Figure 1.
Label retaining assay in murine salivary glands.
A) Label Retaining Assay. At 10 days after birth (P10), FVB mice were pulsed with 4 doses of EdU (100 mg/kg) or BrdU (30 mg/kg) 12 hours apart. Tissue was collected from 10-week old animals. B) LRCs from 3 mice were manually quantified per salivary gland compartment (acinar and ductal). Data are expressed as percentage from the total of cells in each individual compartment for both parotid and submandibular glands. C) Comparison of labeling efficiency between EdU and BrdU. Data are shown as percentage of LRCs per individual compartment. A 2-sided unpaired T-test was performed for analysis (n = 5 per group). D–E) Representative images of parotid and submandibular glands of BrdU-pulsed animals. LRCs in the acinar compartment (a) are shown with white arrowheads. LRCs in ductal compartment (d) are pointed with black arrows. Example of ductal compartment is delineated by dashed line and pointed with black arrowhead F–K) Representative fluorescent images of salivary glands from EdU-pulsed animals. EdU LRCs are shown in green and DAPI in blue.
Figure 2.
Molecular markers in salivary gland LRCs.
Representative images of parotid and submandibular glands of 10-day old and 10-week old animals stained for Keratin 14 (A–D), Keratin 5 (E–L), Smooth Muscle alpha Actin (M–P). Q–T) Fluorescence in Situ Hybridization for kit mRNA. EdU LRCs are shown in green, DAPI in blue, and all other markers in red. White arrowheads point at co-localization of each marker with the LRCs in the acinar compartment. Yellow arrowheads point at co-localization of each marker with the LRCs in the ductal compartment. Full size images of every panel are shown in Figure S2.
Figure 3.
Sphere Assay with murine salivary glands.
Representative microscope images of spheres grown from submandibular (A) and Parotid (B) glands from 10-week old mice.
Figure 4.
Proliferative potential of LRCs.
A–C) Dual staining for EdU and Ki67 in parotid-derived spheres from 10-week old mice. Co-localization is shown with white arrowheads. D–F) Secondary spheres stained for EdU. G–I) Dual staining for EdU and Ki67 in parotid gland tissue sections of 10-week old mice. Zoomed region in yellow square is shown in I’. In both panels, Ki67 is shown in red, EdU in green, and DAPI in blue.
Figure 5.
Differentiation of Salivary gland Spheres.
A–B) Amylase staining (red) of parotid-derived spheres at days 2–3 in culture. C–D) Confocal images at Z = 0.5 um and Z = 6 um of double staining for amylase (red) and EdU (green) at day 4. Areas in yellow dashed squares are shown in C’ and D’. White arrow points at an amylase-positive cell with traces of EdU. Glands were obtained from mice at 10 weeks of age. E–G) Double immunofluorescence staining for Amylase (red) and EdU (Green) of parotid gland of 10-week old mice. White arrowhead points at LRCs in the acinar compartment; yellow arrowhead points at LRCs in ductal structures.
Figure 6.
Effect of radiation on salivary gland LRCs.
A) Experimental setup. A single 5 Gy dose of radiation was given at week 4 to EdU-pulsed animals (n = 7). Tissue was collected at week 10. Representative images of EdU staining of parotid (B) and submandibular (C) glands are shown for irradiated animals and untreated controls. EdU positive cells were quantified manually per individual compartment for both treatments and expressed as percentage of LRCs per compartment for both glands (D–E). P values were obtained with 2-sided unpaired t-test per compartment (n = 7 for irradiated groups, n = 12 for parotid untreated control group and n = 6 for submandibular untreated control group). F) Experimental setup. 5 Gy dose of radiation treatment was given 24-hours prior tissue collection to EdU-pulsed animals (n = 3). G–J) Immunofluorescence staining of Activated Caspase-3 (red) and EdU (green) in parotid and submandibular glands. No co-localization is observed.