EMBO/soda-nel huggingface dataset:
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 1

Figure 1. A homozygous M1AP splicing mutation was identified in a consanguineous Pakistani family with severe oligozoospermia and meiotic metaphase I arrest

The family pedigree. Arrows represent the individuals for whom whole-exome sequencing was performed. Squares represent males, circles represent females, diamonds indicate offspring, and the slash symbols denote deceased family members. Solid squares indicate male patients suffering from severe oligozoospermia. Double horizontal lines indicate consanguineous marriage.

Representative images of hematoxylin and eosin-stained testicular sections from the patient (V:3) and a man diagnosed with obstructive azoospermia, serving as the control. The magnified view of the boxed area is shown in the lower left corner of the image from the patient. The blue arrow indicates unaligned chromosomes in the representative metaphase cells. Scale bars, 50 μm.

Immunofluorescence staining of testicular sections from control and patient V:3 with antibodies against H3S10p (red), a marker of metaphase cells. The nuclei were stained with Hoechst 33342 (blue). The magnified view of the red box is shown in the lower left corner of the image from the patient. Scale bars, 50 μm.

Sanger sequencing chromatograms showing the homozygous M1AP splicing mutation in patients and heterozygous mutation in their fertile brother. The arrowhead indicates the position of the mutation.

RT-PCR products of the pET01 empty vector and vectors expressing the wild-type or c.1074+2T>C M1AP mini gene (containing genomic sequences spanning intron 5-6 and intron 8-9) in HEK-293T cells. The splicing products of wild-type and mutant mini genes were detected by Sanger sequencing. The wild-type transcript is spliced canonically, while the mutant transcript is aberrantly spliced, leading to the skipping of exon 7. M, marker.

Schematic representation of the aberrant splicing and the predicted protein truncation caused by the M1AP mutation. Vertical solid bars indicate exons of the human M1AP gene. The M1AP mutation (c.1074+2T>C) is located in intron 7-8 near exon 7 (Ensembl transcript ID ENST00000421985.2) and is indicated by the arrowhead. Green lines represent canonical splicing, and red lines represent aberrant splicing caused by the M1AP mutation.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 2

Figure 2. M1AP forms discrete foci on the chromosome axes in spermatocytes

Western blot analysis of M1AP expression in various tissues from adult wild-type (WT) mice. GAPDH served as the loading control.

Western blot analysis of M1AP expression in testes from mice at different days postpartum (dpp). GAPDH served as the loading control.

Immunofluorescence staining of M1AP (green) and SYCP3 (red) on spermatocyte spreads of WT or Spo11-/- mice. Scale bar, 10 μm.

Quantification of M1AP foci in WT spermatocytes at the indicated stages. L, leptotene; E/MZ, early/mid zygotene; LZ, late zygotene; EP, early pachytene; M/LP, mid/late pachytene; Z-like, zygotene-like. Data are presented as the mean ± SEM. n shows the number of spermatocytes analyzed from three biological replicates.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 3

Figure 3. Meiotic arrest at metaphase I caused by decreased crossovers in M1apKI/KI male mice

Testicular histology from 8-week-old control and M1apKI/KI mice. The magnified view of the boxed area is shown in the lower left corner of the image of M1apKI/KI mice. The blue arrow indicates unaligned chromosomes in the representative metaphase cells. Scale bars, 50 μm.

Meiotic metaphase I (MMI) spermatocytes stained with Giemsa. The arrows indicate univalent chromosomes. Scale bars, 10 μm.

Number of bivalents per nucleus.

Frequencies of nuclei with univalents.

Frequencies of nuclei with only XY univalents, only autosome univalents, or both XY and autosome univalents.

Representative spread spermatocytes stained for SYCP3 (green) and MLH1 (red). Scale bars, 10 μm.

Number of MLH1 foci per nucleus.

Frequencies of autosomal chromosomes with 0, 1, and 2 or 3 MLH1 foci.

Frequencies of nuclei with an MLH1 focus at the pseudoautosomal region (PAR) of XY chromosomes.

Representative spread spermatocytes stained for the lateral element (SYCP3, green) and the central element (SIX6OS1, red) in early and mid/late pachynema. Miniaturized H1t staining (white) images are shown in the left lower corner of the overlay images. The arrows indicate the untouching XY chromosomes. Scale bars, 10 μm.

Quantification of nuclei with XY untouching in early and mid/late pachynema.

