EMBO/SourceData huggingface dataset:
Figure 7A Mouse cortical neurons (DIV 7) were infected with lentivirus carrying mCherry-Parkin for 45 h, and then the neurons were treated with 5 µg/mL A/O for 5 h and subjected to immunofluorescent assay using anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bar, 2.5 µm.Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. B The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h and then were treated with 1 µg/mL A/O for 48 h. The neurons were stained with DAPI, anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies and visualized. Scale bars, 10 µm. Yellow arrows indicate uncleared mitochondria.Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. F The neurons were infected with lentivirus carrying mCherry-Parkin for 36 h and were treated with 100 ng/mL Antimycin A (Antimycin A alone, AA) and 1 µM DFP for 10 h. The neurons were subjected to immunofluorescent assay using anti-TOM20 (magenta) and anti-LAMP 2 (gray) antibodies. Scale bars, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. Yellow arrows indicate the co-localization between mitochondria and lysosomes.Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. H The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h. Then, the neurons were treated with 100 ng/mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti-MAP2 antibody. Scale bars, 10 µm. I, J The neurite length (I) and cell death (J) in more than 30 neurons were quantified from each group in H. The data from three independent experiments were presented as the means ± SD., *, p<0.05; ***, p<0.001, one-way ANOVA.
Figure 1(A-B) THP1 over-expressing IFITM3, IFITM3-Y20F, IFITM3-∆21 or control were pre-treated or not with Amphotericin B for 1 hour and then transduced with a VSV-gp (A) or Measles-gp (B) pseudotyped LV. Transduction efficiencies were evaluated at FACS five days later. Fold rescue of transduction over the DMSO control was calculated. Data are shown as mean ± SEM (n=5-8 biological replicates run in technical duplicate). P values are for one sample t test versus Oe-Luc DMSO =1, ** for p<0.01, *** for p<0.001.(E-F) THP1 over-expressing IFITM3, IFITM3 lysin-less mutant (4KR) (E), IFITM3 CIL-lysin mutant (3KR) (F) or control were transduced with VSV-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=9-6 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01. **** for p<0.0001 and ns for not significant.(G-H) THP1 over-expressing IFITM1, IFITM1-3KR (G), IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR (H) or control were transduced with Measles-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=4-9 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01.**** for p<0.0001.
Figure 2(A-B) Immunofluorescence images were performed using TCS SP5 Leica confocal microscope, 60x with oil on THP1 over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR or IFITM3-Y20F (A) (n=22 images acquired from 3 biological replicates) and primary monocytes derived macrophages (MDM) over-expressing IFITM3-WT, IFITM3-4KR, IFITM3-3KR (B) (n=12 images acquired from 3 independent donors). Co-localization (purple areas marked by white arrows) of IFITM3 (in red) with the lysosome associated membrane protein 1 (LAMP1) marker (in blue) was evaluated. Representative zoomed images are shown,(D) IFITM3-IFITM3 interactions (red dots) were evaluated by proximity ligation assay (PLA) in THP1 knock out for endogenous IFITM3 and expressing either HA-IFITM3 and His-IFITM3, HA-3KR and His-3KR or His-dimer mutant IFITM3 (D.M) and HA-dimer mutant. Images were acquired on TCS SP5 Leica confocal microscope 60x with oil. Representative images are shown (n=8 images of 4 biological replicates), scale bar 40μm.(F) IFITM3 dimerization was assessed by native PAGE followed by WB analysis in THP1. IFITM3 higher order protein complex bands are highlighted by the asterisk. IFITM3 dimers and higher molecular weight complexes were quantified over the normalizer using ImageJ (mean ± SEM, n=7 biological replicates). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.
