Cell culture and reagents
The squamous cell carcinoma cell line A431 was purchased from American Type Culture Collection (Manassas, Virginia, USA) while the non-tumorigenic immortalized human keratinocyte HaCaT cell was obtained from Dr. Nancy Bigley (Wright State University). A431 and HaCaT cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.
siRNA transfection, transient transfection and lentiviral transduction
siRNA transfection was conducted using Dharma-FECT transfection reagent (Dharmacon, Lafayette, CO, USA) or Lipofectamine RNAi-Max (ThermoFisher Scientific, Carlsbad, CA, USA), following the manufacturer’s instructions. ERK3 siRNA was purchased from Ambion/ThermoFisher Scientific (siERK3, cat # 4390824, assay ID s11148) and DNp63a siRNA or non-silencing control (NSC) were purchased from Qiagen (Valencia, CA, USA) as previously described .
For transient transfection, A431 cells were transfected with empty vector control (EV) or ERK3 plasmids using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), and 48 hours after transfection cells were harvested and tested for protein expression. A431 cells were transduced with lentiviruses expressing an empty vector CDH or CDH-ERK3 as previously described .
Cells were lysed in the lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM complete protease inhibitors and 1 mM phosphatase inhibitor cocktail III (Sigma-Aldrich). Immunoblot analysis was carried as previously described . The following primary antibodies were used in immunoblotting: Mouse monoclonal anti-pan-p63 (4A4), rabbit monoclonal anti-ERK3 (ab53277, 1:1000, Abcam), rabbit polyclonal anti-pRac1 S71 (sc-12924, 1:500, Santa Cruz), mouse monoclonal anti-Rac1(ab33186, 1:5000, Abcam) and mouse monoclonal anti-b-actin (A5316, 1:20,000, Sigma-Aldrich) antibodies were used to detect ΔNp63α, ERK3, pRac1, Rac1 and β-actin, respectively. β-actin was used as a loading control. Appropriate secondary antibodies (HRP-conjugated goat anti- mouse [170-6516, Biorad] or anti-rabbit [170-6515, Biorad]) were used for visualization by chemiluminescence (Thermo Scientific).
RNA extraction and RT-qPCR
Total RNA was extracted from cells 24-hours post-transfection for gene expression analysis using Trizol reagent (Ambion). SuperScript VILO Master Mix (Invitrogen) was used for reverse transcription to generate cDNA as per the manufacturer’s instructions. Quantitative Polymerase chain reaction (qPCR) was performed using TaqMan® Universal Master Mix (Applied Biosystems), designed Roche Universal primers and Universal Probe (Roche Diagnostics) on the 7900HT Fast Real-Time PCR Systems (Applied Biosystems) using the following primers: GAPDH forward primer [AGCCACATCGCTCAGACAC], GAPDH reverse primer [GCCCAATACGACCAAATCC], ERK3 forward primer [TTTGCTGAAATGCTGACTGG], and ERK3 reverse primer [CCAGTCAGCATTTCAGCAAA]. GAPDH was used as internal control, and the relative gene expression level was calculated by the ΔΔCT method .
Trans-well migration assay
Cell migration was analyzed using a modified two chamber trans-well system (8.0 μm pore, BD Biosciences Falcon) according to the manufacturer’s protocol. At 24 hours post-transfection, cells were detached by trypsin/EDTA, washed once with serum-free medium, and then re-suspended in serum-free medium. 0.6 mL of complete medium with 10% FBS was added to the bottom of each well. A total of 1.5x106 cells per well was added in trans-well inserts and cells allowed to migrate for 18-20 hours in a 37°C incubator. Using cotton swabs, cells which failed to migrate in the upper surface of the trans-well were removed. The migrated cells attached on the undersurface of inserts were fixed with 10% formalin, stained with crystal violet solution (0.5% in water) and followed by quick washes with distilled water or PBS. Migrated cells were then photographed under a microscope at 10 X magnification, and five images per condition were taken to count the migrated cells by ImageJ 1.52 software.
Cloning of the ERK3 reporter and luciferase reporter assay
The fragments of ERK3 gene enhancer region containing p63 binding sites was amplified by PCR using the following primers: BS1 forward primer [GCGCGGTACCGTTCTTCTTTGTTTCCTCAG], BS1 reverse primer [GCCACTCGAGCACGTTCAAACCATAGCAAC], BS2 forward primer [GCGGTACCAGGTCTTAGTGCTGTTGTAG] and BS2 reverse primer [GCGTCTCGAGCCTAAACACTATGCAATGCTG] and cloned into the PGL3-promoter luciferase vector via KPN1 and XHO1. H1299 cells were plated on 24-well plates and co-transfected with p63-BS1 or –BS2 luciferase reporters and Renilla luciferase constructs along with empty vector control or 0.1 or 0.3 mg of DNp63a. The luciferase assay was performed as previously described .