Data information: (C-E, G, and H-K), Data are presented as the mean ± SEM. n, the number of cells scored from three biological replicates. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001; two-tailed Student's t-test.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 4

Figure 4. Decreased recombination intermediates in M1apKI/KI male mice

Immunofluorescence staining with antibodies against SYCP3 (red) and MSH4 (green) on spermatocyte spreads. Scale bars, 10 μm.

The mean number of MSH4 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.

Immunofluorescence staining with antibodies against SYCP3 (red) and RAD51 (green) on spermatocyte spreads. Scale bars, 10 μm.

The mean number of RAD51 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.

Immunofluorescence staining with antibodies against SYCP3 (red) and DMC1 (green) on spermatocyte spreads. Scale bars, 10 μm.

The mean number of DMC1 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.

Data information: (B, D and F), Data are presented as the mean ± SEM. n, the number of cells scored from at least three biological replicates. **** P<0.0001; NS, not significant; two-tailed Student's t-test.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 5

Figure 5. M1AP shows cytoplasmic localization in fetal oocytes, and is dispensable for crossover formation in female mice

Western blot analysis of M1AP and TEX11 expression in fetal ovaries (16.5 days postcoitum (dpc)) and testes (14 dpp) from WT mice. GAPDH served as the loading control.

Immunofluorescence staining of M1AP (green) and SYCP3 (red) on meiotic spreads of fetal ovaries (16.5 dpc) and adult testes from WT mice. Scale bars, 10 μm.

Confocal imaging of zygotene/pachytene meiocytes on cell smears of fetal ovaries (16.5 dpc) and adult testes from WT mice after immunofluorescence staining of M1AP (green) and SYCP3 (red). Scale bars, 10 μm.

Immunofluorescence staining with antibodies against SYCP3 (red) and MLH3 (green) on oocyte spreads from control and M1apKI/KI embryos (18.5 dpc). Scale bars, 10 μm.

Number of MLH3 foci per cell in control and M1apKI/KI oocytes.

Representative haemotoxylin-stained ovarian sections of 5-dpp-old control and M1apKI/KI mice. Scale bars, 50 μm.

Follicle counts and total oocyte counts per ovary at 5 dpp.

Data information: (E), Data are presented as the mean ± SEM. n shows the number of cells scored from two biological replicates. (G), Data are presented as the mean ± SEM of three biological replicates. NS, not significant; two-tailed Student's t-test.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 6

Fig. 6. M1AP interacts with the ZZS complex.

Identification of proteins interacting with M1AP by coimmunoprecipitation and mass spectrometry (co-IP/MS). The top three candidates on the ranking list sorted by the number of unique peptides are shown. PSMs, peptide-spectrum matches.

Co-IP using anti-M1AP antibody with whole-testis lysates of WT mice and M1ap-/- mice, followed by western blot analyses of M1AP, SHOC1, and TEX11.

Co-IP detecting the interaction between M1AP (fused with an N-terminal MYC tag) and each of the ZZS complex components (fused with an N-terminal GFP tag) exogenously expressed in HEK-293T cells.
M1AP interacts with the mammalian ZZS complex and promotes male meiotic recombination (2022)
Figure 7

Fig. 7. M1AP colocalizes with TEX11 and facilitates TEX11 recruitment at the recombination intermediates.

Immunofluorescence staining of M1AP (green) and TEX11 (red) on the spermatocyte spreads of WT mice.

Ratios of M1AP-TEX11 co-localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.

Immunofluorescence staining of M1AP (green) and DMC1 (red) on the spermatocyte spreads of WT mice.

Ratios of M1AP-DMC1 co-localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.

Immunofluorescence staining of M1AP (green) and RPA2 (red) on the spermatocyte spreads of WT mice.

Ratios of M1AP-RPA2 co-localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.

Immunofluorescence staining of M1AP (green) and SYCP3 (red) on spermatocyte spreads of control, Dmc1-/- or Spo16-/- mice.

Immunofluorescence staining of TEX11 (green) and SYCP3 (red) on the spermatocyte spreads of control and M1apKI/KI testes.

The mean number of TEX11 foci per cell in control and M1apKI/KI spermatocytes.

Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes.

Schematic diagram showing a proposed model in which M1AP acts as a partner of the ZZS complex in meiotic recombination in mouse meiotic prophase I. M1AP is recruited by SPO16 and facilitates TEX11 recruitment to stabilize recombination intermediates.

