General In Vitro experimental methods

2.3.1. Cell culture

NIH 3T3s (mouse embryonic fibroblasts cell line) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 10% donor bovine serum (DBS, Gibco) and 2% penicillin/streptomycin (P/S, Gibco) in 5% CO2 at 37 °C. Cells were washed prior to all experiments to remove traces of

antibiotics. All experimental media contained no antibiotics. Cells will be referred to as 3T3s hereafter.

2.3.2. Transfection of fibroblasts

A stable 3T3 cell line with Cx43 expression knocked down, and an empty vector control, was previously created by Dr Peter Cormie, a post doc previously of University College London, and kindly made available for my use (Mendoza- Naranjo, Cormie, A. Serrano, et al. 2012). The knocked down cell line was made by transfecting 3T3 cells with a pSuper.retro.puro vector (OligoEngine) containing the Cx43 specific shRNA sequence (GGTGTGGCTGTCAGTGCTC), kindly donated by W.H. Moolenar. An empty vector control (hereafter referred to as EV) was also created using a retroviral pSuppressor vector (Imgenex Corporation). GP2-293 (Clontech) packaging cells were seeded at 20% confluence in DMEM with 10% foetal calf serum (Gibco) and transfected with 15 µg of retroviral plasmid by calcium phosphate precipitation to produce retroviral supernatants. The culture media was collected after 2 days and filtered, and used to infect 3T3 fibroblasts. 3T3 fibroblasts containing the Cx43 shRNA retrovirus were selected and maintained by incubation with 2 µg ml-1 of puromycin (Invitrogen). Cells containing the Psuppressor empty vector were selected and maintained by incubation with 500 µg ml-1 _{of gentamicin (Sigma-} Aldrich).

2.3.3. Live imaging of proliferation and propidium iodide uptake

Cells were grown in flat bottom 96 well plates. Prior to imaging, cells were incubated with media containing 0.5 µg ml-1 Hoechst for 30 minutes. This concentration was selected following a pilot study and was not toxic to the cells. The media containing Hoechst was removed, the cells washed with PBS, and media (DMEM supplemented with 10% DBS) or BCM was added which contained 1 µg ml-1 of propidium iodide. S.aureus or HKSA was added as appropriate. Cells were then live imaged every hour for 24 hours on an ImageXpress® Micro XL system (Molecular Devices). MetaXpress 5.0 software (Molecular Devices) cell count analysis was used to determine the total number of cells in each image (Hoechst positive) and the number of dead cells (cells that had taken up propidium iodide from the media).

2.3.4. Western blotting

Protein extraction

Cells were grown in 6 well plates to 90-100% confluence before commencing experiments. They were incubated in experimental conditions for the time indicated in the figures. Following the incubation period, cells were placed on ice and washed with cold PBS. Samples were harvested using cold RIPA buffer (see table 2.1) supplemented with phosphatase inhibitors and protease inhibitors (Roche), and using a cold cell scraper (Greiner bio-one). Samples were sonicated for 30 seconds in a USC100T Ultrasonic cleaner (VWR), then kept on ice for 30 minutes, before centrifuging for 30 minutes at 15000 x g at 4 °C. Protein concentrations were quantified using a Pierce BCA protein assay kit (ThermoScientific), following the manufacturer’s protocol and determining absorbance with an ELx800 Absorbance Microplate Reader (BioTek) using Gen5 plate reading software (BioTek).