Figure 3(B) K562 were prestimulated with IFNα and then transduced with VSV-gp pseudotyped LV. Transduction was evaluated five days after TD (mean ± SEM, n=4 biological replicates run in technical duplicate). Data are represented as Folds over the Oe-Luc control. p values are for one sample t test versus -IFNα = 1. ns for not significant.(C) Interferon stimulated genes (ISGs) mRNA expression were measured 24 hours post-IFNα stimulation by RT-qPCR in K562 (mean ± SEM, n=3 biological replicates run in technical duplicate).(E) K562 were pre-stimulated or not with IFNα for 24 hours and then inoculated with the infectious ZIKV (mean ± SEM, n=3 biological replicates run in technical triplicates). Viral supernatant was collected at different time points and titered on Vero E6 cells. p values are for Mann Whitney test. ** for p<0.01, *** for p<0.001.(F) K562 over-expressing IFITM1, IFITM1-3KR or control were transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc = 1. **** for p<0.0001.K562 over-expressing IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR or control were pre-exposed or not to Ampho B and then transduced with Measles-gp (G), BaEV-gp (H) pseudotyped LV. Transduction efficiencies were measured by flow cytometry five days after TD (mean ± SEM, n=5-4-4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1 or unpaired t test for Ampho B versus DMSO. * for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001.
Figure 5(A) Interaction between IFITM3 wild-type or 3KR and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay. Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild-type or IFITM3-3KR. Number of foci were counted on imageJ and normalized over the number of nuclei (mean ± SEM, n=5 biological replicates). White arrows indicate foci, scale bar 40μm.(B) Phosphatydinositol-3 phosphate (PIP3) levels were measured by immunofluorescence in THP1 over-expressing the His-IFITM3/HA-IFITM3 and His-3KR/HA-3KR. PIP3 was quantified via Imagej (mean ± SEM, n=5 biological replicates).(C) THP1 over-expressing IFITM3 or control were transduced with VSV-gp pseudotyped LV in presence or absence of Rapamycin (Rapa). Transduction efficiency was measured at FACS five days post-TD (mean ± SEM, n=6 biological replicates run in technical duplicate. p values are for one sample t test versus Oe-Luc DMSO=1 and Mann Whitney test for Rapa vs DMSO. ** for p<0.01, **** for p<0.0001.(D) THP1 over-expressing IFITM3-Y20F-3KR or control were exposed or not to Rapamycin and transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated by flow cytometry five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1. ** for p<0.0.(E) Hematopoietic stem and progenitor cells (HSPC) were transduced with VSV-gp or Measles-gp pseudotyped LV in presence or absence of Rapamycin. Transduction was evaluated at FACS five days later measuring the % of BFP expression (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05.
Figure 6Levels of phosphatidylinositol (PI) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562. The results are presented as log plots of the PI ratio in K562 over HSPC (D).(E) PIP3 levels were measured by immunofluorescence (scale bar 40μm) and quantified as integrated density with ImageJ software in THP1 and K562 over-expressing IFITM3 (mean ± SEM, n=3 biological replicates). p values are for Mann Whitney test, * for p<0.05.(F) Interaction between IFITM3 and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3. Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei (mean ± SEM, n=4 biological replicates run in technical duplicate), scale bar 40μm. p values are for Mann Whitney test, ** for p<0.01, *** for p<0.001.).PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 (G) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC (H) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM (I) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.
Figure 7(A) Calu3 over-expressing IFITM1, IFITM2, IFITM3, PM-mutant IFITM3 Y20A or control were transduced with SARS-CoV2 Spike-pseudotyped LV (mean ± SEM, n>6 biological replicates run in technical duplicate). Transduction efficiency was measured by FACS five days after transduction, p values are for one sample paired T test versus Oe-Luc Ctrl=1, **for p<0.01.(B-C) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3, IFITM3 Y20A or control were infected with SARS-CoV2. Viral titer was calculated by plaque assay in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test, * for p<0.05, *** for p<0.001.(G) Calu3 over-expressing IFITM1, IFITM3 or control were challenged to SARS-CoV2 Omicron pseudotyped LV. Transduction efficiency was evaluated 5 days post-transduction (mean ± SEM, n=7 biological replicates run in technical duplicate). p values are for one sample T test versus Oe-Luc Ctrl=1, **for p<0.01, ****for p<0.0001.(H-I) Calu3 cells over-expressing IFITM1, IFITM2, IFITM3 or control (H) or IFITM3, Y20A or control (I) were infected with Omicron at MOI 1. Viral supernatants were collected at different time points and tittered on Vero E6 cells. Data are presented as mean ± SEM (n=4-4 biological replicates run in technical triplicate). p values are for Mann Whitney test , **for p<0.01, ***for p<0.001, ****for p<0.0001.
