Cell culture and Reagents
786-O, A498, TK-10 ccRCC cells, Renca mouse RCC cell and HepG2 liver adenocarcinoma cell (the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone) and 100 U/ml penicillin and 100 g/ml streptomycin. All these cells were routinely cultured in 5% CO2 at 37 °C. After chemical treatments, cells were collected for western blots or other assays. Sunitinib malate (#S1042) was purchased from Selleck. Anti-mouse PD-L1 antibody (BP0101) was purchased from BioXCell.
Western blots analysis and antibody
Cells or tumor tissues were washed with ice-cold PBS and lysed in RIPA lysis buffer containing a fresh protease and phosphate inhibitor mixture (50 mg/ml aprotinin, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM β-glycerolphosphate). Cell lysates were then prepared for western blots. Protein concentrations were quantified by BCA protein assay. Cell lysates were mixed with 4X loading buffer and heated at 95℃ for 5 min. Equal volumes of lysates were run on 5–15% SDS-PAGE gels and transferred to 0.2-mm nitrocellulose membranes (GE, A29411350). Blots were blocked for 1 h at room temperature in TBS with 0.05% Tween 20 (Sigma Aldrich, P7949) (TBS-T) and 5% nonfat milk. Primary antibodies were incubated overnight at 4℃ in TBS-T with 5% nonfat milk. HRP-conjugated secondary antibodies were incubated 1 h at room temperature. Blots were washed with TBS-T, 3 times, 5 min and TBS, 1 time, 5 min each after both primary and secondary antibody incubations. Blots were developed with Western Lighting Plus-ECL (Thermo, TK275827) and exposed in dark room. Blots were normalized to GAPDH or β-Actin loading controls. Blots were incubated with primary antibodies against GAPDH (Santa Cruz, Sc-32233), Histone H3 (Cell Signaling Tech, 4499), β-Actin (Santa Cruz, Sc-47778), TFEB (Cell Signaling Tech, 37785), TFE3 (Cell Signaling Tech, 14779), and PD-L1 (Abcam, ab213524) overnight at 4°C prior to being probed with the appropriate peroxide-conjugated secondary antibodies.
Real-time quantitative PCR
Total RNAs was extracted using an RNAiso plus kit (TaKaRa, Japan). Complementary DNA was synthesized through reverse transcription using ReverTra Ace qPCR RT Kit (TOYOBO, Japan). Quantitative PCR analysis of cDNA was performed with SYBRGreen reaction master mix on a Real-time PCR System (Eppendorf International, Germany). Target mRNA levels were normalized to the level obtained for GAPDH. Changes in transcript level were calculated using DD△Ct method. The primers used in this experiment were listed in Table 1
Table 1: Primer
Name (Human)
|
Forward primer
|
Reverse primer
|
TFE3
|
CCGTGTTCGTGCTGTTGGA
|
GCTCGTAGAAGCTGTCAGGAT
|
TFEB
|
CCAGAAGCGAGAGCTCACAGAT
|
TGTGATTGTCTTTCTTCTGCCG
|
CD80
|
TGCCTGACCTACTGCTTTGC
|
AGGGCGTACACTTTCCCTTC
|
CD86
|
CGACGTTTCCATCAGCTTGTC
|
CGCGTCTTGTCAGTTTCCAG
|
CD273
|
ACCAGTGTTCTGCGCCTAA
|
CCTGGGTTCCATCTGACTTTG
|
CD274
|
GGTAAGACCACCACCACCAAT
|
TGATTCTCAGTGTGCTGGTCAC
|
CD275
|
CGTCTTCTTGAACATGCGGG
|
TTTTCTCGCCGGTACTGACT
|
CD276
|
CTCACAGGAAGATGCTGCGT
|
CTGTGAGGCAGAACCACAGT
|
VTCN1
|
TCTGGGCATCCCAAGTTGAC
|
TCCGCCTTTTGATCTCCGATT
|
VISTA
|
ACGCCGTATTCCCTGTATGTC
|
TTGTAGAAGGTCACATCGTGC
|
CD155
|
AGGCTATAATTGGAGCACGACC
|
GGTTTGTCCACAGGACGGAT
|
CD270
|
CAAGGTGATCGTCTCCGTCC
|
TCTGTGGGTCAGTGGTTTGG
|
GAL3
|
ATAACCTGCCTTTGCCTGGG
|
AGCAATTCTGTTTGCATTGGGC
|
HMGB1
|
TATGGCAAAAGCGGACAAGG
|
CTTCGCAACATCACCAATGGA
|
CD70
|
GTCACTTGGGTGGGACGTA
|
CAGTATAGCCTGGGGTCCTG
|
CD154
|
ACATACAACCAAACTTCTCCCCG
|
GCAAAAAGTGCTGACCCAATCA
|
CD252
|
GAGCCCCTCTTCCAACTGAA
|
CAGTTCTCCGCCATTCACAT
|
β-ACTIN
|
CATGTACGTTGCTATCCAGGC
|
CTCCTTAATGTCACGCACGAT
|
GAPDH
|
GGAGCGAGATCCCTCCAAAAT
|
GCTGTTGTCATACTTCTCATG
|
xCELLigence
Experiments were carried out using the RTCADP instrument (Roche, Germany) which was placed in a humidified incubator maintained at 37°C with 5% CO2. For time-dependent cell response profiling, 10000 cells/well were added to 16 well E-Plates. The electronic sensors provided a continuous and quantitative measurement of cell index in each well. Cell index is a quantitative measure of cell number present in a well, e.g. lower cell index reflects fewer cells are attached to the electrodes. The E-Plate 16 was monitored over the time frame indicated.
