2.1 Clinical samples The First Affiliated Hospital of Soochow University (Suzhou, China) approved our experimental protocols. A total of 225 colon cancer tissue specimens and 20 adjacent normal tissue specimens were obtained for immunohistochemistry (IHC). Ethics of the clinical sample experiments was approved by the Institutional Review Board of Soochow University. Informed consent was also obtained from the patients for experimentation.
2.2 Immunohistochemistry and immunofluorescent staining For IHC analysis of p-PKCδ, a rabbit anti-human monoclonal antibody for p-PKCδ (phospho S299, ab133456) purchased fron Abcam was used in 1:100 dilution. For B7-H4 IHC staining, the mouse anti-human monoclonal antibody (clone 3C8) for B7-H4 was produced in our lab and used in 1:200 dilution [37]. For IF analysis, the secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100, Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, Invitrogen), respectively.
4-µm sections were cut from paraffin-embedded samples by a Leica microtome. Tissue microarrays were deparaffinized, rehydrated, rinsed, and stained for B7-H4 and p-PKCδ, as previously described [38]. IHC staining was performed using the ChemMate™ Envision/HRP technique (Gene Tech Company Limited). According to the percentage of positive cells and the IHC staining intensity, the signals of B7-H4 or p-PKCδ were classified into four groups: negative, low, medium, or high. The Quickscore was defined as the product of the proportion and intensity scores. Cases for which the Quickscore was ≥ 4 were considered positive, and the other cases were regarded as negative. The staining slides were examined and evaluated by two independent investigators.
For IF analysis, serial sections were incubated with anti-p-PKCδ, anti-B7-H4 and/or an isotype IgG monoclonal antibodies for 1.5 h at room temperature, respectively. Alexa Fluor 488-conjugated secondary antibody was used to detect p-PKCδ. Alexa Fluor 594-conjugated secondary antibody was used to detect B7-H4. IF images were observed under a Leica DM2500 microscope.
2.3 Cell culture and transfection The human CRC cell lines HCT116, SW620, SW480,RKO and NCM460 cells were purchased from Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai). Cells were cultured in RPMI 1640 medium (HyClone) containing 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified atmosphere of 5% CO2. PKCδ activator TPA, PKCδ inhibitor rottlerin (Santa Cruz Biotechnology) and STAT3 inhibitor cryptotanshinone (Selleckchem) were stored at − 80°C.
Human PKCδ-specific siRNA, human B7-H4-specific siRNA and corresponding control siRNAs (con siRNAs) were purchased from GenePharma Co. Ltd (Shanghai, China). siRNA was transfected into HCT116 or SW620 cells using Lipofectamine 2000 reagent (invitrogen). RT-qPCR and Western blot were performed to examine the transfection efficiency.
2.4 Total RNA isolation and RT-qPCR assays Total RNA was isolated using TRIzol reagent (Invitrogen). RNA was quantified with a spectrophotometer (BioDrop-µLite). Total RNA was reverse transcribed into cDNA using PrimeScript ™ RT Master Mix (Takara Bio). SYBR PrimeScript RT-qPCR Kit was used (Takara Bio) to examine individual genes. The PCR procedure was performed as following: 95°C for 2 min, 45 cycles amplification at 95°C for 10 s, at 59°C for 40 s and at 72°C for 45 s. All the genes examined was normalized to GAPDH mRNA level. The primers of individual genes used in RT-qPCR are listed in Supplemental Table 1.
