2.1 Cell culture
Human muscle invasive bladder cancer cell lines T24, TCCSUP and UMUC-3, mouse derived bladder cell lines MB49, and 293T cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Short tandem repeat DNA profiling analysis were performed to ensure the stable and reliable of all cell lines during the experiments. T24, TCCSUP, UMUC-3 and MB49 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.), and 293T cells were cultured in DMEM medium (Thermo Fisher Scientific, Inc.). Mediums mentioned before were pre-supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) without any antibiotics, and all cell lines were incubated under standard conditions (37 °C and 5% CO2).
2.2 Cell counting kit-8 (CCK-8) assay and Colony Formation assay
Each groups of T24, TCCSUP and MB49 cell lines were prepared in 96-well plates (1000 cells/well) under standard conditions. Then, premixed medium with a 10% concentration of CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto Japan) reagent was added into each well and incubated under standard conditions for 1–2 h before measurement at 450 nm. For colony formation assay, different groups of cells were prepared in 6-well plates (1000 cells/well) and cultured for 6–8 days under standard conditions followed by 3 min staining and 6 min fixation (Wright - Giemsa Stain Kit, NJJC bioengineering institution, China) at room temperature before colonies comparison.
2.3 Plasmids construction and transfection
Sequences of short hairpin RNA (shRNA) against CDS of msSTING were validated from Sigma-Aldrich Online. Scramble sequences were designed using Wizard v3.1 (https://www.invivogen.com/sirnawizard/) to ensure the absent of seed sequence matches. msSTING sequences and scramble sequences were synthesized (Tsingke, Hangzhou, China) and inserted into the plko.1-puro vector. Then the vector was co-transfected with pSPAX2 and pMD2G (with a ratio of 4: 3: 1, respectively) into the 293T cell lines cultured in 100 mm plates (30–60% cell density) using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to manufacturer’s instruction. The supernatant, which contained Lentivirus, was harvested and centrifuged (800 × g, 5 min) 48 or 72 h post-transfection and added into MB49 cells cultured in 6-well plates (30–60% cell density) immediately. Successfully transfected shSTING cell lines were screened with an increasing concentration of puromycin (Sigma-Aldrich; Merck KGaA). The siRNA used here were also transfected using Lipofectamine 3000. Finally, the transfection efficiency was validated by western blotting. The msSTING and siSTING sequences used here were showed in Table S1.
C57BL/6 mice were purchased from Jackson Labs. 4 week mice were mainly used in this experiment and all mice were maintained in pathogen-free barrier facilities and were approved by Zhejiang University experimental animal welfare ethics review committee. 2 × 106 of MB49 cells with shSTING or shScr transfection were subcutaneous injected on the flank of mice. Then cisplatin (3 mg/kg) or PBS were injected subcutaneous injected around the tumors in day 13 and day 16. Tumors size were measured regularly by length (L), width (W) and height (H), respectively, and tumor volume was calculated as 1/2 × L × W × H. Then tumors were harvest in day 23, the weight of tumors were normalized by their corresponding body weight. Fresh tumor issues were then used for flow cytometry analysis.
2.5 RNA-sequence (RNA-seq) and Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
TRIzol (Thermo Fisher Scientific, Inc.) was used for cells total RNA extraction according to manufacturer's protocol. Then different groups of RNA were used for RNA-seq or qRT-PCR. T24 cells were treated with cisplatin (2 µg/ml) or PBS for 24 h at standard condition. RNA-sequence was performed by Novogene China. Gene expression levels were quantified as Fragments Per Kilobase Million and then Log2 –transformed as A value. The significant different expression genes were identified as absolute fold change of A value > 1.5 combined Padj (adjust P) value < 0.05 between two groups. Then different expression genes were used for plotting heatmap and volcano map by R soltware (Version 4.0.2, https://cran.r-project.org/src/base/R-4/). GO and KEGG analysis were performed on DAVID online (https://david.ncifcrf.gov). Pathways with Padj value < 0.05 were identified as significant different pathway. As for qRT-PCR, Takara PrimeScript™ RT and SYBR EX Taq™ kits (Takara Bio, Inc., Otsu, Japan) were used for qRT-PCR according to manufacturer's instruction. The standard thermocycling conditions used were as follows: Initiate Step, 95.0 °C: 30 s; cycling Step, 40 cycles of 95 °C: 5 s and 60 °C: 30 s; melt curve analysis Step, 65 °C to 95 °C, increasing in 0.5 °C increments for 5 s. The specific primers used in this experiment were listed in Table S1. Control groups were conducted to confirm the absence of the agent pollution or primer dimers. Targeting genes were finally normalized to GAPDH expression, and the relative mRNA expressions were calculated using the ΔΔCq method.
2.6 Enzyme linked immunosorbent assay (ELISA) and IFN reporter assay
Secreted supernatant IL-6 of T24 and TCCSUP cell lines was measured using the Human IL-6 ELISA kit (DAKEWE, China) according to manufacturer’s instructions. IFN-β in cell supernatants with biological activity was compared using a reporter 293T cells stably expressing a pISRE (Genechem Co. Shanghai, China). Reporter cells were co-cultured with supernatant from different groups for 24 h before measured by a fluorescent enzyme meter Varioskan™ Flash at 488 nm/520 nm (Thermo Fisher Scientific, Inc.). Intensities of cellular fluorescent were normalized to the total number of T24 and TCCSUP cells.
