C-src Genes

Primary murine and human AEC were infected with PR/8 at MOI 0.5, as described previously^{11 (link)}. A/PR8 was diluted in PBS^-/- containing BSA and was added to the cells for 1 h, until the inoculum was removed and changed to infection medium (DMEM supplemented with BSA, pen/strep, L-Glutamine and trypsin) for further incubation. For co-culture experiments, AEC were seeded first, allowed to reach confluence, and were infected with A/PR8 or mock infected. 24 h p.i., BMDM were flow-sorted from the BALF of A/PR8-infected mice (BMDM1 were sorted at D7 p.i., BMDM2 were sorted at D21 p.i.) and added directly to the monolayer for 24 h. Cells were stained with Annexin V for apoptosis and Ki67 for proliferation assays and quantified by FACS. For western blot analysis, murine AEC were cultured in medium DMEM supplemented with pen/strep, L-glutamine and 10% heat-inactivated FCS. Cells were incubated at 37 °C for 5 days and thereafter treated with rPLET1 (40 ng/ml) for 12 h. Cell lysates were prepared in the same manner, as those used for the kinase activity assay described below. For the proliferation assays, murine AEC were cultured in DMEM enriched with pen/strep, L-glutamine and 2% FCS. Cells were incubated at 37 °C for 4 days and thereafter treated with rPLET1 (40 ng/ml) and/or MEK inhibitor U0126 (Promega, #V1121, 10 µM) for 12 h. For qPCR analysis, murine AEC were cultured in DMEM, pen/strep, L-glutamine and 10% FCS, after reaching the confluence they were infected with PR/8 at MOI 0.5, as described previously^{11 (link)} and treated with rPlet1 (20–40 ng/ml) and/or Src activator (Santa Cruz Biotechnology, Cat No #sc-3052, 10 µM) for 12 h. Cells were stored RLT buffer and stored in –80 °C for further qPCR analyses. For qPCR analyses of Src family-related genes, murine AEC were cultured as described above and treated with rPlet1 for 1 and 2 h. Cells were taken into RLT buffer and stored in –80 °C for qPCR analysis. In the stimulation assay of TR-AM with the AEC supernatant, AEC were cultured as mentioned above. When the cells reached confluence, they were infected with PR/8 at MOI 2.0 or mock infected, as described above and incubated at 37 °C for 12 h/24 h. The supernatant of these cells was used to stimulate the TR-AM (300,000 cells/cm² in a 24-well plate, seeded in RPMI medium supplemented with pen/strep, L-glutamine, 2% FCS and 2.5% HEPES, 6 h prior to the treatment) for 12 h. The supernatant of TR-AM cultures was used for soluble Plet1 quantification by ELISA.

Full-length human PANX1 in the pEGC Bacmam vector from our previous study was used(14 (link)). Mouse PANX1 (UniprotID: Q9JIP4) was synthesized by GenScript and subcloned into the pEGC Bacmam vector(45 (link)). The translated product contains the human or mouse PANX1 protein, a thrombin digestion site (LVPRGS), an enhanced GFP protein, and an 8x His tag. pCMV5 mouse Src was a gift from Joan Brugge & Peter Howley (Addgene plasmid # 13663; http://n2t.net/addgene:13663; RRID:Addgene_13663). The mouse Src gene was subcloned into the pEGN Bacmam vector(45 (link)). The translated product contains a mCherry protein, a thrombin digestion site, and the Src protein. Primers for site-directed mutagenesis were designed using QuikChange Primer Design website (https://www.agilent.com/store/primerDesignProgram.jsp) and synthesized by Eurofins Genomics. The QuikChange mutagenesis protocol was used to generate all the mutants of the study. Sanger sequencing was performed to identify positive clones.