Western blots

Equal amount of protein were re-suspended in 4x Laemmli buffer (Bio-Rad), supplemented with β-mercaptoethanol as per the manufacturer’s instructions. Samples were then boiled at 95 °C for 5 minutes. Samples were separated on 10% Mini-Protean® _{TGX™ gels (Bio-Rad), alongside a Precision Plus Protein™} Dual Colour Standard (Bio-Rad) in a mini protean Tetra cell tank (Bio-Rad) in T/G/S running buffer (Bio-Rad) at 100 volts for up to 2 hours. Samples were then transferred to a Hybond™ C nitrocellulose membrane (Amersham Biosciences) for 1 hour at 100 volts in western blot transfer buffer (see table 2.1. for recipe). Membranes were blocked in western blot blocking buffer (see table 2.1) for 30 minutes. Blots were incubated with primary antibodies (see table 2.2) made in western blot blocking buffer (see table 2.1) for the time indicated. They were then washed 3 times in PBS containing 0.1% Tween 20, before incubating with the relevant secondary antibody (also made in western blot blocking buffer) for the time indicated. Secondary antibodies were horse-radish peroxidase (HRP) conjugated and protein levels were visualised using an enhanced chemiluminescence (ECL) system (ThermoScientific) on a ChemiDoc™ MP Imaging System (Bio-Rad). Densitometry was used to quantify protein bands using Quantity One® Version 4.6.3 software (Bio-Rad). α-tubulin or GAPDH

expression were used as loading controls, and the ratio of Cx43 to α-tubulin or GAPDH was determined.

Table 2.2. Table of primary and secondary antibodies used to do western blots.

Concentrations and incubation periods are shown.

Antibody antigen Species raised in Company and code Concentration Incubation time

Anti-Cx43 Rabbit Sigma-Aldrich (C6219) 1:4000 1 hour at room temperature Anti-α- tubulin Rat Abd (MCA77G) 1:2500 1 hour at room temperature Anti-GAPDH Rabbit Sigma-Aldrich

(G9545) 1:1000 1 hour at room temperature Anti-rabbit HRP Goat Sigma-Aldrich (A6154) 1:1000 1 hour at room temperature Anti-rat HRP Goat CalbioChem

(401416)

1:2000 1 hour at room temperature

2.3.5. Immunofluorescence and auto-fluorescence

Immunofluorescent staining

Cells were grown on Millicell EZ slides (Millipore) to 90-100% confluence before commencing experiments. They were incubated in experimental conditions for the time indicated in the figures. Following the incubation period, cells were washed with PBS then fixed with 4% paraformaldehyde (PFA) for 5 minutes. They were washed twice more with PBS, then blocked in immunofluorescence blocking buffer for 30 minutes (see table 2.1). Samples were incubated with rabbit anti-Cx43 antibody (Sigma-Aldrich, C6219, 1:2000) at room temperature for 1 hour. Samples were washed with immunofluorescence blocking buffer, then incubated with goat anti-rabbit Alexa Fluor® 488 (Invitrogen, A11008, 1:400) for 1 hour at room temperature. Cells incubated with BCM for 7 days had high levels of auto-fluorescence when excited with a 488 laser, so were instead incubated with goat anti-rabbit Alexa Fluor® 633 (Invitrogen, A21070, 1:400). Samples were counterstained with Hoechst (Sigma-Aldrich, H33342, H33258, 1:50,000), washed in PBS and mounted in Citifluor® (Glycerol/PBS solution, Citifluor Ltd).

Confocal Imaging

Immunofluorescence was imaged on a Leica SPE upright or inverted confocal microscope. Optimal gain and offset were set in advance and kept constant during image acquisition. A 488 nm laser was used to excite Alexa 488, a 633 nm laser was used to excite Alexa 488 and a 405 nm laser to excite the Hoechst stain. Where cells were incubated with BCM for 7 days a 488 nm laser was instead used to determine the extent of auto-fluorescence.

The regions imaged were selected at random by only observing the Hoechst channel, and single optical sections were obtained. A minimum of three regions was imaged of each biological replicate. A negative control, using no primary antibody but following all other conditions, was done for each experiment and used to threshold images during quantification.

Quantification of immunofluorescence and auto-fluorescence

Images were imported into Image J (National Institute of Health) for quantification. 8 bit images were identically thresholded, to create a binary image. The total area of connexin expression or auto-fluorescence was measured and was normalised to the number of nuclei in each image. The results from experimental replicates were averaged.