Cell line
Immortalized mouse macrophages (IMM) (a generous gift from Dr. Eicke Latz 47,48) were cultured in complete DMEM (Gibco, Grand Island, NY, USA) complemented with 10% FBS (Gibco), in a 5% CO2 incubator at 37oC, maintained at low densities and passaged until reaching the confluent state, usually every 3–4 days on sterile tissue culture plates.
Real-time PCR
The RNeasy kit (QIAGEN, Hilden, Germany) was used for isolation of total RNA from cell culture. Total RNA (1ug) was converted to cDNA using MultiScribe reverse transcriptase (Thermo Scientific, Rockford, IL, USA), and real-time PCR was performed using Power SYBR Green PCR Master Mix with specific primer to determine gene expression level. The primers are listed in supplementary Table 2. The ΔΔCt method was used for determining relative gene expression and beta-actin was used as a housekeeping gene.
MARCKS cloning
Mouse MARCKS was amplified by PCR using primer containing 15 bp overlap complementary to the vector ends at the 5’ end of the forward and reverse primers (5’-AGATCTGCCGCCGCGATCGCATGGGTGCCCAGTTCTCC-3’) and (5’- GCGGCCGCGTACGCGTTTACTCGGCCGTTGGCGC-3’). The PCR products were cloned into third-generation lentiviral vector, pLenti-C-mGFP (OriGene, Rockville, MD, USA) using infusion enzyme (TAKARA, Kusatsu, Japan). Plasmid sequences were verified by Sanger sequencing at Psomagen Inc (Rockville, MD, USA).
Stable expression by lentiviral transduction
HEK293FT cells (Thermo Scientific) were seeded on 6 wells cell culture plates (250,000 cells/well) overnight prior to transfection. pLenti-C-mGFP with MARCKS sequence were transfected to HEK293FT cells via TranIT-TKO (MirusBio, Madison, WI, USA). Conditioned media containing lentiviral particles were harvested 24h and 48h post-transfection and filtered using 0.45 µM filters (Millipore, Carrigtwohill, Ireland) before being used to infect IMMs cells. Cells were monitored under fluorescence microscopy. Stable cell transfectants were selected using puromycin (5 µg/mL) (Gibco) containing media. Single cells were isolated and cultured for 10–14 days to obtain homogenous clonal populations. MARCKS expression levels were confirmed using western blots.
CRISPR CAS 9-mediated MARCKS gene knockout
CRISPR Cas9-mediated MARCKS knockout was generated by Cas9 ribonucleoprotein using the Neon Transfection System Starter Pack (Invitrogen) according to the manufacturer’s instruction. Briefly, three single guide RNA (sgRNAs) that contain MARCKS targeting sequences and a Cas9 nuclease-recruiting enzyme were designed using CRISPR-Cas9 guide RNA design checker (www.idtdna.com/CRISPR-Cas9). IMMs were collected and washed once with PBS. Then, 4x106 cells were electroporate with Cas9 protein v.3 (Integrated DNA technologies, San Diego, CA, USA), complexed with 3 sgRNAs (seed sequences: 5’-CACGTCGTCGCCCAAGGCGG-3, 5’-TGGCCACGTAAAAGTGAACG-3’, 5’-AGCAAGAAGGAGTCGGGCGA-3’) in the nucleofector buffer (Invitrogen, Waltham, MA, USA) and cultured for 3–4 days. The knockout candidates were screened using western blot and mass spectrometry.
Western blot analysis
IMMs were lysed in Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with Halt protease inhibitor (Thermo Fisher Scientific) and phosphatase inhibitor cocktails (Thermo Fisher Scientific). Total protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (20 µg total protein) were loaded into NuPAGE 4 to 12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and run at 200 V for 1 h. The proteins were transferred to the PVDF membrane (Thermo Fisher Scientific). The membranes were blocked in 5% BSA overnight and then incubated with specific primary antibodies for one hour followed by incubation with secondary antibodies for one hour. The antibodies are listed in supplementary Table 3. The blots were developed using ECL substrate (Thermo Fisher Scientific) The results were visualized using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). The densitometric analysis of western blot results was performed using Image J.
