Fig 2: MK2 inhibitor elicits model-specific transcriptomic changes(A) DEGs were identified comparing transcriptomic profiles of inflamed TCT and TNFΔARE mice with vehicle or ATI-450 (adjusted p value < 0.1, LFC > 0.25, Wald test with Benjamini-Hochberg correction).(B) Hallmark GSEA analysis of data from ATI-450 treated animals showed common unidirectional changes in 9 pathways between TCT and TNFΔARE animals. Three pathways with opposing enrichment between the two models were also identified (p value < 0.05, adjusted p value < 0.1, adaptive multi-level split Monte-Carlo with Benjamini-Hochberg correction). GSEA analysis also identified model-specific pathway enrichments for (C) TCT and (D) TNFΔARE. Note that PI3K-AKT-mTOR signaling is uniquely down-regulated in inflamed TCT animals treated with ATI-450.See also Figure S1.

Fig 3: MK2 target gene expression is enriched in murine inflammatory monocytes and neutrophils(A) Single cell UMAP clustering of CD45+ enriched cell populations from the small intestine of mice at homeostasis or 4 days post-infection of H. polygyrus. Raw data from (Biton et al., 2018) (GEO: GSE106510). Original cell annotations of the 12 cell types were used.(B) MK2 target genes are commonly detected and highly expressed in inflammatory monocytes and neutrophils.(C) UMAP display of MK2 Target gene score for all cells demonstrates high MK2 target gene signature in inflammatory monocytes and neutrophils.(D) Overlay of violin plot and box plot shows that MK2 target gene expression is significantly enriched in intestinal inflammatory monocytes and neutrophils (∗∗∗ p value < 2.2 × 10−16, Wilcoxon rank-sum test).(E) FACS-mediated quantification of myeloid cell recruitment to colonic (TCT) or (F) ileal (TNFΔARE) mucosa in mice after ATI-450 or vehicle treatment. Myeloid cells are defined as CD45 + CD11b+ cell. Neutrophils and Inflammatory Monocytes are defined within myeloid cells gate based on GR1 and Ly6C surface staining. Data are represented as mean ± SEM. ∗ p value < 0.05; ∗∗ p value < 0.01 (Student's t-test vehicle vs. ATI-450).See also Figures S3 andS4.

Fig 4: Torin treatment disassembles PBs.A: HUVECs were treated with either Torin (250 nM) or a DMSO control for 2 h prior to fixation in 4% paraformaldehyde. Samples were permeabilized in 0.1% Triton X-100. Immunofluorescence was performed for Hedls (PBs, white) and DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, **** = P<0.0001. B: HUVECs were treated with either Torin (250 nM) or a DMSO control for 90 min prior to the addition of sodium arsenite (0.25 mM) for 30 min. Cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. Immunofluorescence was performed for Hedls (PBs, white) and DAPI (nuclei, blue). Scale bar = 20 μm. Hedls puncta were quantified using CellProfiler and presented as number of Hedls puncta per cell, all cells counted are displayed. Results were plotted in GraphPad and a 2-way ANOVA was performed on the main column effects with a Tukey’s multiple comparison test, bar represents the mean; n = 3, * = P<0.05. C: HUVECs were transduced with recombinant lentiviruses expressing either KapB, constitutively active MK2 (MK2EE), or an empty vector control and selected with blasticidin (5 μg/mL). Cells were treated with either Torin (250 nM) or a DMSO control for 2 h prior to harvest in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for phospho-S6 (Ser235/236) and total S6. D: HUVECs were transduced and treated as in C and harvested in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for phospho-ULK (Ser757), phospho-ULK (Ser555), and total ULK. E: HUVECs were transduced as in C and harvested in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for phospho-Beclin (Ser90) and total Beclin. Samples were quantified by normalizing total and phospho-Beclin protein levels to the total protein in each lane and then using Image Lab (BioRad), normalizing to the vector control, and then dividing phospho- over total Beclin. Results were plotted in GraphPad and a one-way ANOVA was performed with a Dunnett’s test, ±SEM; n = 4, * = P<0.05.

Fig 5: KSHV-mediated autophagy utilizes MK2.A: HUVECs were transduced with recombinant lentiviruses expressing shRNAs targeting MK2 or a non-targeting control (NS) and selected with puromycin (1 μg/mL). After selection, transduced cells were infected with wild-type (WT) or delete KapB (delB) BAC16 KSHV via spinoculation and infection was allowed to proceed for 96 h. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for MK2. B: Samples from A were quantified for MK2 protein level and normalized to the total protein in each lane using ImageLab software (BioRad). MK2 levels were then graphed by normalizing to the matched NS for each infection condition in GraphPad, and a one-way repeated measures (RM) ANOVA with a Šidák’s multiple comparison test was performed, ±SEM; n = 3, *** = P<0.001, **** = P<0.0001. C: HUVECs were transduced with shMK2-expressing lentiviruses or NS controls, selected, and infected as in A. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for total hsp27 and phospho-hsp27 (S82). D: Samples from C were quantified by normalizing phospho- and total hsp27 protein levels to the total protein in each lane using Image Lab (BioRad) then by dividing phospho-hsp27 over total hsp27 and normalizing to the matched NS for mock, WT KSHV and delB infection, respectively. Results were plotted in GraphPad, a one-way RM ANOVA with a Šidák’s multiple comparison test was performed, ±SEM; n = 3, * = P<0.05. E: HUVECs were transduced, selected, and infected as in C. Cells were treated with Bafilomycin A1 (BafA1, 10 nM) or a vehicle control (DMSO) for the indicated times prior to harvest in 2X Laemmli buffer. Protein lysates were resolved by SDS-PAGE and immunoblot was performed for LC3. F-G: Samples from E were quantified using Image Lab (BioRad) software and then normalized, first to total protein and then to their respective starting time points (0 h). In F, the accumulation of LC3-II in control cells was compared with accumulation in MK2-silenced cells in either uninfected (top) or WT KSHV infected (bottom) cells. Results were plotted in GraphPad and a linear regression statistical test was performed between control or silenced cells. ±SEM; n = 3, * = P<0.05. In G, results were plotted in GraphPad to show accumulation of LC3-II in MK2-silenced cells to compare between the following groups: WT-infected vs uninfected (left), uninfected vs delB infected (middle) and WT infected vs delB infected (right). A linear regression statistical test was performed between infection conditions in each group as indicated. ±SEM; n = 3, * = P<0.05.