Materials
Rabbit polyclonal antibodies recognizing cleaved Caspase 3 and the N-terminal of GSDME were purchased from Abcam(Catalogue No.ab13847 and No.ab175614,Cambridge, UK). Rabbit monoclonal antibodies to GSDME-N-terminal or JNK1+JNK2+JNK3 or JNK1 + JNK2 + JNK3 (phospho T183+T183+T221) also from Abcam(Catalogue No.ab215191 or No.ab179461 or No.ab124956,Cambridge, UK).TNF-α was purchased from InvivoGen (Catalogue No.rcyc-htnfa,San Diego, USA).Cycloheximide(CHX) was purchased from Sigma-Aldrich (Catalogue No.66-81-9,St.Louis, MO, USA).TNF-α+ CHX acts as an inducer of apoptosis[11].The JNK inhibitor SP600125 was purchased from Selleck(Catalogue No.S1460, Houston, TX, USA).
Cell culture
Human tubular epithelial cell line (HK2) was from Bio-Rad Laboratories (Shanghai, China) and the cells cultured in DNME/F12 media containing 10% FBS and 1% penicillin streptomycin. The cells were stimulated with TNF-α(20 ng/ml) and CHX(10μg/ml) to induce cell death, with or without pretreatment with SP600125 (30μM) for 1 h.
LDH release assay
After TNF-α and CHX treatment, the activity of LDH released into cell culture supernatants was measured by using the LDH release kit (Promega) according to the manufacturer’s protocol. The supernatant LDH activity was expressed as a percentage of total LDH in the cell lysate.
Microscopy
To examine the morphological changes of apoptotic or secondary pyroptotic cells, HK2 cells were seeded in the 6-well plates and subjected to indicated treatments. Bright field cell images were captured using the Optical inverted microscope (Olympus, Japan).All the image data displayed represents at least three random field of view.
HK2 cell Hoechst/PI fluorescent staining
HK2 cells were seeded in the 24-well plates and treated with TNF-α and CHX. Then, cells were stained with Hoechst (Beyotime, China) and PI(BD, USA).Images were collected with a fluorescence microscope(Carl Zeiss, Germany).
Immunofluorescent analysis of GSDME
After treatment with TNF-α and CHX, the expression of GSDME was detected by immunofluorescence. Cells were fixed with 4 % paraformaldehyde, permeabilized with 0.2 % Triton X-100, and blocked with 5 % fetal bovine serum (FBS). Then, the cells were stained with rabbit anti-GSDME antibody, followed by incubation with a Cy3-conjugated goat secondary antibody against rabbit IgG (Servicebio,China,).Finally, the cells were stained with DAPI(Thermo Fisher
Scientific, USA). Images were captured and analyzed with a fluorescence microscope (Carl Zeiss, Germany).
Western blot
Cells or tissues were lysed in RIPA protein lysate (Beyotime, China). Cell lysates were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Roche, Swit). Blots were probed with appropriate antibodies. Expressions of full length-GSDME and GSDME-N-terminal were detected using anti-GSDME antibodies. Data were analyzed by Image J Software.
Pristane induced lupus (PIL) models
As described previously,female BALB/ C mice at 6-8 weeks were intraperitoneally injected with 0.5ml Pristane(Sigma-Aldrich)[16]. Urine protein was detected at 6 weeks after modeling, and urine protein score measured once every two weeks. Albustix test paper was used to determine the urinary protein. The mice were stimulated to urinate by gentle massage of the abdomen, and urine was collected. The fresh urine was dropped into the reaction area of the test paper, and the results were read within 1min. The urine protein score was determined according to the comparison between the color degrees of the reaction area and the standard color band. If urine protein score was more than 1 point in two consecutive tests, it was decided that the modeling was successful.
PIL mice were divided into three groups, named vehicle only control group, PIL group (n=9), and PIL+SP600125 group(n=9) respectively. Six normal mice were treated with vehicle as control group. In the PIL group, mice received the same amount of vehicle solution (DMSO and PBS). In PIL+SP600125 group, mice were injected intraperitoneally with SP600125 dissolved in 2% dimethyl sulfoxide (DMSO) in PBS at a dose of 30 mg/kg body weight once per day [17].
Immunofluorescence analysis of GSDME/Caspase-3 p17
The frozen kidney sections were blocked with 5% fetal bovine serum and then stained with rabbit anti-GSDME antibody(Abcam, Catalogue No.ab215191) and rabbit anti-caspase3 p17 antibody (Abcam, Catalogue No.ab13847), followed by staining with a second Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (Servicebio, Catalogue No.GB21303).For assessment of mouse IgG deposition in kidneys, the frozen kidney sections were stained with Alexa Fluor 555-conjugated goat anti-mouse IgG(Abcam, Catalogue No.b150114).
Assessment of histopathological changes
Renal tissues were fixed with 10% formalin and paraffin-embedded for tissue sectioning. The sections were then stained with hematoxylin and eosin (H&E). Histopathological changes were examined by pathologists unaware of the experimental information. Austin score to grade lupus disease activity was used as described previously [18].
Statistical analysis
All data were expressed as mean ± standard deviation, analyzed by SPSS 20.0 software, and plotted by GraphPad Prism 7.0 . One-way ANOVA was used for the comparison of the mean between groups.The difference was considered statistically significant where p<0.05 between comparisons.