Tissue immunofluorescence staining
Formalin-fixed, paraffin-embedded human skin tissue microarrays described previously  were used for co-immunostaining studies. Human tissue samples consisted of normal skin (N=53), actinic keratosis (AK) (N=66), cutaneous squamous cell carcinoma (SCC) (N=59), and basal cell carcinoma of the skin (BCC) (N=57). Skin tumor and non- tumor skin tissue slides were co-stained for p63 and ERK3 as previously described with some modifications [6, 10]. Briefly, the staining of both p63 and ERK3 were performed using heat-based antigen retrieval processes with a citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Tissues were incubated with anti-p63 (4A4, 1:800) and anti-ERK3 (ab53277, 1:50, Abcam) primary antibodies at 4 C. Tissue sections were visualized and imaged using a Leica CTR 6000 Microscope (Leica Microsystems, Wetzlar, Germany) and ImagePro 6.2 software (Media Cybernetics, Bethesda, MD). Three representative pictures were taken under a microscope at 20 X magnification for each tissue sample to measure the mean fluorescence intensity (MFI). Background intensity first was subtracted, and then nine measurements of fluorescence intensity at 9 different areas with the same size were taken for each tissue sample. Average mean fluorescence intensity was calculated using ImageJ 1.52 software.
A431 cells transduced with CDH or CDH-HA-ERK3 lentiviral vectors were grown on sterile glass coverslips, and at 24-hours post plating, cells were washed with 1xPBS prior fixation with 4% paraformaldehyde for 15 minutes. After three washes with 1xPBS for 5 minutes, cells were permeabilized with 0.2% triton- X-100 for 5 minutes followed by three washes with 1XPBS for 5 minutes. Cells were blocked with 5% normal goat serum in 1XPBS for 1 hour at room temperature and then incubated with mouse monoclonal anti-HA antibody (Sigma-aldrich, MO, USA) for overnight at 4 C. After three washes with 1XPBS for 5 minutes, cells were incubated with Alexa Fluor 555 Phalloidin (1:40, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor goat anti-mouse 488 (1:500, Invitrogen, Carlsbad, CA, USA) for 90 minutes at room temperature. Cells were washed with 1XPBS for four times 5 minutes each, and mounted with Vecta-Shield plus DAPI Mounting media (Vector Laboratories, Burlingame, CA, USA). Cells were visualized and imaged at 63 X magnification using a Leica CTR 6000 Microscope (Leica Microsystems, Wetzlar, Germany) and ImagePro 6.2 software (Media Cybernetics, Bethesda, MD).
Generation of stable cell lines expressing ERK3 shRNA by lentiviral transduction
A431 and HaCaT cell lines with stable knockdown of endogenous ERK3 were generated by lentiviral expression of a short hairpin RNA (shRNA) specifically targeting ERK3 mRNA (shERK3) in the presence of 5μg/ml polybrene. As a control, cells with stable expression of the non-targeting shRNA (shGIPZ) were used. Cells were split and selected two days post-transduction by puromycin (0.8 μg /mL for A431, 1 μg /mL for HaCaT) for 14 days. The knockdown was confirmed by Western blotting analysis and RT-qPCR.
Cell proliferation assay
Cell proliferation was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI), following the manufacturer’s instructions. Stable A431 or HaCaT cells were plated in five 96-well plates and allowed to grow at 37°C in the incubator. At specific time points, MTS-containing reagent was added to the cells. After 2 hours incubation, the absorbance was measured by Synergy H1 microplate reader (BioTek, Winooski, VT) at 490 nm.
Statistical analysis for stained tissue
Adjusted mean fluorescence intensity and standard error of mean (SEM) levels of p63 and ERK3 from all type of skin tissue (normal skin samples, basal cell carcinoma samples, squamous cell carcinoma samples and actinic keratosis samples) were plotted. A mixed effects (ANOVA) tests were used to determine the correlation between the two response variables (MFI of p63 and ERK3) in nine measurements per sample. Post-hoc multiple comparison methods using Dunnett’s test were performed to compare between MFI values of p63 or ERK3 between control samples (i.e., normal skin samples) and all other samples (i.e., AK, SCC, BCC) (Dunnett, 1955, 1980). PROC MIXED procedure (SAS/STAT®, Ver 9.4, SAS Institute Inc., Cary, NC) was used for analyses [30, 31]. A P-value of less than 0.05 was considered statistically significant.