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1.
Figure 7

Figure 7. Naphthalene injury selectively ablates mucin-expressing cells. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

WT mice sensitized with OVA (controls), and Munc13-2-deficient non-OVA-treated mice were subjected to naphthalene injury, and the airways examined 2 days later for AB/PAS+ staining. Images shown are representative of 3 mice for each condition. Scale bars, 15 μm.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
2.
Figure 9

Figure 9. Mucin secretory defects in Munc13-2-deficient mice are widespread. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

Tissues of several mucin-secreting organs in WT Munc13-2-deficient mice, as indicated, were examined for AB/PAS+ staining. Images shown are representative of 2–3 mice each. For salivary gland: SL, sublingual; SM, submandibular. Nasal septum, scale bars, 10 μm; other scale bars, 50 μm.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
3.
Figure 3

Figure 3. Mucin stores and secretion from OVA-sensitized, metaplastic mouse airways. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

A and B, AB/PAS-stained lung sections before (A; bronchus) and after (B; trachea) tracheal perfusion and agonist challenge (scale bars in A = 30 μm, in B = 10 μm). C, tracheal mucin secretion, time course (left), integrated response (right). Experiment per Fig. 2C (n = 5).

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
4.
Figure 8

Figure 8. CCSP lipoprotein secretion is affected in Munc13-2-deficient mice. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

CCSP mRNA (A; NS, n = 4) and protein (B; n = 3) levels in whole lung extracts were assessed by qRT-PCR and Western blotting, respectively, in non-OVA-sensitized WT and Munc13-2-deficient mice. A sample blot is shown above the graph in B;*P < 0.05, Student's t test. C, bronchial epithelium was assessed for CCSP immunostaining in the tracheas of the same mice. Images shown are representative. Scale bars, 10 μm.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
5.
Figure 1

Figure 1. Munc13 domain maps and mRNA expression in mouse tissues. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

Left, domain maps representing Munc13. The N-terminal splice variants for Munc13-2 are indicated in green (b, brain) and blue (ub, ubiquitous). Note the lack of an N-terminal C2A domain in some genes, including the brain splice-variant of Munc13-2. MUN, Munc13 homology domain. Right, conventional RT-PCR was used to identify the Munc13 isoforms expressed in tracheal epithelium (luminal extraction), the residual tracheal tissue, and nasopharynx. Thymus, thyroid and cerebellum were used as a source of positive control RNA for all Munc13 isoforms, and cerebrum (inset, upper right), additionally, was used for Munc13-1.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
6.
Figure 5

Figure 5. Muc5b glycoprotein expression in mouse lung. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

The presence of Muc5b was assessed in whole lung extracts by agarose Western blotting. The arrow, left, indicates the Muc5b glycosylated monomer band in submucosal gland extract (S. Gland), which served as a positive control. ‘Immersion extraction’ (left-hand blots) is lungs immersed in GuCl buffer. ‘Luminal Extraction’ (right-hand blot) is GuCl buffer injected into airways prior to immersion in GuCl buffer. U, unreduced; R, reduced (DTT). Samples from non-OVA-sensitized and OVA-sensitized animals are indicated at the bottom. The bands at positions in the right-most blot indicated by * are interpreted as the non-glycosylated, Muc5b dimer (unreduced) and monomer (reduced), precursors of the mature mucin. Blots are representative of 3 or more prepared for each condition.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
7.
Figure 2

Figure 2. Mucin stores and secretion from control, non-OVA sensitized Munc13-2-deficient mouse airways. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

A and B. AB/PAS-stained lung sections before (A) and after (B) tracheal perfusion and agonist challenge (T, trachea; B, bronchus; scale bars in A = 30 μm, in B = 10 μm) C, tracheal mucin secretion, time course (left) and integrated response (right). Isolated, perfused trachea were challenged with purinergic agonist (ATPγS, 100 μm) during the period indicated by the bar. Perfusates were collected at 5 min intervals and sampled for contained mucins by ELISA. Data (mean ±s.e.m., n = 4) are normalized to the mean baseline period (30 min) preceding the agonist challenge. Integrated response (right). Supra-baseline agonist-stimulated data for WT and Munc13-2-deficient mice were summed, after subtracting the mean baseline, to estimate the integrated mucin secretory response; *P < 0.05.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
8.
Figure 4

Figure 4. Airway epithelium of Munc13-2-deficient mice is not metaplastic. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

WT and Munc13-2-deficient mice, without and with OVA treatment as indicated, were assessed for indications of mucous metaplasia. A, BALF inflammatory cell profile. Mouse BAL fluids were assessed for total cells (inset) and for relative cell composition (bar codes in inset). Data presented as the mean +s.e.m. (n = 4–6). Note the effects of OVA treatment, but not of Munc13-2 genetic status. B, mucin gene expression in mouse lung. Whole lung extracts were assessed for relative Muc5AC and Muc5B mRNA expression levels by qRT-PCR. Data are normalized to the WT, control (OVA–) mice, and presented as the mean +s.e.m. (n = 3). C, electron micrographs of bronchial Clara cell apical pole region. G, secretory granule; M, mitochondria; rER, rough ER; scale, 0.5 μm. Micrographs shown are representative of those examined from 3 different mice for each condition.

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.
9.
Figure 6

Figure 6. Mucin and CCSP expression in mouse airways. From: Munc13-2−/− baseline secretion defect reveals source of oligomeric mucins in mouse airways.

A, Muc5B and CCSP co-localization. Sections of bronchial epithelium from WT and Munc13-2-deficient mice, without or with OVA sensitization as indicated, were stained for Muc5B (green) and CCSP (red), and examined by confocal microscopy. The images shown, representative of sections from 3 mice each, are overlays of green, red and DIC images, acquired simultaneously. Note that Muc5b staining in the WT control mouse appears sparse with use of the FITC conjugated secondary antibody (scale bars, 10 μm). B, Muc5b distribution in mouse lung. A more sensitive IHC staining technique using a peroxidase conjugated secondary antibody revealed a broad distribution of Muc5b in the airways. The background image shows a tissue section taken through the entire axial bronchus of a 90-day-old wildtype C57BL/6J mouse. Note the dark staining of the airway epithelium deep into the lung. Insets left show that Muc5b is expressed in the axial bronchus (a) and the proximal portion of minor daughter branches (b), but it is absent in the terminal bronchioles (c). a′, demonstration of immunolabel specificity, with normal rabbit serum used in place of anti-Muc5b. The image shown in a′ is taken from the same portion of the axial bronchus in a neighbouring section as shown in a. Scales bars, 250 μm (low magnification) and 25 μm (high magnification).

Yunxiang Zhu, et al. J Physiol. 2008 April 1;586(Pt 7):1977-1992.

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