EMBO/SourceData huggingface dataset:
Figure 5A,B. MDA-MB-231 (A) and MDA-MB-436 (B) tumor xenografts were treated with LNPs-empty, LNPs-miR-Control or by LNPs-miR-182-3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs-miR-Control, LNPs-miR-182-3p or equivalent volume of LNPs-empty as indicated in the scheduling. The mean of tumor volumes (n=5 per group) is shown. Data information: For A, B data are shown as mean ± SD. P values are determined by unpaired two-tailed t testC,D. Tumors from mice treated in A and B were processed to measure miR-182-3p expression by TaqMan qPCR. Data information: For C, D the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. , P values are determined by unpaired two-tailed t test;E. Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA-MB-231 human breast cancer xenografts. Scale bar: 50μm. F. The histograms show: the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, Tunel or CD31 staining. The analyses were performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For F, data are shown as mean ± SD. (F), P values are determined by Mann-Whitney test.G, H. Luminescent MDA-MB-436 cells were injected into the brain and monitored by IVIS imaging system. After one week from implant, treatment with LNPs-miR-Control and LNPs-miR-182-3p was performed as indicated in A and B. Representative images from in vivo (upper panel) or ex-vivo (bottom panel) brain tumors are shown in G. Boxplots (H) show the measurement of photons for each brain tumor (n=5 per group) acquired at the indicated times. Data information: For H, the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. For H, P values are determined by unpaired two-tailed t test;
Figure 6A,B. PDTCs #1 and #2 underwent two rounds of transfection with miR-Control or miR-182-3p. Three days after the second transfection, miR-182-3p and TRF2 expression were analysed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in A, B two or three biological replicates were performed, respectively.C,D. Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50μm. At least 85 3D cells were analysed for each experimental condition. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in C, D two or three biological replicates were performed, respectively.E. NSG mice implanted with breast PDTX (#2) were treated with LNPs-empty, LNPs-miR-Control or LNPs-miR-182-3p as indicated in the scheduling. Caliper measurement of tumors were taken at the indicated days. The mean of tumor volumes (n=5 per group) is shown. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test;F. miR-182-3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t testG. Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E. Scale bar: 50μm. H. The histograms show: the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and Tunel. The analysis was performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For H, data are shown as mean ± SD. For (H), P values are determined by Mann-Whitney test.
Figure 7A. Representative images of intestine sections from mice previously treated with LNPs-Empty or LNPs-miR-182-3p. H&E staining (scale bar: 200μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50μm). B,C. Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX positive cells (%) (C) on intestine samples. D. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of skin samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). E,F. Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX positive cells (%) (F) on skin samples. G. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). H,I. Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX positive cells (%) (I) on bone marrow samples. Data information: For B,C,E,F,H,I, data are shown as mean ± SD. A Mann-Whitney test t test was used to calculate statistical significance. Four mice per group were analysed the points represent the number of field analysed for each condition.
Figure 2(B) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting.(C) (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells.(D) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting.(E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting.(F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.
Figure 3(A) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FVmutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody.(B) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.(C) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I.(D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm.(E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.
Figure 4(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies.(B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells.(C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm.(D) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown.(E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.
Figure 5(A) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux.(B) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A).(C) atg1Δpep4Δ cells containing YFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown to log phase and starved in SD‐N. Cells were monitored by time‐lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in 'Materials and methods'. Data are plotted as the mean±s.e.m. from at least 90 quantified cells. Additional images are shown in Supplementary Figure S3H. Bar=5 μm.(D) atg1Δatg13Δ cells expressing GFP‐tagged wild‐type Atg13, and either wild‐type Atg1 or the Atg1‐VE mutant (left panel) and wild‐type cells containing Atg13‐GFP and either wild‐type Atg13 or Atg13‐FV were grown to mid log phase followed by starvation for 6 h in SD‐N media. GFP cleavage was analysed by western blotting. Figure source data can be found with the Supplementary data.
Figure 1A, B Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125 A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125 C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008 D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)
Figure 2A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015) B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)  C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice  D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)  E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439) F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm
Figure 6 A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)  B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm  C Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)  D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice  E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells  F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm  G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)  H Representative images of cultured organoid on Day 3  I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)  J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049 J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049 J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049
Figure 7 A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice  B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown  C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots  F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting  G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later  J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)
Figure 8 A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445  C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117) D-G Albumin levels in feces (D on day 4 of recovery (n = 5)colon length (E) on day 4 of recovery (n = 5)H&E staining of colon tissues (F on day 4 of recovery (n = 5). Scale bars = 100 μmRT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301
Figure 2(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.(B) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains.(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.(D) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb CDC 1551 strains (E) ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb Erdman strains (F). ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).qRT-PCR analysis for IFN-β mRNA (G) in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).IFN-β protein (H) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(I) ELISA analysis for IFN-β secreted by BMMs treated with EVs plus liposomes containing M.tb RNA. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.qRT-PCR analysis for IFN-β mRNA (J) in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.IFN-β protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.
Figure 4(A) Immunofluorescence microscopy analysis for colocalization of M.tb with Lamp-1 in WT BMMs pre-treated with EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. The cells were pre-treated with EVs supplemented with recombinant mouse IFN-γ for 5 hr and subsequently infected for 24 hr with GFP-expressing M.tb. (B) quantitative analysis for the colocalization of M.tb with Lamp-1. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM ; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.(C) M.tb CFU in WT BMMs pre-pretreated with recombinant mouse IFN-γ and EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.(D) and (E) liposomes containing RNA isolated from EVs released from BMMs infected with WT M.tb were also added to cells 5 hr prior to infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant(F) liposome-encapsulated RNA isolated from EVs released from BMMs infected with WT M.tb was included during EV and IFN-γ pre-treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.
Figure 6(A and B) Quantitative analysis of immunofluorescence microscopy images for colocalization of M.tb with Lamp-1 in WT (A) and Mavs -/- (B) BMMs infected with M.tb for 24 hr and then treated for an additional 24 hr with moxifloxacin (MFX) and/or EVs from M.tb-infected BMMs (EVs+MFX) in the presence of recombinant mouse IFN-ɣ. Mock, no EVs or moxifloxacin treatment; EVs_Control, EVs from uninfected BMMs. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).LC3 (C), colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).NOX2 (D) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).p47phox (E) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with LC3 (F) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with NOX2 (G) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with p47phox (H) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(I and J) M.tb CFU analysis in WT (I) and Mavs -/- (J) BMMs infected with M.tb for 24 hr followed by treatment with EVs, moxifloxacin or combination in the presence of IFN-ɣ for 24 and 72 hr. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).