Culture of human EndoC-βH1 cells, HeLa cells and human islets and cell treatment

EndoC-βH1 cells, a human pancreatic beta cell line provided by Dr. R. Scharfmann (University of Paris, France) [16], were cultured in Matrigel-fibronectin-coated plates as previously described [17]. These are slowly proliferating cells, dividing every 5 days [16]. EndoC-βH1 cells have been shown to be chronically infected by a xenotropic mouse retrovirus [18]. However, our RNAseq data indicate that they do not present an increased basal innate immune response and have also a similar immune response when treated with IFNα as compared to primary human islets [19]. The human cervical cancer cells HeLa were grown in Dulbecco’s modified Eagle’s medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and 2% penicillin–streptomycin (Lonza). These cell lines were free of mycoplasma infection, as determined by monthly testing using the MycoAlert Mycoplasma Detection kit (Lonza).

Human islets from 6 non-diabetic organ donors (see Additional file 1) were isolated in Pisa, Italy, following a protocol previously described [20] with the approval of the local ethics committee and sent to Brussels for dispersion and experiments [21].

Each experiment considered as n = 1 corresponds to one independent biological observation, i.e. EndoC-βH1 cells and HeLa cells from different passages or human islets from different donors.

As positive controls for some of the parameters studied, the cells were treated with human IFNα (PeproTech, Rocky Hill, NJ, USA) at 2000 U/ml or transfected with 1 µg/ml of the synthetic dsRNA analog polyinosinic-polycytidylic acid (Sigma-Aldrich, Saint-Louis, MO, USA) as previously described [22].

RNA interference

EndoC-βH1 cells or dispersed human islets were transfected overnight with 30 nmol/l of different siRNAs, the medium was then changed, and cells kept in culture for 48 h to 6 days to allow gene silencing. Transfection was performed using siRNA targeting PNPT1 (PNPT1#1: 5′-GCUGCACUACGAGUUUCCUCCUUAU-3′, PNPT1#2: 5′-CCUUUGGUGGUUGACU ACAGACAAA-3′, and PNPT1#3: 5-GGGCAGUACGAAUAGGAAUAAUUGA-3′; HSS131758, HSS131759 and HSS131760 respectively, Thermo Fisher Scientific, Waltham, MA, USA), SUV3 (5′-GGCCUCUGGACAAGAAUGAAGUAAA-3′; HSS110378, Thermo Fisher Scientific) or ADAR (5′-TTCCGTTACCGCAGGGATCTA-3′; 1027416, Qiagen, Venlo, The Netherlands) using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA). In experiments with double transfection, we mixed 30 nmol/l of each siRNA used. Allstars Negative Control siRNA (siQ; Qiagen) was used as a negative control.

Viral infection

The prototype strain of enterovirus (CVB5 / Faulkner) was obtained from the American Type Culture Collection (ATCC, Old Town Manassas, Virginia, USA). GMK cells were used to amplify viruses and their identity was confirmed by a plaque neutralization assay with CVB5 anti-sera. EndoC-βH1 cells were inoculated for 1 h at 37 °C with CVB5 at a multiplicity of infection (MOI) of 5 based on our previous studies [23]. The inoculum virus was then removed, and cells were cultured in medium containing FBS during 24 h to allow viral replication.

mRNA extraction and real-time PCR

The Dynabeads mRNA DIRECT purification kit (Invitrogen) was used to purify polyadenylated mRNA from cultured cells, and reverse transcription was performed using the Reverse Transcriptase Core kit (RT-RTCK-03, Eurogentec, Liège, Belgium). We used the SsoAdvanced Universal SYBR Green Supermix (BIO-RAD, Hercules, CA, USA) to perform the real-time PCR amplification reactions. We generated standard curves to allow adequate quantification of our data, and corrected gene expression by the housekeeping gene β-actin, as its expression is not affected by the conditions used in this study (data not shown). The list of primers used in this study can be found in Additional file 2.

Protein extraction and western blot analysis

Cells were washed with PBS and lysed using 1X Laemmli Sample Buffer (60 mM tris(hydroxymethyl)aminomethane pH 6.8, 10% Glycerol, 1.5% Dithiothreitol, 1.5% 2-mercaptoethanol, 2% Sodium dodecyl sulfate and 0.005% bromophenol blue). To confirm PNPT1 and ADAR knockdown, we used a rabbit polyclonal anti-PNPT1 antibody diluted 1/2000 (ab96176, Abcam, Cambridge, UK) and a rabbit monoclonal anti-ADAR1 antibody diluted 1/1000 (#14175, Cell Signaling Technologies, Danvers, MA, USA). To evaluate the type I IFN activation, we measured STAT1 and STAT2 phosphorylation using rabbit anti-phospho antibodies (pSTAT1: #9167 and pSTAT2: #8841, Cell signaling Technologies) diluted 1/1000, total STAT1 and STAT2 expression using a rabbit anti-STAT1 (sc-346, Santa Cruz Biotechnology, Dallas, USA) and a monoclonal rabbit anti-STAT2 (#72604, Cell Signaling Technologies) antibody, both diluted 1/1000. We have previously shown that total STAT1 and STAT2 expression are up-regulated in beta cells following exposure to a type I IFN [19]. We normalized our data for the expression of the housekeeping gene β-actin using a polyclonal rabbit anti-β-actin antibody diluted 1/2000 (#4967, Cell Signaling Technologies). A secondary donkey anti-rabbit antibody coupled with the horseradish peroxidase (711-036-152, Jackson ImmunoResearch Laboratories, Wes Grove, PA, USA) was used to detect the signal. Immunoreactive bands were visualized using the SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific), detected using ChemiDoc XRS + (BIO-RAD), and quantified with the Image Studio Lite v5.2 software (LI-COR Biosciences).

Immunofluorescence

After different periods post-siRNA transfection or viral infection, cells were fixed with PFA 4% for 15 min followed by permeabilization with Triton X-100 0.3% for 10 min. Cells were blocked in PBS-BSA 3%—Tween20 0.02% during 30 min and then incubated overnight with the monoclonal mouse J2 anti-dsRNA antibody diluted 1/200 in blocking buffer (10010200, Scicons, Hungary). Cells infected with CVB5 were stained for the viral capsid protein VP1 using the enterovirus-specific polyclonal antiserum KTL-510 [24]. In dispersed human islets, beta cells were also stained for insulin using a polyclonal guinea pig anti-insulin ready-to-use antibody (IR00261-2, Agilent, Santa Clara, CA, USA) and fibroblasts were stained using the rabbit polyclonal anti-vimentin antibody diluted 1/200 (ab137321, Abcam). The signal was detected using Alexa Fluor 568 rabbit anti-mouse, Alexa Fluor 488 goat anti-guinea pig and Alexa Fluor 647 donkey anti-rabbit (A11061, A11073 and A31573 respectively, Thermo Fisher Scientific) secondary antibodies at 1/200. Hoechst 33342 (Sigma-Aldrich, Saint-Louis, MO, USA) was used for nuclear staining. The coverslips were mounted with the Glycergel mounting medium (C056330-2, Agilent) and immunofluorescence was visualized using a Zeiss microscope equipped with a camera (Zeiss-Vision, Munich, Germany). Images were acquired at ×40 magnification and analysed with the AxioVision software.

Statistical analysis

Data are expressed as means ± SEM. A normality test was performed to evaluate the distribution of the observed determinations. Paired or unpaired t test were performed to assess statistical significance and results with p < 0.05 were considered statistically significant.