ELISA
TNF and IL6 were quantified using ELISA kits (Invitrogen, Vienna, Austria) kit according to the manufacturer's instructions. Briefly, IMMs were treated with 100 ng/mL LPS (LPS from Salmonella minnesota R595, Enzo Life Sciences) for 6 hours in quadruplicate and the supernatants were collected at the indicated time points. Capture antibody was applied to 96 well plates overnight followed by blocking with 1x diluent buffer for one hour at room temperature, then supernatants with appropriate dilution factor were put in each well for two hours at room temperature. Detection antibody and streptavidin-HRP were sequentially added to the assay plate and incubated one hour and 30 minutes respectively. ELISA washing steps were performed 4 times with 0.05% tween 20 (Thermo Fisher Scientific) in PBS (PBS-T). ELISAs were developed with TMB substrate for 10 mins followed by a stop solution. Absorbances were determined using a microplate reader. The results were interpreted in comparison to the standard curve.
Seahorse Assay
IMMs in different experimental groups (control vs LPS 6 hours) were dispersed into monolayers for the measurement. Mitochondrial stress tests were performed at 37°C using the Seahorse XFe96 bioanalyzer (Seahorse Bioscience). IMMs were seeded at 2× 105 cells per well on the Seahorse analysis plates. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) for the mitochondria were measured in XF media (containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) under basal conditions and in response to 1.5 µM oligomycin, 1 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP), and 0.5 µM rotenone and antimycin A. All of the values were normalized with total protein using Wave software (Agilent).
Mass spectrometry
Sample preparation. WT and MARCKS knockout IMMs were seeded on a 10 cm dish and cultured overnight, the cells were unstimulated or stimulated with 100 ng/mL LPS for 6 hours. The indicated samples were lysed using RIPA buffer (Thermo Fisher Scientific). For each sample, 500 ug of total protein mass was run on Bis-Tris NuPAGE gel (Invitrogen) as described above. The gels were fixed using fixing solution (47.5% methanol and 5% glacial acetic acid) for 30 minutes at room temperature. The fixed gels were stained using PageBlue® protein staining solution (Thermo Fisher Scientific) for one hour at room temperature, and then destained overnight with ddH2O at 4°C. Each lane was cut into 1 mm3 pieces using razor blades and the gel pieces were put in 1.5 mL Eppendorf tubes and processed using in-gel digestion according to the published protocol and summarized below49.
In-gel protein digestion: Briefly, 500 µL of acetonitrile (ACN) were added to the gel pieces and incubated for 10 minutes at room temperature before removing all the supernatant from the tube. For reduction of disulfide bond, 50 µL of DTT in 100 mM ammonium bicarbonate (ABC) was added to the tube and incubated at 56 ° C for 30 minutes. Then, 50 µL of 55 mM of 2-chloroacetamide (CA) in 100 mM ABC solution were added and incubated for 20 minutes at room temperature in the dark. Then, trypsin solution (Promega, Madison, WI, USA) (1:25 w:w ratio) was added and the samples were incubated at 37°C for 17 hours. Following incubation, peptides were cleaned and desalted using C18 ZipTip tips (Millipore) according to the manufacturer’s instruction.