Data information: (A, C, E, G, and H), Scale bars, 10 μm. (B, D, F, I, and J), Data are presented as the mean ± SEM. n shows the number of cells scored from at least two biological replicates. (I and J), **** P<0.0001; NS, not significant; two-tailed Student's t-test.

Expanded View Figure Legends
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 2

Figure 2 - NLRP3 inflammasome activation is impaired in Lamtor1-deficient macrophages

A, B Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs (A) or WT and Lamtor1 KO THP-1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL, for 4 h (BMDM) or 2 h (THP-1)] stimulation. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 3 biological replicates.

C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation. Data are shown as the means ± SEM. * indicates P < 0.05 by Student's t-test, n = 3 biological replicates.

D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation and the percentage of propidium iodide positive cells were counted by flow-cytometry. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 3 biological replicates.

E Effects of Lamtor1 deficiency on IL-1β dynamics. Western blot shows IL-1β and β-actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.

F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h). Data are shown as means ± SEM. n.s. indicates P > 0.05 by one-way ANOVA, n = 3 biological replicates.

G Confirmation of the reconstitution of the active form Caspase-1 (p10 and p20) or GSDMD (GSDMD-NT).

H Effects of Lamtor1 deficiency on the priming phase. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with treated with LPS (200 ng/mL, 4 h). Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 3 biological replicates.

I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h). Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test and n.s. indicates P > 0.05 by one-way ANOVA, n = 3 biological replicates.
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 3

Figure 3 - mTORC1 activity is not involved in the inflammasome activation phase

A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1. Scale bars = 5 μm.

B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as the means ± SEM. * indicates P < 0.05 and *** indicates P < 0.001 by one-way ANOVA, n = 3 biological replicates.

C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).

D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Data are shown as the means ± SEM. ** indicates P < 0.01 and n.s indicates no significant difference by one-way ANOVA, n = 3 biological replicates.

E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h). Data are shown as the means ± SEM. n.s indicates no significant difference by one-way ANOVA, n = 3 biological replicates.

F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel). Data are shown as the means ± SEM. n.s indicates no significant difference by one-way ANOVA, n = 3 biological replicates.
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 4

Figure 4 - Interaction between Lamtor1 and HDAC6 is necessary for inflammasome activation

A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM). Data are shown as the means ± SEM. *** indicates P < 0.001 by one-way ANOVA, n = 3 biological replicates.

B-F Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids (B, C), Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D) Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells (E, F). Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids. Data are shown as means ± SEM. * indicates P < 0.05 and ** indicates P < 0.01 by one-way ANOVA, n = 3 biological replicates.

G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test. The data are representative of three experiments that showed consistent results. Scale bars, 10 μm.

H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega). Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 3 biological replicates.

I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.

J Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as the means ± SEM. *** indicates P < 0.001 and n.s. indicates no significant difference by Student's t-test, n = 3 biological replicates.
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 5

Figure 5 - Lamtor1 interacts with NLRP3 in the presence of HDAC6

A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.

B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test. The data are representative of three experiments that showed consistent results. Scale bars = 5 μm.

C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.

E, F Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.

G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids. Data are shown as the means ± SEM. ** indicates P < 0.01 by Student's t-test, n = 3 biological replicates.
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 6

Figure 6 - ASC speck originates near the lysosomes

A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s). Scale bars = 1 μm.

C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm. Data are shown as the means ± SEM. *** indicates P < 0.001 and n.s indicates no significant difference by one-way ANOVA, n=4 independent experiments.
The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 7

Figure 7 - DL-all-rac-α-tocopherol inhibits Lamtor1 and HDAC6 interactions and suppresses inflammasome activation

A Schematic showing the use of a NanoBRET assay to search for inhibitors of Lamtor1-HDAC6 interactions in a library of natural compounds.

B-F Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B) or D -α-tocopherol (E) (10 μM, overnight). Data are shown as means ± SEM. ** indicates P < 0.01 by Student's t-test, n = 3 biological replicates. IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) or D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as means ± SEM. * indicates P < 0.05 by one-way ANOVA, n = 3 biological replicates. D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight). Data are shown as means ± SEM. n.s indicates no significant difference by one-way ANOVA, n = 3 biological replicates.

G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints. Data are shown as means ± SEM. ** indicates P < 0.01 by Student's t-test, n = 6 mice. Scale bars = 30 μm.

H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS. Data are shown as means ± SEM. * indicates P < 0.05 by Student's t-test, n = 4 mice (control) and n = 5 mice (DL-all-rac-α-tocopherol).