Gemcitabine

Gemcitabine treatment: BALB/c mice were injected intraperitoneally with Gemcitabine (Selleckchem, Houston, TX, USA) on days -1, 1, and 3 at the dose of 40 mg/kg; IMQ was applied from day 1 to their shaved backs for 5 consecutive days topically. The mice were photographed and sacrificed for skin lesion analysis on day 8 (mice divided into 3 groups: vehicle (IMQ+vehicle), Gemcitabine (IMQ+GEM), and untreated (normal)). Anti-IL-21R antibody treatment: BALB/c mice were injected intraperitoneally with anti-mouse IL-21R antibody (4A9) (BioXCell, West Lebanon, NH, USA) on days -2, 0, 1, 3, and 5 by i.p. injection of 140 μg anti-IL-21R antibody; IMQ was applied from day 1 to their shaved backs for 6 consecutive days topically. The mice were photographed and sacrificed for skin lesion analysis on day 8 (mice divided into 3 groups: vehicle (IMQ+vehicle), anti-IL-21R antibody (IMQ+Anti-IL-21R), and untreated (normal)).

The MTT assay was performed as previously described [30 (link)]. After different treatments, the pancreatic cancer cells were seeded into 96-well plates and further incubated with various concentrations of gemcitabine (Selleck) for 48 h. Then, 20 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well. The plates were incubated for 4 h, after which the medium was replaced with 150 μL of dimethyl sulfoxide (Sigma-Aldrich). The optical density was detected at 490 nm. Each concentration of gemcitabine was set up in five replicate wells.

Gemcitabine (cat# S1714) and Cisplatin (cat# S1166) were purchased from Selleck Chemicals (Houston, TX, USA), and LCL161 (cat# HY-15518) and Birinapant (cat# HY-16591) were from purchased from MedChemExpress (Monmouth Junction, NJ, USA). Drug response tests were performed as previously described (17 (link)).

For the cell survival assay, 1000 cells/well were plated in 96-well plates. Cells were cultivated with media supplemented with 10% FBS. After 24 h, the cells were treated with different concentrations of cisplatin (Selleck, S1166) or gemcitabine (Selleck, S1714) for 24 h. CCK-8 (Meilunbio, MA0218) was then added to the plate and incubated at 37 °C for 2 h. The optical density (OD) was read on a Bio Tek Eon Multi-Mode Microplate Reader. For the colony formation assays, 1000 cells/well were plated in 6-well plates, and the cells were cultivated with media supplemented with 10% FBS. After 10 days, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde for 2 h, the paraformaldehyde was discarded, and the cells were stained with crystal violet for 30 min and then photographed. An appropriate number of cells was seeded in 96-well plates and cultured to the normal growth stage. After incubation with 10 µM EdU solution for 2 h, the cells were fixed with 4% paraformaldehyde. ApolloR staining reaction solution (1×) was incubated for half an hour, and then 1 × Hoechst 33,342 reaction solution (Click-iT EdU Assays, Thermo Fisher, USA) was added. After staining, photos were taken using a fluorescence microscope. All the above operations were performed in the dark.

TNBC cell lines (TNBCC), MDA-MB-231 and MDA-MB-468 were purchased (ATCC, CA, U.S.A.). The Gemcitabine-resistance cell lines (GRC), MDA-MB-231-R and MDA-MB-468-R were produced by treating TNBCC with increasing Gemcitabine concentrations (0.1–15 nm) for 12 months (GI50 < 7.0) [18 (link)]. Gemcitabine was bought from AbMole (Beijing, China). Cells were cultured in DMEM (PM150210) containing 4500 mg/l glucose, 10% fine FBS (164220-100) and 100 U/ml penicillin–streptomycin (PB180122) in 5% CO₂, 95% O₂ condition at 37°C (Procell, Wuhan, China).
Each cell line was also incubated with NLRP3 agonist Nigericin sodium salt (NSS) and antagonist CY-09 separately (Selleck, Shanghai, China) to differentiate NLRP3 expression in both TNBCC and GRC. Wnt inhibitor Wnt-C59 was added into subgroups of GRC so as to inactivate signaling pathway (Selleck, Shanghai, China).
All the cell lines were exposed to the 0, 1, 3, 5 nM Gemcitabine, respectively, for 72 h for the following assays.