Ab7800

For histological analysis, the bladders of xenograft mice were fixed with 4% paraformaldehyde for 24 h. After cryoprotection in 30% sucrose for 24 h, each bladder was sliced into 20 μm sections using a cryostat (Leica, Lussloch, Germany) and stained with hematoxylin and eosin (H&E). For immunofluorescence (IF) staining, bladders were stained with antibodies against ID2 (NBP-88630; NovusBio), TFCP2L1 (OAAB09732; Aviva Systems Biology), CDK1 (ab131450; Abcam, Cambridge, MA, USA), CD44 (ab78960; Abcam), and cytokeratin 14 (KRT14; ab7800; Abcam). Alexa Fluor 488-conjugated (A11001 and A11008) anti-mouse and anti-rabbit antibodies or an Alexa Fluor 546-conjugated anti-rabbit antibody (A11010) were used as secondary antibodies (Molecular Probes). Nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI; D9542; Sigma–Aldrich). Three representative areas per slide were randomly selected for each animal. The stained samples were imaged using an inverted fluorescence microscope (EVOS® FL Color Imaging System, Life Technologies).

Pre-designed small interfering RNA (siRNA) directed against human TCHHL1 (s43057 and s43059) and Kruppel-like factor 4 (KLF4; s17795) and negative control siRNA were purchased from Life Technologies (Carlsbad, CA). An epidermal growth factor receptor (EGFR) inhibitor (AG 1478) was purchased from Abcam (Cambridge, UK). An antibody against an oligopeptide (HPQRERLVLQREASTTKQ) corresponding to part of the C-terminal region of TCHHL1 was previously generated^{12 (link)}. Antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2; #4695), phospho-ERK1/2 (#4370), stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK; #9252), phospho-SAPK/JNK (#4668), p38 mitogen-activated protein kinase (p38 MAPK; #8690), phospho-p38 MAPK (#4511), v-akt murine thymoma viral oncogene homolog (AKT; #9272), phospho-AKT (#9271), signal transducers and activator of transcription 3 (STAT3; #9132), phospho-STAT3 (#9145), EGFR(#4267), phospho-EGFR (#4407) and β-Actin (#4967) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Antibodies against cytokeratin-10 (M7002), Ki67 (M7240), and p53 (M7001) were purchased from DAKO (Carpinteria, CA). Antibodies against human cytokeratin-14 (ab7800) and filaggrin (ab218863), and antibodies against transglutaminase1 (PA5-59088) were purchased from Abcam and Thermo Fisher Scientific Inc. (Waltham, MA), respectively.

Sections were deparaffinized in Histoclear and rehydrated through a series of ethanols. Antigen retrieval was performed in citrate buffer pH 6 (Sigma‐Aldrich) at 95 °C for 20 min, followed by blocking and permeabilization for 1 h with 20% neonatal calf serum (NCS, Sigma‐Aldrich) and 0.4% Triton X‐100 (Sigma‐Aldrich) in phosphate buffered saline (PBS). Samples were then incubated overnight at 4 °C in primary antibody diluted in blocking buffer (gp100, Abcam, ab137078, 1:100) (Keratin‐14, Abcam, ab7800, 1:100). Slides were washed three times in PBS and incubated with the appropriate secondary antibody diluted in blocking buffer for 1 h at room temperature (donkey anti‐rabbit Alexa Fluor 488 or donkey anti‐mouse Alexa Fluor 594, ThermoFisher Scientific, 1:1000) and washed three times in PBS. Finally, slides were mounted using Vectashield Hardset with DAPI (Vector Laboratories, Peterborough, UK).

The Indiana University Animal Care and Use Committee approved the use of animals in this study and all procedures were performed as per NIH guidelines. Transformed cells with 50% basement membrane matrix (BME) type 3 (3632-005-02, Trevigen) (total 100 µL volume) were implanted into the mammary fat pad of 5–6-week-old female NSG (NOD/SCID/IL2Rgnull) mice. A 60-days slow release 17β-estradiol (0.72 mg) pellet (SE-121, Innovative Research of America) was implanted at the time of mammary fat pad injection. Tumor growth was measured weekly and tumor volume was calculated using the formula- sagittal dimension (mm) × [cross dimension (mm)]2/2^{43 (link)}. The maximal tumor burden permitted by the Indiana University Animal Care and Use Committee is 2 cm × 2 cm. The maximal tumor burden was never exceeded during this study. After 2–4 months, tumors and lungs were collected and processed for hematoxylin and eosin (H&E), ERα, GATA3, FOXA1, EpCAM (MA5-29698, Invitrogen,1:500), CK5/6 (IR 780, Dako, Ready-to-use), CK8 (35bH11, N1560, Dako, 10 µg/ml), CK14 (LL002, ab7800, Abcam, 1 µg/ml), and CK19 (IR 615, Dako, Ready-to-use) staining^{43 (link)}.

To examine the 4-NQO-induced tongue tumor, it was removed after lethal anesthesia and fixed in 10% buffered formalin. Paraffin-embedded tissues were sliced into 4-μm-thick serial sections by a microslicer. H&E staining and immunohistochemical staining were performed on serial sections, as previously described.³⁵ (link)^,36 (link) In brief, H&E staining was conducted using conventional Mayer’s H&E stain. For immunohistochemical staining, rabbit monoclonal anti-CK14 antibody (ab7800, Abcam, Cambridge, UK), rabbit polyclonal anti-p63 antibody (GTX102425, GeneTex, Irvine, CA, USA), and mouse monoclonal anti-vimentin antibody (TX100619, GeneTex) were used as primary antibodies, goat anti-rabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer (414141F, Nichirei, Tokyo, Japan) was used as secondary antibody, and 3,3′-diaminobenzidine (DAB⁺, K3467, Dako A/S, Glostrup, Denmark) was used to visualize the complex. Specificities of the antibodies were verified using a universal negative control immunoglobulin (IS600, Dako A/S). Images of serial sections were obtained through microscopy (Olympus, Tokyo, Japan) with a CCD camera (Nikon, Tokyo, Japan).

Cells were seeded into 8-well μ-Slides (Ibidi) at a density of 80,000 cells/well. After overnight incubation, cells were washed with pre-warmed PBS, fixed with 4% formaldehyde solution for 15 min at room temperature, washed, and then blocked with complete blocking solution (0.5% BSA, 0.1% TritonX-100, 5% goat serum in sterile PBS) for one hour at room temperature. Next, samples were incubated overnight at 4 °C with the relevant primary antibodies (anti-E-Cadherin antibody (ab11512-Abcam), anti-Vimentin Antibody (V9) (sc-6260-SantaCruz), anti-gamma H2A.X (phospho S139) antibody (ab11174-Abcam) anti-cytokeratin 8 (ab53280-Abcam) and anti-cytokeratin 14 (ab7800-Abcam). After incubation, the cells were washed with PBS, and the secondary antibodies (Alexa Flour 488, Alexa Fluor 546, Alexa Fluor 555) were added in complete blocking solution, followed by incubation for two hours at room temperature. Nuclei were labeled with DAPI. Imaging was carried out using a ZEISS LSM-710 system (Carl Zeiss microscopy Gmbh, Jena, Germany) with a 40×/1.4 Plan-Apochromat oil immersion objective. Images were processed with ZEN (Carl Zeiss microscopy Gmbh, Jena, Germany). γ-H2AX foci were counted with FindFoci, an automated ImageJ plugin [96 (link)].