Adherent HEK293T (ECACC, Catalogue Number: 96121229) cells were grown in DMEM media supplemented with 10% fetal bovine serum. Transient transfection was conducted using lipofectamine-2000 by following the manufacturer’s protocol. Specifically, the cells were cultured in 60mm Petri dishes until 80% confluency. Transfection solution was made by mixing 500ng of plasmid DNA, 4uL of lipofectamine-2000 reagent, and 100uL Opti-MEM media. After 10min incubation at room temperature, the DNA-lipid complexes were added to the cell culture and incubated at 37°C. The next day, 10mM sodium butyrate was added to the cells to boost protein expression. The cell culture was then grown at 30°C for another day before harvesting. The cell pellet was flash-frozen with liquid nitrogen and stored at −80°C. For co-transfection experiments, equal amounts of hPANX1 (250 ng) and mSrc (250 ng) plasmids were used in the transfection mixture. For Neuro2A cell culture (ATCC, Catalogue Number: CCL-131), EMEM media is used instead of DMEM. All the other procedures described above are the same for expressing proteins in Neuro2A cells.
For the western blot experiment, the cell pellet was lysed in TBS buffer (20 mM Tris pH 8.0, 150 mM NaCl) with protease an inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 2 mM pepstatin, 0.8 μM aprotinin and 2 μg/ml leupeptin), 1mM sodium orthovanadate and 1% glycol-diosgenin detergent for 30 min on ice. The lysate was clarified by centrifugation at 21,000rpm for 20 min and the soluble portions were mixed with 2x SDS loading buffer supplemented with 5% 2-Mercaptoethanol. The samples were resolved in precast gradient SDS gel (4–20%) or 7.5% PhosTag gel. A Chemidoc instrument was used to directly image the in-gel fluorescence signal after electrophoresis. Subsequently, the protein in the gel was transferred to the nitrocellulose membrane using the semi-dry transfer buffer (48 mM Tris base, 39 mM glycine, 20% methanol). The membrane is blocked in the TBST buffer (20 mM Tris pH8.0, 150 mM NaCl, 0.1% Tween 80) with 4% non-fat milk for 1 h at room temperature. Afterward, primary antibodies (1:2000 dilution) were incubated with the membrane overnight at 4°C. The next day, the membrane was washed with TBST buffer for 4 times, 10 min each before goat anti-rabbit IgG secondary antibodies were added (1:25000 dilution). After 1h, the secondary antibodies were discarded and the membrane was washed again with TBST for 4 times, 10 min each. The western blot signal was detected using ECL Pierce substrate and imaged using a Chemidoc instrument. A brightfield image was overlaid with the illuminance signal to visualize the position of protein markers in the membrane. The anti-PANX1 antibody is obtained from abcam (Catalogue number: ab124131). The anti-Src (Catalogue number: 36D10) and anti-pY100 (Catalogue number: 9411) antibodies are obtained from Cell Signaling. The anti-PANX1-pY198 (Catalogue number: ABN1681) and anti-PANX1-pY308 (Catalogue number: ABN1680) antibodies were obtained from Millipore Sigma.
For de-glycosylation experiment, Neuro2A/HEK293T cells expressing designated genes were solubilized in TBS buffer with protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 2 mM pepstatin, 0.8 μM aprotinin and 2 μg/ml leupeptin), 1mM sodium orthovanadate and 1% glyco-diosgenin detergent for 30min on ice. The samples were clarified by centrifugation at 20,000g for 30 min. The de-glycosylation reaction was made by mixing 16 uL of the supernatant with 2uL of PNGase F enzyme and 2 uL of GlycoBuffer 2 (10X). The control reaction replaced the PNGase F enzyme with water. The reaction was allowed to occur at room temperature overnight. The next day, the samples were mixed with 20 uL 2× SDS sample-loading buffer (Sigma) supplemented with 5% βME and resolved by SDS-PAGE. The gel was imaged in the ChemiDoc system by probing the GFP and mCherry fluorescence signal.