Figure 8(A-B) VSV (A) or ZIKV (B) viral supernatants were collected one day post-infection in THP1 (A) or Calu3 (B) over-expressing IFITM3, IFITM3 lysin-less mutant (4KR), IFITM3 CIL-lysin mutant (3KR) or control and titered on VERO E6 cells. Data represent the mean ± SEM (n=5-3 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05, ** for p<0.01.(C-D) K562 were inoculated with the infectious VSV (C) or ZIKV (D) (mean ± SEM, n=3 biological replicates run in technical duplicate). Viral supernatant was collected at different time points and titered on Vero E6 cells.(I) IFITM3 and PIP3 level were measured by immunofluorescence (scale bar 100µm) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron (n=3 biological replicates). p values are for Mann Whitney test, ** for p<0.01. (J) PIP3 coupled to a specific PIP carrier (PIP3+Carr Ctrl) or Carr Ctrl were provided to Calu3 over-expressing IFITM3, control or KO for IFITM3 prior to infection with SARS-CoV2 Omicron. Infectious supernatants were collected after three days and tittered in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Wilcoxon signed ranked test, * for p<0.05. The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Mann Whitney test, * for p<0.05.
Figure 1Western blot analysis was performed using SETD1A fragment-expressing 293T cells. These constructs were used for the following rescue experiments. Representative images from 2 independent experiments are shown.Setd1afl/fl;CreER cells were transfected with SETD1A fragment and endogenous Setd1a was deleted by tamoxifen, then assayed for colony-forming potential. Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).qRT-PCR was performed at 3 days post-tamoxifen to analyze Fancd2 expression in human SETD1A-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).293T cells were transfected with SETD1A-GFP fusion constructs. Cell nucleus was visualized with DAPI. The percentage of GFP nuclear localization is indicated in each image. Asterisks in images indicate the cells with nuclear specific SETD1A-GFP signals. Scale bars: 10 µm. G−H. Mouse Setd1afl/fl;CreER MLL-AF9 leukemia cells were transfected with FO3-GFP fusion constructs. (G) Subcellular localization of GFP fusion constructs was visualized by immunofluorescence and nucleus was stained with DAPI. The percentage of GFP nuclear localization is indicated in each image. Scale bars: 10 µm. (H) FO3-GFP fusion expressing cells were treated with tamoxifen and colony assay with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).
Figure 2Relative expression of Mlh1 in SETD1A rescued MLL-AF9 leukemia cells. qRT-PCR with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test for (D)Percentage of GFP+ SETD1A mutant-expressing leukemia cells in PB was analyzed at 3 weeks post-transplant (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 One-way ANOVASurvival of recipient mice harboring SETD1A mutant-expressing leukemia cells after transplantation (TX) was plotted (n = 5 / group). Data information: **P ≤ 0.01, *P ≤ 0.05 Kaplan-Meier for (F)).
Figure 3SETD1A FLOS domain-binding proteins were identified by Co-IP MS. Full-length SETD1A and FO3 fragment were used as baits and overlapped proteins (red dots) were identified as SETD1A FLOS domain-binding proteins.293T cells were transfected with full-length SETD1A alanine mutants and protein extracts were used for the Co-IP experiments.F. MOLM-13 leukemia cells were examined by ChIP-seq analyses against HA-tagged SETD1A, BuGZ, BUB3, H3K4me3 and H3K4me1. Heatmaps of SETD1A(+)/BuGZ(+) regions (upper) and SETD1A(-)/BuGZ(+) regions (lower) are shown.
Figure 4Relative expression levels of Bugz were analyzed in Bugz shRNA-expressing mouse MLL-AF9 leukemia cells at day 3 post-treatment, respectively. Data from 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01 (Student's t-testSurvival of recipient mice harboring dox-inducible shRNA-expressing leukemia cells was plotted (n = 8-10 / group). Data information: **P ≤ 0.01 Kaplan-Meier for (H)).Images are shown of representative karyotypes in control, Bugz and Bub3 shRNA-expressing leukemia cells. Asterisks in images indicate the trisomy 15 in all cells analyzed. Red arrows indicate chromosomal aneuploidies. Representative images of sister-chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels.
