.

(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.

(H) Unsupervised hierarchical clustering.

(I) Venn diagram showing the overlap between the genes that failed to be activated in SCNT 2-cell embryos and derepressed in D-SCNT. Heatmap, GO terms showing the expression pattern of 257 overlapping genes.

(J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.
Transient Dux expression facilitates nuclear transfer and induced pluripotent stem cell reprogramming (2020)
Figure 2

Figure 2. Dux facilitates chemical-mediated somatic cell reprogramming.

(A) Schematic diagram for the induction of CiPSCs from MEFs using the "three-step" method (Zhao et al., 2018).

(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's  t-test. n.s., not significant.

(C) Morphological changes at distinct time points during D-CiPS induction. Scale bar, 100 μm.

(D) Representative images of the number of AP+ positive colonies. n = 5 biological replicates per condition.

(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.

(F) Representative immunostaining images of D-CiPSCs expressing pluripotency markers. n = 5 biological replicates. Scale bar, 50 μm.

(G) Histology by H&E staining of teratoma tissues derived by D-CiPS cells. n = 3 biological replicates. Scale bar, 200 μm.

(H) Chimeric mouse generated from D-CiPSCs.

(I) Karyotype analysis of D-CiPSCs and canonical CiPSCs. The majority of D-CiPSCs and CiPSCs cells possessed a normal karyotype. Scale bar, 10 μm.

(J) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D-CiPSCs or canonical CiPSCs. Filled and empty squares represent methylated and unmethylated CpGs, respectively.

Expanded View Figure Legends
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 1

Figure 1. TRPV2 was functionally expressed in mouse differentiated brown adipocytes.

A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.

B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.

C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.

D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.

E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mouse brown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student’s t-test.

F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.

G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ## P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 2

Figure 2. Brown adipocytes from TRPV2KO mice exhibited facilitated differentiation.

A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student’s t-test.

B. The number of 6 day-differentiated mouse brown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 3

Figure 3. Basal expression of genes related to BAT function and their changes.

A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptor γ Pparγ) and β-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student’s t-test.

B and C. Changes in Ucp1 (B) and Pgc1α (C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 4

Figure 4. Energy imbalance in TRPV2-deficient mice.

A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student’s t-test.

B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).

D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).

E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1α, Pparγ, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student’s t-test.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 5

Figure 5. Comparison of adipocyte sizes between WT and TRPV2KO iBAT.

A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.

B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.

C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.

D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student’s t-test.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 6

Figure 6. TRPV2KO mice show impaired cold-induced thermogenesis.

A, B, C and D. Changes in mRNA expression of Ucp1 (A), Pgc1α (B), Pparγ (C) and Trpv2 (D) in iBAT of WT and TRPV2KO mice before (25oC), after 1 day and 3 days cold (4oC) exposure. Mean ± SEM, n = 5 - 8; * P < 0.05; ** P < 0.01 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

E and F. Western blot results of UCP1 protein in iBAT from WT and TRPV2KO mice before (25oC) and after 3 days cold (4oC) exposure (E). Comparison of UCP1 protein levels before and after cold exposure in iBAT from WT and TRPV2KO mice (F). Mean ± SEM, n = 6; * P < 0.05 vs. 25oC group; # P < 0.05 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

G and H. Averages of core body temperatures (G) and activities (H) of WT and TRPV2KO mice with or without cold (4oC) exposure. Data are presented as mean ± SEM, n = 5; * P < 0.05 vs. WT group. Unpaired Student’s t-test.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 7

Figure 7. Comparison of thermogenic functions of iBAT in WT and TRPV2KO mice.

A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 – 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.

D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student’s t-test.

F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.
Lack of TRPV2 impaired thermogenesis in mouse brown adipose tissue (2016)
Figure 8

Figure 8. Comparison of the changes upon high fat diet treatment between WT and TRPV2KO mice.

A. Body weight changes between WT and TRPV2KO mice treated with high fat diet (HFD) for continuous 8 weeks from 13-week-old.

B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.

D. Weights of tissues related to energy metabolism in WT and TRPV2KO mice after 8-week HFD treatment.

E. mRNA expression of Ucp1 and Pgc1α in iBAT from WT and TRPV2KO mice after 8-week HFD treatment. All data are presented as mean ± SEM, WT mice (n = 6), TRPV2KO mice (n = 8); * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student’s t-test.