U937 Cells

Pooled, genome-wide CRISPR deletion screens were performed in three cell lines: K562 stably expressing SFFV-Cas9-BFP, Ramos cells lentivirally infected with SFFV-Cas9-BFP, and U937 cells lentivirally infected with EF1a-Cas9-Blast34 (link). The library was synthesized, cloned and lentivirally infected into cells as previously described20 (link). Briefly, the parent vector for the libraries was derived from a pSico lentiviral vector which expresses GFP and a puromycin-resistance cassette separated by a T2A sequence45 (link)58 (link); we replaced GFP with mCherry to make the final parent vector, pMCB320. Sublibraries were PCR-amplified from pooled-oligo chips (CustomArray, Agilent), digested with BstXI and BlpI restriction enzymes, and ligated into BstXI/BlpI-cut pMCB320 using T4 ligase. Libraries and vectors will be made available via Addgene. Three days after infection, cells were placed under puromycin selection (0.7 μg ml⁻¹, Sigma) for an additional 3 days after infection, then split at time 0. Throughout the screen, the pooled libraries were maintained at 1,000 cells per guide or a total of ∼250 million cells in large spinner flasks. K562 and U937 were grown for ∼2 weeks, and Ramos cells were growth for ∼3 weeks due to their slower division time. Genomic DNA was extracted following Qiagen's Blood Maxi Kit, and the guide composition was sequenced and compared to the plasmid library using casTLE20 (link) version 1.0 available at https://bitbucket.org/dmorgens/castle. Briefly, casTLE compares each set of gene-targeting guides to the negative controls, selecting the most likely maximum effect size which explains the distribution of targeting guides. It then determines the significance of this maximum effect by permuting the results20 (link). Both safe-targeting and non-targeting controls were used for this analysis. For the ricin sensitivity screen, cells were treated with ricin toxin (Vector Labs) at 0.25 ng ml⁻¹ for 24 h, ricin was removed and then cells were allowed to recover to normal doubling rate. This treatment occurred four times over 2 weeks.

L. pneumophila serogroup I parental strain AA100/130b and the mutants dotA, ankB, and complemented ankB mutants were grown as described previously [10] (link). Escherichia coli strain DH5α was used for cloning purposes. Isolation and preparation of human monocyte-derived macrophages (hMDMs) and maintenance of the macrophage-like U937 cells were performed as previously described [9] (link). Cultures of A. polyphaga and D. discoideum were performed as described previously [9] (link),[10] (link).
For intracellular proliferation studies, infections were performed as we described previously [9] (link). Briefly, macrophages were infected at a multiplicity of infection (MOI) of 10 for 1 h followed by treatment with 50 µg/ml gentamicin for 1 h to kill extracellular bacteria. At each time point, the macrophages were lysed and dilutions were plated on agar plates. For single cell analysis studies, infections were performed as we described above and at 12 h cells were fixed and processed for confocal microscopy. Phagosomes were isolated from post nuclear supernatants (PNS) of infected cells as described previously [29] (link). Samples were then fixed and probed as described below. The cya constructs were generated by PCR using specific primers (Table 1). Measurement of cAMP in cell lysates for adenylate cyclase fusion assays was performed using the Direct Cyclic AMP Enzyme Immunoassay kit (Assay Designs), as we described previously [10] (link).

For SILAC experiments, THP-1 or U937 cells were cultured in RPMI 1640 deficient in L-arginine and L-lysine, and supplemented with 10% dialyzed FBS. Two linages of cells were cultured in light medium (L-arginine (Arg 0) and L-lysine (Lys 0)) and heavy medium (L-arginine 13C6-15N4-HCl (Arg 10) (#89990, Thermo) and L-lysine 13C6-15N2-HCl (Lys 8)) (#88209, Thermo). Prior to infection, cells were treated with PMA (Phorbol 12-myristate-13-acetate) (#P1585, Sigma-Aldrich) for 24 h, then heavy-labeled cells were infected at 90% confluency with S. Typhimurium 14028S at MOI 100, while light-labeled cells were mock infected. After incubation for 1 h at 37°C in a 5% CO₂ atmosphere, extracellular bacteria were killed at 100 mg/ml gentamicin for 2 h, and then switched to medium containing 25 mg/ml of gentamicin for the remainder of the experiment. Cells were lysed at indicated time points in lysis buffer containing 8M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 2 g/l Aprotinin, 10 g/l Leupeptin, 1×protease inhibitor cocktail (#CW2200, Cwbio), and 5 mM sodium butyrate. Protein concentration was determined using a BCA protein assay and 5 mg proteins in two states mixed at equal ratio. Samples were digested with trypsin, and peptides were enriched for lysine acetylation using the anti-KAc antibodies noncovalently coupled to protein A agarose beads (#13416, Cell Signaling Technology). Desalted peptides were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Q Exactive mass spectrometer. Heavy arginine (Arg10) and lysine (Lys8) were selected for SILAC quantification. The eXtracted Ion Current (XIC) of all isotopic clusters associated with the identified amino acids sequence in the light label cluster was summed up, and calculated the ratio between two heavy and light label partners. Then the ratio was normalized, the median of the total ratio population was shifted to 1 and data was analyzed using the MaxQuant software version 1.3.0.5. We used Significance A to assess the significance of outlier ratios. Only peptides with average 1.5-fold change of acetylation in two states and significance A values less than 0.05 were considered up or down regulated. The score at lysine acetylation of proteins greater than 20 is considered to be reliable.

Cell viability assays were performed using MTT/MTS as previously described^{8 (link)}. For propidium iodide uptake assays in serum-free medium, U937 cells (after centrifugation at 300 g, 5 min) were washed once in serum-free RPMI-1640, pelleted again (as previous centrifugation) and resuspended in serum-free media. 4×10⁴ cells were seeded and incubated with a range of CpoBD13 concentrations (diluted in serum-free medium) for 30 min at 37°C. PI (diluted in ice-cold PBS) was added to a final concentration of 1 µg/mL before analysis by flow cytometry using a BD FACSCanto II with FACSDiva 8.0.1 (BD Biosciences).

RNA sequencing was performed for the whole transcriptome analysis. U937 cells were treated with 8 μM CHI or 0.1 μM CLA or DMSO control for 48 h. Cells were harvested and total RNA was isolated with Trizol reagent. Sequencing libraries were generated using NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. The resulting libraries were sequenced by Novogene (Beijing, China) on a HiSeq device (Illumina, San Diego, CA, USA) to yield a transcriptome. The index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. featureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. Significant differences in expression were analyzed using DESeq2. Genes with absolute fold changes > 1 and a Benjamini–Hochberg adjusted p < 0.05 were validated further. The enrichment analysis utilized the online website tool Metascape (http://metascape.org/) referred to different databases.