Figure 5293T cells were transfected with SETD1A F2 alanine mutant constructs. Myc-DDK-tagged SETD1A was used for Co-IP.293T cells were transfected with BuGZ mutants. Myc-DDK-tagged BuGZ mutants illustrated on Figure S5B were used for Co-IP.293T cells were transfected with BUB3 mutants and protein extracts were used for the Co-IP experiments. Myc-DDK-tagged BUB3 mutants illustrated on Figure S5C were used.293T cells were transfected with Myc-DDK-tagged human BuGZ constructs and the protein extracts were used for a Co-IP experiment.
Figure 6293T cells were transfected with Myc-DDK-tagged BuGZ variant constructs and the protein extracts were used for the Co-IP experiment.293T cells were transfected with Myc-DDK-tagged BuGZ constructs and the protein extracts were used for a Co-IP experiment.293T cells were transfected with Myc-DDK-tagged wild-type BuGZ and the cells were treated with 1,6-hexanediol for 3 h. The protein extracts were used for the Co-IP experiments.
Figure 7SETD1A-GFP fusion-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, then assayed for colony-forming potential. The FUCCI reporter (mCherry-Cdt1(30-120)/Citrine-Geminin(1-110))-expressing cells were used as control. Data from 3 biological replicates are shown. Data are presented as mean ± SD.SGC-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, and RNA-seq analysis was performed with sorted cells. Data are shown as log2 fold change over tamoxifen non-treated GFP-expressing control.
Figure 1D. Quantity of Ki67+ cells in tumour and representative histology images analysed by Immunohistochemistry (IHC) that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10, Muc1 of colon tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.G. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein expression (right) in the tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.
Figure 2representative histological images (B) of wild-type control mice or BTNL2-KO mice treated with 2% DSS for 8 days were shown (n=15).E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β, IL-6 and IL-10 of colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.
Figure 3representative H&E images (H) of wild-type control mice or BTNL2-KO mice treated with Fc or mIL-22-Fc (ip. 5 μg/mouse) at day 0, 2, 4, 6 during 2% DSS treatment were shown (n=12). Data information Data are representative of three biological replicates H).
Figure 4B. IL-22 and IL-17a expression in lamina propria lymphocytes (LPLs) after cultured in plate-coated Fc, mBTNL2-Fc (30 μg/mL) with or without IL-23 (10 ng/mL) for 20 h was analyzed by ELISA. C. IL-22 production from cells of LPLs cultured in the presence of plate-coated Fc, mBTNL2-Fc with anti-control Ab or anti-BTNL2 mAb for 20 h was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shownD. CD4+ T cells, γδ T cells, ILC3s, CD8+ T cells sorted by FACS were cultured in the plate-coated Fc, mBTNL2-Fc with or without IL-23 (10 ng/mL) for 20 h, and IL-22 production was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shownF. IL-22 production was analyzed by Flow cytometry in LPLs of wild-type mice treated with Fc or mBTNL2-Fc (ip. 50 μg/mouse) on day 0, 2, 4, 6 during 2%DSS treatment. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=12 for the Fc group and n=14 for the mBTL2-Fc group (F). Pooled data from three biological replicates were shown F.G. IL-22, IL-17a, RORC and HIF-1α expression in splenocytes after cultured in plate-coated Fc or mBTNL2-Fc (30 μg/mL) for 20 h was analyzed by real-time PCR. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Data are representative of three biological replicates
Figure 5E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 from colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=15 Pooled data from three biological replicatesJ. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 of colonic tissue described in (F) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=12 Pooled data from three biological replicates
Figure 6Representative colonic tumour burden (B) that described in (A) were shown (n=12). Data information: Data are representative of three biological replicates (BF. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10 and Muc1 of colonic tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for F, Each dot represents one mouse, n=12. Pooled data from three biological replicatesG. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein (right) expression of tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for G, Each dot represents one mouse, n=12. Data are representative of three biological replicates left panel of G). Pooled data from three biological replicates are shown in right panel of GH, Quantity of Ki67+ cells in tumours and representative histology images that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for H). Each dot represents one mouse, n=12. Pooled data from three biological replicates are shown in H.