EMBO/SourceData huggingface dataset:
Figure 2A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)B: Representative images of WT, Cnot6l-/- and Btg4-/- oocytes showing PB1 emission at 16 h after culture. Arrows indicate PB1. Scale bar, 100 μmC: Representative images of chromosome spreads made from WT and Cnot6l-/- oocytes after 16 h of in vitro maturation culture. Immunofluorescent staining of topoisomerase II (TOP2) and the centromere antigen CREST were performed to indicate chromosome arms and centromeres, respectively. Numbers of paired sister chromatids are indicated. Scale bar, 5 μmD: Percentage (%) of aneuploidy among in vitro cultured WT and Cnot6l-/- oocytes that have released PB1s. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicateE: Rates of GVBD, PB1 emission, and normal spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes cultured in vitro. Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicateF: Confocal microscopy results showing spindle assembly in WT, Cnot6l-/- and Btg4-/- oocytes at metaphase I (MI) and metaphase II (MII). Scale bar, 20 μmG: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm
Figure 3A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μmB: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n)C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μmD: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μmE: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)
Figure 4A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicatesB: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signifC: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student"s t test. n = 3 biological repliD: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectivelyE: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the textF: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples
Figure 6Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins from 50 (A oocytes are loaded in each lane. DDB1 (A is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shownWestern blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins fro 100 (B) oocytes are loaded in each lane α-tubulin (B) is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shownImmunofluorescenc of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001E: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicatesF: Scatter plot comparing polysome-bound transcripts between WT and Cnot6l-/- oocytes at GV, MI and MII stages, respectively. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectivelyI: Quantitative RT-PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l-/- oocytes at GV, MI and MII stages. Error bars, standard deviations (n = 3 biologica repeats)
Figure 7A: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytesB: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stageC: Co-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shownD: RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student"s t
Figure 8fluorescence microscopy results (B) showing the translation activities of the Cnot6l 3"-UTR (WT and CPE mutated) in GV- (maintained by 2 μM milrinone) or MII- (released from milrinone) arrested oocytes. GFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive control. Scale bGFP signal indicated translational activation of Cnot6l 3"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive co Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicatefluorescence microscopy results (E) showing the expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U0126 treatment (20 μM). Scale bar, 1e expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U01 F: Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated (n)G: Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126. Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L, CNOT7 and/or BTG4 and are released from milrinone at 12 h after microinjection. Then the oocytes were further culture for 24 h with or without adding U0126 to the medium. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate
Figure 1WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F) (E Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) (F, Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E) from three independent experiments are shown as fold change (F Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for GSDMD or LC-3 G). Representative immunoblots showing GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). H, Quantification of band intensities for GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change , H) Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.
Figure 3Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable. Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test.
Figure 4WT or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B were infected with flagellin-expressing STm and fixed A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm and fixed C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E were infected with flagellin-expressing STm and fixed (E, Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.SLC15A4feeble BMDCs transduced with SLC15A4-GFP F, were infected with flagellin-expressing STm and fixed Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.SLC15A4feeble BMDCs transduced with SLC15A4-GFP G) were infected with flagellin-expressing STm and lysed (G) 1 h after infection. G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands.
Figure 5WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) were infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (E, Cell pellets were probed for caspase-1, GSDMD, actin (E, Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.WT or SLC15A4feeble BMDCs transduced with non-target control, ATG5 or ATG7 shRNAs G) G). Cell pellets were probed for caspase-1, GSDMD, actin , ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) H, I) and mcherry-LC3 (H, I), (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.
Figure 1A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01.E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.
Figure 2A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice.C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice.G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.
Figure 3A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.D. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.E. Centrosome (PCNT) separation was scored per spermatocyte. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two-tailed Student's t-test comparing the percent of unseparated centrosomes in each mouse mutant to control. n.s. (not significant), ***P<0.001 .
Figure 4A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.B. The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates (n = 30 total cells) in control, Aurka cKO, Plk1 cHet, and Plk1 cKO spermatocytes at the pachytene-stage of meiotic prophase and metaphase I.C. Quantification of gamma-tubulin positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 137, 169, 198, and 198, respectively.D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.Western blots of gamma-tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.
Figure 5A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.
Figure 6A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications
Figure 1(A, B) B-ALL progression measured as (A) absolute number or (B) percentage of OFP+ cells in peripheral blood of transplanted mice (mean ± SD, each dot represents an individual mouse, CTRL = 6 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA; mice in blue and yellow (B) were also analyzed by single-cell RNA sequencing(F, G) Percentage of CD4+ and CD8+ cells within OFP- BM cells (F) and maturation state (G) of CD8+ lymphocytes (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) in the BM immune infiltrate at 12- and 17-days after B-ALL (mean ± SD, each dot represents an individual mouse; ** p = 0.005, **** p ≤ 0.001, Two-Way ANOVA).
Figure 2(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice).(C) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA).(E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse).