Pa1 108

All tissues were paraffin-embedded and acquired from the Department of Pathology at the Affiliated Hospital of Nantong University. The paraffin-embedded tissues were sliced into 4-μm sections. The tissues slices were dewaxed, dehydrated and rehydrated. A rabbit anti-Pax4 polyclonal antibody (PA1-108, ThermoFisher) was added and incubated into sections overnight at 4°C. The SP-9000 HistostainTM Plus kits (ZSGB-BIO) were utilized as per the manufacturer’s protocol. Tumor slices were then evaluated in a blinded manner. Ten fields were chosen for examination of cell-staining intensity and proportion of positive cells. Immunohistochemical staining was evaluated according to the immunoreactive score (IRS), and then evaluated using the staining intensity and proportion of positive cells. The staining intensity was graded as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). The proportion of positive cells were scored as 0 (negative), 1 (<10%), 2 (10–50%) or 3 (>50%). Both scores were multiplied and the IRS was determined. Values ≥3 were defined as positive cytoplasmic expression, and values <3 were regarded as negative.

The transfected cells were collected after 24 h, and the cells were lysed using RIPA lysis buffer in order to extract total protein. The protein concentration was determined using the BCA method. Proteins were then separated by 10% SDS-PAGE gel electrophoresis, and transferred to a membrane, blocked with 5% skimmed milk powder at room temperature for 1 h. The membranes were then incubated with primary antibodies targeting Pax4 (PA1-108, ThermoFisher), Grb2 (ab32111; Abcam), p-Raf (ab157201; Abcam), T-Raf (ab230850; Abcam), p-MEK (ab278562; Abcam), t-MEK (ab32576; Abcam), p-ERK (ab136926; Abcam), t-ERK (ab184699; Abcam), Vimentin (ab92547; Abcam), Bax (ab32503; Abcam), CyclinD1 (ab16663; Abcam) and GAPDH (ab181602; Abcam). The membranes were incubated at 4°C overnight, washed with TBST three times, five min each time. Then, the membranes were incubated with secondary antibodies for 1 h at room temperature. The membranes were washed with TBST three times, five min each time. Finally, ECL luminescent solution was added to the dark room, exposed and developed.