Mass Spectrometry. An Orbitrap Fusion Eclipse with an EASY-Spray ion source (Thermo Fisher Scientific, San Jose, USA) coupled to a Thermo UltiMate 3000 (Thermo Fisher Scientific) was used for LC-MS/MS experiments. 1 ug of total peptides were injected for LC-MS/MS analysis. Peptides were trapped on an Acclaim C18 PepMap 100 trap column (5 µm particles, 100 Å pores, 300 µm i.d. x 5 mm, Thermo Fisher Scientific) and separated on a PepMap RSLC C18 column (2 µm particles, 100 Å pores, 75 µm i.d. x 50 cm, Thermo Fisher Scientific) at 40 ⁰C. The LC steps were: 98% mobile phase A (0.1% v/v formic acid in H2O) and 2% mobile phase B (0.1% v/v formic acid in ACN) from 0 to 5 min, 2–35% linear gradient of mobile phase B from 5 to 155 min, 35–85% linear gradient of mobile phase B from 155 to 157 min, 85% mobile phase B from 157 to 170 min, 85–2% linear gradient of mobile phase B from 170 to at 172 min, 2% of mobile phase B from 172 to 190 min. Eluted peptides were ionized in positive ion polarity at a 2.1 kV of spraying voltage. MS1 full scans were recorded in the range of m/z 375 to 1,500 with a resolution of 120,000 at 200 m/z using the Orbitrap mass analyzer. Automatic gain control and maximum injection time were set to standard and auto, respectively. Top 3sec data dependent acquisition mode was used to maximize the number of MS2 spectra from each duty cycle. Higher-energy collision-induced dissociation (HCD) was used to fragment selected precursor ions with normalized collision energy of 27. MS2 scans were recorded using an automatic scan range with a resolution of 15,000 at 200 m/z using the Orbitrap mass analyzer. Data analysis, label free quantification and statistical analysis were performed using Proteome Discoverer 2.5 (Thermo Fisher Scientific). Briefly, the raw files were searched against the mouse uniport database with the list of common protein contaminants. The groups and conditions were specified to obtain the quantification ratios and adjusted p-values were calculated using Benjamini-Hochberg method. The data visualization was performed using R packages. The differentially expressed proteins were identified using log2 fold change less than − 0.5 (0.7-fold change) for downregulated proteins and log2 fold change more than 0.5 (1.4-fold change) for upregulated proteins combined with the adjusted p-value less than 0.05. Volcano plots were generated by ggplot2. All the significant proteins were used as input to identify the significant enriched Go and pathway analysis. KEGG pathway analyses were search against mouse KEGG genome database and the bubble chart was generated by pathfindR. The adjusted p-value were calculated using Bonferroni method. The heatmap was generated by pheatmap. Go enrichment analysis was performed using shiny v. 0.77 by searching against the mouse string database with the FDR cutoff 0.05. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium50 via the PRIDE51 partner repository with the dataset identifier PXD042097 (temporary reviewer account details: Username: [email protected] Password: cyYoee0O ).
Confocal microscopy
IMMs were seeded in 24 well dishes with an inserted glass coverslip at 2x104 cells per well and incubated overnight to recover. The cells were then treated with 1 mg/mL LPS for 5, 10, 15, 20, 30, 45, 60 minutes or left untreated for the phospho-MARCKS colocalization study, 15, 30, 60 minutes and untreated for the endosome colocalization study and 1, 5 minutes and untreated for Golgi colocalization study. After LPS treatment, the cells were washed with ice cold PBS three times and fixed with 2% paraformaldehyde for 20 minutes at room temperature. The coverslips were washed three times with PBS-T, the primary antibody was added in PBS-T with 0.1% BSA and incubated overnight at 4oC. After extensive washing with PBS-T, the secondary antibody was added and incubated in the dark on the coverslips for 1 hour at room temperature. The coverslips were washed once with PBST, Hoechst dye was added (1:15000) and incubated in the dark for 15min after which the coverslips were again extensively washed with PBST. The coverslips were mounted on slides using ProLong Gold (Life Technologies, Grand Island, NY) and kept in the dark at 4oC until visualization. The visualization of the image was performed using confocal Imaging was performed using a Leica SP8 confocal microscope and data quantification including colocalization analysis was performed using Imaris software.
Statistical Analyses
Statistical analyses were performed using Prism 9.0 (GraphPad software, Inc., La Jolla, CA, USA).