Journal: Journal of leukocyte biology

Article Title: ATF2 orchestrates macrophage differentiation and activation to promote antibacterial responses.

doi: 10.1093/jleuko/qiad076

Figure Lengend Snippet: Fig. 4. ATF2 overexpression limits intracellular Mtb survival in M1 macrophages. Macrophages were polarized with IFNγ and LPS toward the M1 state and subsequently infected with Mtb for downstream analyses. (A) Representative Western blot analysis (of 3 independent experiments) of non- polarized (M0) or M1-polarized THP-Vector and THP-ATF2 macrophages infected with Mtb (MOI 10) for 24 h. β-tubulin was used as the loading control. A.U., arbitrary units. (B) The percent phagocytosis of GFP-expressing Mtb (Mtb-GFP, MOI 10) by M1 macrophages was analyzed by flow cytometry after 4 h infection. (C and D) Macrophages were infected with Mtb expressing a lux operon (Mtb-lux, MOI 5), and Mtb viability was measured as a luminescence readout at indicated days post-infection. (E) Macrophages were infected with Mtb (MOI 10), and cell culture supernatant was collected at 6 and 12 h post-infection. The concentration of IL-1β in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA). (F) The cell viability of Mtb-infected macrophages (MOI 5) at day 3 post-infection was quantified using Cell Titer-Glo and expressed relative to non-infected (–Mtb) cells. (G and H) Concentration of (G) IP-10 and (H) arginase from cell culture supernatant of Mtb-infected macrophages (MOI 10) after 12 h post-infection was measured using antibody-based bead multiplex assays. Data in this figure are the mean ± SD of 3 biological replicates. MOI, multiplicity of infection; P.I., post-infection. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Membranes were developed using the Clarity Western Enhanced Chemiluminescence Substrate from Bio-Rad, and imaging analysis was performed with the ImageQuant LAS4000 imaging system (Cytiva; GE Healthcare Lifesciences).

Techniques: Over Expression, Infection, Western Blot, Plasmid Preparation, Control, Expressing, Flow Cytometry, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay

Journal: Journal of leukocyte biology

Article Title: ATF2 orchestrates macrophage differentiation and activation to promote antibacterial responses.

doi: 10.1093/jleuko/qiad076

Figure Lengend Snippet: Fig. 7. ATF2 is precisely regulated in response to various physiologic conditions in hMDMs, and its activity is correlated with improved antibacterial responses. (A) Representative Western blot analysis of hMDMs for indicated days post-differentiation (dpd) in response to either GM-CSF (5 ng/mL) or M-CSF (50 ng/mL). After 8 d of differentiation, hMDMs were recovered in media without GM-CSF or M-CSF for 2 d (8 + 2 dpd). Quantification of total ATF2 levels is expressed as arbitrary units (A.U.). The fold change of phosphorylated ATF2 (pT69/72-ATF2 or p-ATF2) levels is expressed relative to cells prior to differentiation (0 dpd). (B) hMDMs (differentiated in GM-CSF for 8 d) were infected with live or heat-killed (HK) Mtb at indicated MOIs. Cell lysates were prepared after 24 h post-infection, and the total and phosphorylated levels of ATF2 were analyzed by Western blot. The fold change of total and phosphorylated ATF2 levels is expressed relative to cells prior to infection (MOI 0). (C) Following 8 d of differentiation in GM-CSF, hMDMs were stimulated with IFNγ, LPS, anisomycin (100 nM), or a combination of IFNγ and LPS (M1, 20 ng/mL IFN and 100 ng/mL LPS) for 24 h. The fold change of phosphorylated ATF2 is expressed relative to non-stimulated cells (N.S.). The blots shown in (A to C) are representative of hMDMs obtained from 3 independent donors. (D to J) After 8 d of differentiation with GM-CSF, hMDMs were pre-treated with IFNγ (100 ng/mL), LPS (200 ng/mL), HK-Mtb (MOI 20), or anisomycin (100 nM) for 24 h and maintained throughout the experiments. (D) Pretreated hMDMs were infected with Mtb expressing luciferase (Mtb-luc, MOI 10), and Mtb burden was measured by luminescence readout at indicated days post-infection. Results are shown as relative Mtb burden compared to the start of infection (day 0). (E) The viability of Mtb-infected macrophages (MOI 10) at day 3 post-infection was measured using the Cell Titer-Glo assay. Data are expressed relative to non-stimulated macrophages. Data in (D) and (E) are the mean ± SD of 3 replicates from 1 representative donor of 2. (F to I) OCR kinetics of hMDMs infected with Mtb (Mtb-luc, MOI 10) for 24 h was measured in response to sequential injections of Oligo (1.5 μM), FCCP (1 μM), and Rot/AA (1 μM each) at the indicated time points. The data were normalized to micrograms of protein in each well after the assay. (J) Basal ECAR measurement of Mtb-infected macrophages prior to the injection of Oligo. Data in (F to I) are the mean ± SD of 4 replicates from 1 representative donor out of 2. GAPDH: loading control in (A) and (B). β-tubulin: loading control in (C). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 relative to (D) Mtb burden at 0 d post-infection or (E to J) the non-stimulated condition.

Article Snippet: Membranes were developed using the Clarity Western Enhanced Chemiluminescence Substrate from Bio-Rad, and imaging analysis was performed with the ImageQuant LAS4000 imaging system (Cytiva; GE Healthcare Lifesciences).

Techniques: Activity Assay, Western Blot, Infection, Expressing, Luciferase, Glo Assay, Injection, Control