For the dephosphorylation experiment, mSrc-mCherry WT, Y529F, and K297M were expressed in HEK293T cells. The cells were lysed in TBS buffer (20mM Tris pH8, 150mM NaCl) with 1% glyco-diosgenin detergent for 30min on ice. After clarifying the non-soluble debris by centrifugation at 21,000rpm for 20min, 20 μL supernatant was mixed with 2.5 μL of 10X NEBuffer for Protein MetalloPhosphatases, 2.5 μL 10 mM MnCl₂ and 2 μL Lambda Protein Phosphatase (NEB). The dephosphorylation reaction was allowed to occur at 30 °C overnight. Control samples without adding Lambda Protein Phosphatase are placed on ice. The next day, the samples were mixed with 2× SDS sample-loading buffer (Sigma) supplemented with 5% βME and resolved by SDS-PAGE or Phos-tag gel. The gel was imaged in the ChemiDoc system by probing the mCherry fluorescence signal or western blot analysis.
For in vitro phosphorylation and mass-spectrometry analysis, the hPANX1 WT or mutants were expressed alone or co-expressed with mSrc Y529F mutant in HEK293T cells. Specifically, 50 μg of plasmid DNA (in the case of the co-expressing experiment, 25 μg of human PANX1 plasmid and 25 μg of mouse Src Y529F mutant plasmid was used) was mixed with 150ug of PEI 25K (Polysciences) in a and incubated at room temperature for 30 min. The DNA-PEI complex was then added to the suspension cell culture of HEK293T cells at a density of 2×10⁶ cells/ml. After growing at 37 °C for 8 h, 10 mM sodium butyrate was added, and the temperature was changed to 30°C for 40 h. The cells were then harvested and stored at −80 °C until purification. The protein purification procedure was described in our previous study(14 (link)).
The in vitro phosphorylation is conducted using the human Src-GST protein (Sigma-Aldrich, Catalogue number: S1076) according to the manufacturer’s protocol. Specifically, 1.5 μg of purified PANX1 protein is mixed with kinase assay buffer (25 mm MOPS, pH 7.2, 20 mM MgCl₂, 12.5 mM MnCl₂, 5 mM EGTA, 2 mM EDTA, 0.25 mM DTT), diluted 1:5 with 50 ng/μl bovine serum albumin (BSA). The reaction was supplemented with 0.25 mM ATP, and 0.3 μg recombinant human (active) Src-GST kinase. Samples were incubated for 1 h at 30 °C using a PCR thermocycler. After 1 h, 2x SDS buffer is mixed with the sample to stop the reaction. The sample is then resolved by an SDS-PAGE gel for subsequent western blot analysis.
Purified hPANX1 protein w- or w/o- co-expressing mSrc Y529F mutant was resolved in SDS-PAGE gel and the band corresponding to hPANX1 protein was cut and subjected to in-gel digestion with trypsin. Half of each digested sample was analyzed by nano LC-MS/MS with a Waters M-Class HPLC system interfaced with a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75μm analytical column at 350nL/min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with the Orbitrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS respectively. The instrument was run with a 3 s cycle for MS and MS/MS.
Data were searched using a local copy of Mascot (Matrix Science) with the following parameters. Enzyme: Trypsin/P; Database: SwissProt Human (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C) Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N/Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. Mascot DAT files were parsed into Scaffold (Proteome Software) for validation, filtering and to create a non-redundant list per sample. Data were filtered using 1% protein and peptide FDR and requiring at least two unique peptides per protein.
TsA201 cells were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol. The transfected cells were incubated at 37°C for 18–24 hours before electrophysiological measurements. Whole-cell recordings were performed using a Multiclamp 700B (Axon Instruments) and Clampex software, with pipettes of 3–5 MOhm resistance filled with an internal solution containing 145 mM NaCl, 10 mM Hepes, 10 mM EGTA, pH adjusted to 7.4. The external bath solution contained 160 mM NaCl, 10 mM Hepes, 3 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂, also adjusted to pH 7.4. During the recordings, voltage steps ranging from −100 mV to +140 mV were applied, each lasting 100 ms with a 20 mV increment between steps, and membrane currents were digitally recorded at 10 kHz and filtered at 2 kHz. To precisely measure the PANX1 channel's current, carbenoxolone disodium salt (CBX), a blocker of the channel, was added to the bath solution at a final concentration of 0.1 mM, and CBX-sensitive currents were subsequently calculated by comparing the difference in current amplitude in a cell with and without the presence of CBX.