ethynyl-2'-deoxyuridine (EdU) incorporation assay
EdU cell proliferation kit (17-10527) was purchased from Millipore (Massachusetts, USA). Pretreatment with siRNA, the cells were incubated 16 hours at 37℃ in complete media supplemented with 10 μmol/L EdU. After washing in PBS, the cells were fixed and permeabilized. Reaction cocktail and DAPI (Beyotime, Shanghai, China) were then added. The fluorescence change of cells was detected with flow cytometry or microscope.
Subcellular fractionation
Cells were lysed in NP-40 lysis buffer containing 20 mM Tris-HCL (pH 7.9), 150 mM NaCl, 0.5 mM EDTA and 0.5% NP-40 supplemented with protease and phosphatase inhibitors. Lysed cells were kept on ice for 15 min. The lysates were then centrifuged at 2,000 ×g for 5 min. The resulting supernatants represented the cytosolic and membrane fractions. The corresponding pellets representing the nuclear fractions were washed one time in NP-40-containing lysis buffer and sonicated in nuclear lysis buffer (20 mM Tris-HCl (pH 7.4), 450 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 0.1% SDS). The lysates were then centrifuged at 12,000 ×g for 15 min to obtain the cytosolic and nuclear fractions.
Microscopy
To measure TFE3 nuclear translocation, cells following TFE3-GFP transfection and Sun (5uM) treatments were incubated with DAPI for 10 min. The cells were then washed with PBS, and nuclear translocation fluorescence was measured using confocal microscopy (Carl Zeiss).
Patient samples
Clear cell renal cell carcinoma and benign samples were obtained from surgical excision specimens at the Shandong Provincial Hospital. Utilization of the clinical samples was approved by the Ethical Committee of the Shandong Provincial Hospital affiliated to Shandong First Medical University.
Immunohistochemistry (IHC)
Heat-induced epitope retrieval was performed in 10 mM citric acid buffer (pH 7.2) using a microwave. The slides were incubated at 4 °C overnight with primary antibodies (anti-PD-L1, 1:200 dilution; anti-TFE3, 1:200 dilution). An HRP-conjugated antibody and 3,30-diaminobenzidine (DAB) staining were used to visualize primary antibody binding. High-resolution pictures were obtained on a digital electron microscope, and images were recorded using Case Viewer software. Immunohistochemical results are expressed as a mean score that considers both the intensity of the staining and a positive reaction.
Transfection
Cells were transfected with specifically targeted TFEB (AGACGAAGGUUCAACAUCA), TFE3 (CGCAGGCGATTCAACATTAAC), and the plasmid of TFE3-GFP, using Lipofectamine 2000 Transfection Reagent.
Flow Cytometry
The PD-L1 expression in the cells were determined using flow cytometry. Cells following various treatments, were collected by centrifugation. After two washes with ice-cold PBS, Cells were stained with antibodies against PE-conjugated anti-human PD-L1 antibody. The antibodies used to stain tumor infiltrating lymphocytes (TILs) were listed as followed:anti-CD3-FITC (100203, Biolegend), anti-CD4-APC (100516, Biolegend), anti-CD8-Percp/Cy5.5 (100734, Biolegend), anti-GZMB-PE (104508, Biolegend).
Xenograft mouse tumor models
C57BL/6 mice (6 weeks old) were obtained from the Animal Center of the China Academy of Medical Sciences (Beijing, China). Murine RCC cell Ruca were injected into the right flanks of the mice and allowed to establish tumors. When the tumors reached 50~100 mm3, the mice were given the clinical chemotherapeutics sunitinib (40 mg/kg, i.p.) daily, anti-PD-L1 (200 μg/mouse, i.p.). Tumor volumes (mm3) were calculated from the formula 0.5×L×W2 (L=length, W=width). All animal experiments were approved by the Ethics Committee of the Shandong University School of Medicine.
Statistical analysis
Western blots and fluorescent images were analyzed with Image Pro Plus 6.0. The data are presented as the mean ± SD and were analyzed with GraphPad Prism software (GraphPad). Student’s t-test or one-way ANOVA was used for comparisons among different groups. Kaplan-Meier and Cox proportional hazards analyses were used for survival analysis. All the experiments were repeated at least three times. Values of p<0.05 denoted statistical significance and are indicated as *p≤0.05, **p≤0.01, and ***p≤0.001 in the figures.