Igg2a

Immunostaining of duodenal muscularis externa was performed as previously described with minor modifications (65 (link), 66 (link)). Briefly, duodenal muscularis externa isolated from WT and IP6K2^−/− mice were blocked with 3% bovine serum albumin blocking solution containing the corresponding isotype control antibodies (IgG2a, R&D systems, Cat# MAB003; IgG2b, Dako) for 2 days after fixation with 4% paraformaldehyde overnight. The muscle layers were then washed with PBS containing 0.05% Triton X-100 and incubated with diluted primary antibodies against HuC/D (Thermo Fisher Scientific) or βIII-Tubulin (Biolegend, Cat# 801201, RRID: AB_2313773) for 3 days. After rinsing thrice with 0.05% Triton X-100 in PBS, the muscularis externa were then incubated with secondary goat anti-mouse IgG Alexa 594 (1:350, A-11020, Thermo Fisher Scientific) for 3 h at room temperature. The samples were then washed thrice with 0.05% Triton X-100 in PBS and mounted using antifading medium (12.5 mg/ml DABCO, 90% glycerol, pH 8.8 in PBS). Fluorescence images were obtained using an LSM 880 confocal microscope (Carl Zeiss).

Cells were stained with conjugated CD44-PE (G44-26, mouse, IgG2b, BD), CD24-APC (ML5, mouse, IgG2a, R&D), CD44v6-APC (2F10, mouse, IgG1, R&D) antibodies or matching isotype controls. Samples were acquired and analyzed by Accuri (BD Biosciences) flow cytometer. FACS sorting, using a FACS aria (BD Biosciences), was performed on cells stained with CD44-PE and CD24-APC, whose quality was monitored by flow cytometry. ALDH activity was measured by using ALDEFLUOR assay (Stem Cell technologies). All data were analyzed using FlowJo software (Tree Star). Sorted cells were collected and used for ΔNp63 and TAp63 mRNA expression.

Paired antibodies for immunoglobulin (Ig)E, IgG1 and IgG2a (R&D Systems, Abingdon, UK) were used to measure serum and lung antibody levels, as per the manufacturer’s instructions.

CMT93 cells in 100 µl PBS were injected subcutaneously (s.c.) into 6-week-old male CCR6^−/− mice (1×10⁶ cells/mouse), or CT26 cells in 100 µl PBS were injected s.c. into 7-week-old male BALB/c mice (1×10⁶ cells/mouse). Ten days after tumor cell inoculation, when the tumor cells formed solid tumor tissues, grafted CCR6^−/− mice or BALB/c mice were randomly divided into two groups for treatment with IgG control or anti-CCR6. Twenty micrograms of IgG_2A (R&D System, Clone #54447) dissolved in 100 µl PBS or 20 µg CCR6 (R&D System, Clone #140706) dissolved in 100 µl PBS was separately injected into the tumor sites every other day for 18 days. After 28 days, the mice were sacrificed, and the xenografts were removed, photographed and weighed. For Western blot analysis, polyclonal anti-mouse CCR6 (#ab78429, Abcam) was used as the primary antibody.

To analyze maturation and activation of DCs, cells were tested by flow cytometry for the cell surface expression of CD14 and CD83. Cells were harvested and washed with PBS. Next, 2 × 10⁶ cells were resuspended in 100 µl PBS and incubated for 1 hour with the PE-labeled (CD14, CD83 from Miltenyi Biotec) antibodies or appropriate PE-labeled isotype controls (IgG1, IgG2a from R&D Systems, MN, USA) on ice. Following wash steps with PBS served to remove unbound antibodies. Finally, DCs were analyzed with an Attune NxT Flow Cytometer (Invitrogen, CA, USA).