Fig 1.
SMG acinar cells of Bmi-1-/- mice were more strongly stained with AB at pH 2.5, as compared with those of WT mice.
Hematoxylin and eosin (top panels), AB at pH 1.0 (upper middle panels), and AB at pH 2.5 (lower middle panels) staining analysis of SMG sections from WT and Bmi-1-/- mice (12 weeks). Arrowheads indicate acinar cells. The structures of granular convoluted ducts are marked by asterisks and dashed lines. Scale bars, 100 μm (top, upper middle, lower middle panels) and 20 μm (magnified views in bottom panels).
Fig 2.
SMME analysis of SMG mucins of WT and Bmi-1-/- mice.
Comparison of SMG mucins of WT and Bmi-1-/- mice (5 weeks) separated by SMME. All bands are from different individuals. Reference mucin: porcine gastric mucin (PGM). The SMME membrane was stained with AB (pH 4.0).
Table 1.
Signals assigned to O-glycans in the MS of permethylated alditols obtained from excised bands.
Fig 3.
Increased mucin levels in the SMG tissues of Bmi-1-/- mice.
(A) Quantitative analysis of mucins in the SMG tissues of WT and Bmi-1-/- mice. The bars represent the average amount of mucin in the SMG tissues of six individual WT and Bmi-1-/- mice (Fig 2, n = 2 and S1A Fig, n = 4). Data are presented as mean ± standard error (SE). *P < 0.05. (B) The expression of mucin genes in the SMG of WT and Bmi-1-/- mice. The mRNA levels of each mucin gene relative to gapdh in SMG of WT and Bmi-1-/- mice (4–6 weeks). Data are presented as mean ± SE; n = 5 or 6. * P < 0.0028. N.D; not detected (C) Antibody staining of SMME membranes from SMG fractions of WT and Bmi-1-/- mice with antibodies against Mucin 1, Muc 10/Prol 1, Muc 19, and Smgc. SMG samples and SMME experimental conditions are the same as in Fig 2. (D) Immunohistochemical analysis of Smgc (red) expression of SMGs from WT and Bmi-1-/- mice (12 weeks). DNA was stained with 4’, 6-diamidino-2-phenylindole (blue). Arrowheads indicate positive acinar cells. Ductal structures are marked by dashed lines and asterisks. The granular convoluted ducts were stained non-specifically positive. Scale bars, 20 μm (E) Quantitative analysis of areas stained by immunohistochemistry in (D). Data represent means ± SE of three independent images for each SMG; n = 3. * P < 0.05.
Fig 4.
Glycan profiles of SMG mucins from WT and Bmi-1-/- mice.
Average glycan profiles in MALDI MS of SMG mucin bands of six individual WT and Bmi-1-/- mice (Fig 2, n = 2 and S1A Fig, n = 4). Bars represent signal intensities of permethylated glycans. Data are presented as mean ± SE. Statistical significance was defined at * P < 0.0083 following Bonferroni correction.
Fig 5.
Structural analysis of mucin major glycan observed in SMG of Bmi-1-/- mice.
(A) MS/MS spectrum of the signal at m/z 1140. The signal 3 in S1B Fig was used as the precursor ion. Glycan structures are depicted using Consortium for Functional Glycomics (CFG) graphical notation for glycans. (http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf). (B) Linkage analysis of sialic acids of the glycan by SALSA. Enlarged MS spectra of the modified sialoglycans are shown. The glycan from fetuin was used as a reference for [NeuAc(ɑ2–3)]Galβ1-3GalNAc. Left: (Hex)(HexNAc)(NeuAc), right: (Hex)(HexNAc)2(NeuAc). Indicated m/z values are observed values. The peak at m/z 1338.69 is an artifact signal. (C) The expected chemical structures and their predicted m/z values of the modified sialoglycans by SALSA. “WR” at the reducing end indicates tryptophanyl arginine. (D) The mRNA levels of each glucosaminyltransferase and sialyltransferase gene relative to gapdh in SMG tissues of WT and Bmi-1-/- mice (4–6 weeks). Data are presented as mean ± SE; n = 5 or 6. Statistical significance was defined at *P < 0.0167 for glucosaminyltransferase and * P < 0.0125 for sialyltransferase following Bonferroni correction. (E) Quantification of neuraminidase activity in WT and Bmi-1-/- mice (7–11 weeks) as mU per mg protein. Data are presented as mean ± SE; n = 5 or 6.
Fig 6.
ChIP analysis using SMGs of WT and Bmi-1-/- mice.
(A) Schema of the mouse Smgc locus and ChIP analysis used to detect binding of Bmi-1 to the Smgc locus in the SMG of WT and Bmi-1-/- mice. Amplified regions are shown as red bars. Data are presented as means ± SE; n = 4 or 6. * P < 0.025 (B) Schema of the mouse Gcnt3 locus and ChIP analysis used to detect binding of Bmi-1 to the Gcnt3 locus in the SMG of WT and Bmi-1-/- mice. Amplified regions are shown as red bars. Data are presented as means ± SE; n = 4 or 6. * P < 0.025 (C) A model demonstrating how Bmi-1 may control mucin synthesis.