2.5 Protein extraction and Western blot analysis Human CRC cell lines were cultured in 6-well plates and then lysed with RIPA lysis buffer (Beyotime). Protease inhibitor cocktail was added into the RIPA buffer. After determining the protein concentrations of all samples by BCA protein assay kits (Beyotime), each sample was loaded at equal amounts of total protein and separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The protein bands on gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: mouse anti-human B7-H4 (3C8), rabbit anti-human PKCδ (CST, #9616T), rabbit anti-human/mouse p-PKCδ (Abcam, ab133456), rabbit anti-human/ mouse STAT3 (CST, #12640), rabbit anti-human/ mouse Phospho-STAT3 (p-STAT3, Tyr705) (CST, #9145), rabbit anti-human/ mouse GAPDH (CST, #5174) and rabbit anti-human/ mouse β-actin (CST, #4970). After three times washing with PBST, the PVDF membranes were incubated with secondary antibodies at room temperature for 2 h. The secondary antibodies were as follows: HRP-conjugated goat anti-mouse/anti-rabbit IgG (H + L) (Thermo) and rabbit anti-goat IgG (H + L) (Thermo). The membranes were washed with PBST five times. Then, the membranes were immersed in electrochemiluminescence (ECL) detection reagent (CST). The images were captured by the Gel DocTM EZ System (Bio-Rad). Image Lab 4.0.1 software (Bio-Rad) was used to analyze the intensities of the bands.
2.6 Flow cytometry analysis and IF analysis To examine B7-H4 expression in HCT116, cells were first washed in PBS buffer containing 1% FCS three times. Then cells were stained with a PE-conjugated anti-B7-H4 antibody (358104, Biolegend). PE-labelled mouse IgG1 isotype controls (eBioscience) were used as the control antibody. To examine the intracellular B7-H4 expression, cells were permeabilized using Intracellular Fixation & Permeabilization Buffer (eBioscience). Flow cytometry was performed on a Beckman flow cytometer. Data were analyzed using FlowJo software (Version 7.6, Tree Star Inc.).
For IF analysis of B7-H4 expression in cells, cultured cells were fixed in cold acetone for 10 min, washed in PBS buffer containing 1% FCS, and stained with a PE-conjugated anti-B7-H4 antibody.
2.7 Cell Proliferation Assay Cell viability was assessed by Cell Counting Kit- (CCK-) 8 (Tongren, Shanghai, China). After 16 h of transfection, cells were digested and re-suspended in 2% FBS culture medium with a drug or vehicle, and 5×103 cells were seeded in each 96-well plate and further incubated for 24 hours, respectively. CCK-8 reagent was added to each well and further incubated 2 hours. Then the cells were determined by the optical density (OD) 450 nm values using a microplate reader. Experiments were repeated three times each time in triplicate.
2.8 Transwell invasion assay After 16 h of transfection, cells were digested and re-suspended in 10% FBS culture medium, and 5×104 cells were seeded in 24-well transwell chambers with a pore size of 8 µm (FALCON), the inserts were pre-coated with 40 µL 1: 4 dilution of Matrigel (Corning, USA). Each well of the culture medium was changed to 2% FBS culture medium with a drug or solvent after 6–8 hours. Then, 700 µl of medium containing 20% FBS was added into the bottom chamber, acting as the chemoattractant. After cultured for 24 h, the cells invaded to the underside of the transwell membrane were fixed in methanol and stained with a 0.1% crystal violet solution. Then the staining cells were observed under a Nikon TI-SR inverted microscope and imaged using a Nikon DS-Fi2 camera. Then cells were destained with 30% glacial acetic acid and the cell number was quantified by detecting OD 570 nm using a microplate reader.
2.9 Wound-healing assay For a wound-healing migration assay, 12-well plates were seeded with cells at a density of 3 × 105 /well. After overnight growth and attachment, the cultured cells were scratched with 10-µl pipette tips. After the subsequent 24h culture, wound closure was observed and imaged using a Nikon DS-Fi2 camera. The percentage of wound closure was calculated using Image J software.
2.10 Statistical analysis Statistical analyses of experimental results were performed with GraphPad Prism version 5.0. The associations between p-PKCδ, B7-H4 and various clinicopathological parameters were evaluated by the χ2 test. Correlation was evaluated by the Spearman rank correlation coefficient. Differences between groups were evaluated using a two-tailed unpaired Student’s t test. Replicated experiments were analyzed using a paired Student’s t test. All significance tests were two sided, and P < 0.05 was considered as significance.