2.7 Subcellular fractionation and Western blotting assay
Subcellular fractionation protein was extracted by Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific, Inc.) and NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc.) according to the kit instruction. As for total protein extraction, T24, TCCSUP and MB49 cells were washed using cold PBS before being lysed using RIPA Lysis buffer with 1% cocktail protease inhibitor (Thermo Fisher Scientific, Inc.). After ultrasonic spallation at 4 °C, the protein samples were purified by centrifugation (4 °C, 15,000 × g, 15 min) and the clear supernatant extracts were quantification using a BCA protein assay (Thermo Fisher Scientific, Inc.) and 25 µg/10 µl denatured protein samples were prepared and loaded in 4–12% Tris-acetate gels (Invitrogen; Thermo Fisher Scientific, Inc.) and then separated by electrophoresis. Then, the proteins were transferred onto a polyvinylidene fluoride membrane with 0.45 µm pore size. Then, the membrane was blocked with 5% bovine serum albumin (BSA, Fdbio Science, Hangzhou, China) in Tris-buffered saline containing 1% Tween-20 (TBST) for 1 h at room temperature and further incubated with primary antibodies for 12 h in a shaker at 4 °C. Subsequent to washing with TBST 5 min for three times, the membrane was incubated with secondary antibodies for 1 h at room temperature. The information of abovementioned primary antibodies: Human-Reactive STING Pathway Antibody Sampler Kit (1:1, 000; cat no. #38866; Cell signaling technology, CST), Mouse-Reactive STING Pathway Antibody Sampler Kit (1:1, 000; cat no. #16029; CST), γ-H2A.X (1:1, 000; cat no. ab81299; Abcam), Rad51(1:1, 000; cat no. ab133534; Abcam), GAPDH (1:5, 000; cat no. ab8245; Abcam) and Histone H3 (1:1, 000; cat no. ab1791; Abcam). The information of abovementioned secondary antibodies: Goat anti Rabbit- HRP (1: 3, 000; cat no. PDR007; Fdbio Science) and Goat anti Mouse-HRP (1: 3, 000; cat no. PDM007; Fdbio Science). The blotting results were illustrated by Dura ECL detection kit (Fdbio Science) and specific protein bands were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH was selected as a total control protein or cytosolic control protein, histone H3 was selected as total nuclear control protein or chromatin bound control protein.
2.8 Flow cytometry analysis
Tumor tissues were digested and incubated with biotinylated anti-CD3, anti-CD8 antibodies, followed by incubated with streptavidin-PE/Cy7 (Cat no.SA1012; Invitrogen™), anti-CD45.2-APC for 10 min at room temperature in darkroom. For analyzing DCs, tissue cells were incubated with anti-MHCⅡ-APC and anti-CD11c-PE for 10 min at room temperature in darkroom. To analyze macrophage cells, tumor cells was collected and stained with anti-CD11b-APC and anti-F4/80-PE for 10 min at room temperature in darkroom. The antibodies used in flow cytometry analysis were: anti-CD3 (1:200; cat no. 13-0032-82; eBioscience™), anti-CD8 (1:200; cat no. 13-0081-82; eBioscience™), anti-CD45.2-APC (1:200; cat no. 17-0454-82; eBioscience™), anti-MHCⅡ-APC (1:200; cat no. 17-5321-82; eBioscience™), anti-CD11c-PE (1:200; cat no. 12-0114-82; eBioscience™), anti-CD11b-APC (1:200; cat no. 17-0112-82, eBioscience™) and anti-F4/80-PE (1:200; cat no. 17-4801-82; eBioscience™).Then the infiltration percentages of different cell types in tumors were calculated by BD FACSCanto™ II (BD Biosciences) and then analyzed by FlowJo 10.0 (FlowJo LLC, Ashland, OR, USA).
Different groups of T24, TCCSUP, UMUC-3 and MB49 cells were pre-seeded the day before immunofluorescence assay. 4% paraformaldehyde was used for cell fixation and 0.5% Triton-X 100 was used to increase cell membrane permeability for 10–15 min at room temperature. Then 3–5% BSA was used for 30–60 min at room temperature to block antibodies. Cells were further incubated with primary antibodies overnight at 4 °C and subsequently incubated with corresponding fluorescence antibodies combined with DAPI staining 30–60 min in darkroom and then wash twice by PBS before observed by NIS A1 laser confocal microscope (Nikon, Japan). The primary antibodies used for immunofluorescence assay were: cGAS (1:200; cat no. A8335; Abclonal), F-actin (1:1, 000; cat no. A12380; Invitrogen™), Rad51(1:300; cat no. ab133534; Abcam), γ-H2A.X (1:300; cat no. ab81299; Abcam). The secondary antibodies used for immunofluorescence assay were: Goat Anti-Rabbit Alexa Fluor 488 (1:200; cat no. ab150077; Abcam) and Goat Anti-Mouse Alexa Fluor 594 (1:200; cat no. ab150116; Abcam). The results were analyzed by NIS-Elements Viewer 4.50 (Nikon, Japan).
2.10 Statistical analysis
SPSS 22.0 (IBM Corp. USA) was used to normalize and analyze raw data in this experiment. Data was presented as the mean ± standard deviation. Kaplan-Meier survival method and log-rank test analysis were using to plot (OS) overall survival and (DFS) disease-free survival rates curves. A student’s t-test was used to assess the different between two groups. One-way ANOVA test was performed to assess the differences in multiples groups followed by Student-Newman-Keuls post hoc test. IC50 of MB49 cell lines were measured by Probit regression analysis. P < 0.05 was considered to have statistical significance. Data in experiments was repeated at least 3 times.