Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Ultra-pure LPS (upLPS) from Escherichia coli (O111:B4) was obtained from Invivogen (San Diego, CA, USA). Anti-LC3, anti-TLR4 and anti-Beclin-1 were from Abcam (Cambridge, UK). Vimentin was from Boster Biological Technology (Wuhan, China). Secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-cytokeratin 18 (CK-18), 3-methyladenine (3-MA), wortmannin (Wm), monodansylcadaverine (MDC), 3-[4, 5- dimethylthiazol −2 -yl]-2, 5-diphenyltetrazolium bromide (MTT), 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI), Polymyxin B (PMB) and gentamicin were from Sigma-Aldrich Co.. Fluorescent E.coli (K-12 strain) BioParticles, Lipofectamine 2000 and Annexin V-FTIC Apoptosis Detection Kit were from Invitrogen Life Technologies (Carlsbad, CA, USA). The green fluorescent protein (GFP)-LC3 fusion plasmid was kindly provided by Professor Xiaofeng Zhu. Beclin-1 specific small-interfering RNA (siRNA) and TLR4 specific siRNA was from Shanghai GenePharma Co., Ltd. (Shanghai, China).
Cell culture and viability studies
The simian virus 40 (SV40)-immortalized human peritoneal mesothelial cell line (HMrSV5) has been described previously [17, 18]. HMrSV5 cells were cultured in DMEM/F12 medium containing 10% FBS in a humidified atmosphere consisting of 95% O2 and 5% CO2 at 37°C. The cell line was identified by phase contrast microscopy and immunofluorescence analysis. The effect of LPS on the viability of cultured HMrSV5 cells was determined by MTT assay [17, 19] and flow cytometric analysis .
Immunofluorescence co-staining of CK-18 and vimentin
After fixed in 4% paraformaldehyde for 15 min at room temperature, cells were permeabilized with 0.1% Triton X-100, followed by incubating with 5% BSA in PBS for 60 min at room temperature to block nonspecific binding. Then cells were stained with mouse anti-vimentin and mouse anti-cytokeratin 18 in PBS containing 5% BSA at 4°C overnight. Cells were incubated with secondary antibody for 1 hour at room temperature. Finally, coverslips were sealed with mounting medium. Images were collected by an LSM 510 confocal immunofluorescence microscope (Carl Zeiss, Inc., Jena, Germany).
Measurement of autophagy by immunoblotting
Equal amounts of protein were separated on 15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline for 60 min at room temperature, the membranes were incubated at 4°C overnight with primary antibody. Following incubation with secondary antibodies, the protein bands were detected by an enhanced chemiluminescence system. Densitometric quantification of band intensities was determined using an image analysis program (FluorChem 8900; Alpha Innotech Corp, San Leandro, CA, USA).
Transfection of HMrSV5 cells with GFP-LC3 plasmid
HMrSV5 cells at 50-70% confluence were transiently transfected with 2 μg/ml GFP-LC3 plasmid DNA per dish which was performed with Lipofectamine 2000. After treatments as shown in the figure legends, the cells were fixed with 4% paraformaldehyde and nuclei were labeled with DAPI. Autophagy was assessed by the formation of fluorescent autophagosome puncta. Cells with more than 10 puncta indicated the GFP-LC3 positive cells. Values were calculated from 100 cells/sample.
Detection of autophagic vacuoles by MDC
Treated cells were washed 3 times with PBS and then incubated with 0.075 mM MDC in DMEM/F12 at 37°C for 10 min. The cells were then immediately observed under a fluorescence confocal microscope equipped with the appropriate filters, where MDC exhibits autofluorescence at wavelengths of 365 and 525 nm for excitation and emission, respectively.
SiRNA gene silencing of Beclin-1 or TLR4
Knock down of Beclin-1 or TLR4 in HMrSV5 cells was obtained by utilizing complementary sense and antisense oligonucleotides to human Beclin-1 or TLR4 (Beclin-1 siRNA: sense, 5′-CUCAGGAGAGGAGCCAUUUTT-3′, antisense, 5′-AAAUGGCUCCUCUCCUGAGTT-3′; TLR4 siRNA: sense, 5′-CCACCUCUCUACCUUAAUATT-3′, antisense, 5′-UAUUAAGGUAGAGAGGUGGTT-3′). A non-targeting siRNA pool was applied as a control (negative control siRNA for Beclin-1 siRNA: sense, 5′-UUUAGCCGAUACUGCCUAGTT-3′, antisense, 5′-CUAGGCAGUAUCGGCUAAATT-3′; negative control siRNA for TLR4 siRNA: sense, 5′-UUCUCCGAACGUGUCACGUTT -3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′). HMrSV5 cells were transfected with 1 μg of each duplex using Lipofectamine 2000.
Bacterial killing assay
The E. coli strain (ATCC: 25922) was resuspended in saline without antibiotics prior to infection of HMrSV5 cells. HMrSV5 cells were plated at a density of 5.0 × 105 cells per well and then treated as shown in the figure legends. E.coli was added at a MOI of 20 and incubated at 37°C for 1 hour (t = 0). Then, HMrSV5 cells were washed with cold PBS to remove non-adherent bacteria and stop additional bacterial uptake. Meanwhile, gentamicin (10 μg/ml) was added to limit the growth of extracellular bacteria. The cells were lysed at further 30 min, 60 min and 90 min respectively (t = 30, 60, 90) with sterile distilled water. The number of viable bacteria (colony forming units, c.f.u.) released from cells was detected by plating serial dilutions of bacteria on Luria Bertani (LB) agar plates. Bactericidal activity was analyzed by the percentage of remaining E.coli (%) which was was calculated as (remaining bacteria at each time point/bacteria present at 0 min) × 100.
Analysis of E. coli co-localization with autophagosomes by immunofluorescence
Cells were infected with E. coli (K-12 strain) BioParticles at a MOI of 20:1 for 1 hour. Following phagocytosis, cells were treated as shown in the figure legends. Subsequently, the cells were washed 3 times with PBS and incubated with 0.075 mM MDC in DMEM/F12 at 37°C for 10 min. The cells were observed under a fluorescence confocal microscope equipped with the appropriate filters where MDC exhibits autofluorescence at wavelengths of 365 and 525 nm for excitation and emission, respectively.
Transmission electron microscopy
Cells were fixed at room temperature with former fixative (0.1 mol/l PBS containing 2.5% glutaraldehyde, and 2% paraformaldehyde). The samples were postfixed with 1% osmium tetroxide, subsequently incubated with 1% uranyl acetate, then dehydrated through increasing concentrations of ethanol, and gradually infiltrated in LX-112 medium. Thin sections of each sample were stained with 2% uranyl acetate and lead citrate, and then analyzed under a JEM 1010 transmission electron microscope (JEOL, USA, Inc., Peabody, MA).
Quantitative data were expressed as means ± standard deviations. The statistical differences in multiple groups were determined by one-way ANOVA followed by Student–Neuman–Keuls test. Statistical differences between two groups were analyzed by two-tailed unpaired Student’s t-test. All calculations were performed using SPSS 13.0 statistical software (Armonk, NY, USA). A value of p < 0.05 was considered significant.