Fig 2: Comparison of intracellular signaling in myDC and moDC after TLR stimulation and effect of blockade of MAPK pathways in healthy donors. (a) MAPK/Rsk/GSK3β signaling. (i) Activation of Rsk by both ERK and p38 following TLR ligation in murine splenic and bone-marrow derived DC (arrow = activation), (ii) Signaling downstream of ERK and p38 in all other cells after TLR ligation where Rsk is only activated by ERK, (iii) Rsk drives GSK3β from the active form (GSK3β*) to the inactive form (GSK3β) with downstream effects on IL-12p70/IL-10 secretion, (iv) Sequence of phosphorylation/activation of Rsk by phospho-ERK (p-ERK) and phospho-p38 (p-p38). (b) Timecourse of MK2 activation following TLR stimulation in myDC and moDC. Donor-matched myDC and moDC (105) were stimulated with poly I:C/R848 for the indicated times before Western blotting. To allow cell-for-cell comparison between myDC and moDC, entire samples were used in a single Western blot and probed for phospho-MK2 (p-MK2) with β-actin as a loading control. Infrared secondary antibodies were used to allow for semi-quantitative analysis normalized to loading control. (i) Scanned images for one donor of two. (ii) Normalized p-MK2 for two donors, open circles = myDC, filled circles = moDC, dotted line = upper p-MK2 band, solid line = lower p-MK2 band. (c) Effect of blockade of p38 or MEK on MK2 activation following TLR stimulation in moDC. MoDC were pre-treated with p38 inhibitors SB203580 10 µM (S), BIRB0796 (B) 0.1 or 1.0 µM or MEK inhibitor UO126 10 µM (U) for 1hr followed by poly I:C/R848 stimulation for 30 min then Western blotted for p-MK2 with β-actin loading control as in (b). Shown are scanned images for three independent donors and graphs of normalized p-MK2 (gray bars = upper p-MK2 band, black bars = lower p-MK2 band). (d) Effect of blockade of p38, MEK or both on ERK, p38 and Rsk activation in moDC. MoDC were treated and analyzed as in (c) for phospho-ERK 1/2 (p-ERK 1/2), phospho-p38 (p-p38), phospho-Rsk (p-Rsk, T359/S363, T573 and S380 residues) with total Rsk as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0 µM (B), MEK inhibitor UO126 10 µM (U) or both (BU). Shown are scanned images for three independent donors and graphs of normalised p-RskSer380 for each donor. (e) Effect of blockade of p38, MEK or both on activation of MK2 and Rsk at Ser380 in myDC. MyDC (three donors) were treated, harvested and analyzed as in (c) for p-MK2 and p-RskS380 with β-actin as loading control. Pre-treatment was with p38 inhibitor BIRB0796 1.0µM (B), MEK inhibitor UO126 10µM (U) or both (BU).

Fig 3: Effects of ATF3 on the ERK and JNK signaling pathways.(A and B) The levels of total and phosphorylated MEK1/2, ERK1/2, JNK, P38, P90RSK and AKT in the heart tissues of mice in the indicated groups (n = 6). A, Representative blots. B, Quantitative results. *P<0.05 vs WT/sham. # P<0.05 vs WT/AB after AB. (C and D) The levels of total and phosphorylated MEK1/2, ERK1/2, JNK and p90RSK in cardiac myocytes treated with Ang II at the indicated time points. C, Representative blots. D, Quantitative results. *P<0.05 vs WT group at the 0 time point. # P<0.05 vs WT group at the same time point.

Fig 4: PP2AC determines differential programming of p38-to-mTORC1 signaling. A. The protein level of PP2A-A, PP2A-B and PP2A-C in different CRC cells as analyzed by immunoblot. B. HCT116 cells were transfected with four different PP2AC siRNAs (1, 2, 3 and 4), and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, MK2, ERK and RSK was analyzed by immunoblot. C. SW620 cells were transfected with four different PP2AC siRNAs (1, 2, 3 and 4), and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, S6, MK2, ERK and RSK was analyzed by immunoblot. D. RKO and SW480 cells were transfected with PP2AC plasmid and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, S6, MK2, ERK and RSK was analyzed by immunoblot. E. Effect of p38i on ERK-PP2AC interaction in sensitive and resistant CRC cells. HCT116, SW620, SW480 and RKO cells were treated without or with 10 μM SB202190 for 2 h. Endogenous PP2AC was immunoprecipitated with a PP2AC-specific antibody and analyzed for binding to ERK by Western blot. F. Immunohistochemistry (IHC) staining of primary human CRC tissue microarray. Shown are staining of tumor tissue sections (400 ×) representative of high PP2AC, medium PP2AC and low PP2AC staining. G. PP2AC is differentially expressed in CRC. Scatter plot shows PP2AC staining level in individual tumor as H-score.

Fig 5: p38 targets distinct phosphorylation sites of TSC2 in different CRC cell lines. A.WT, TSC1−/− or TSC2−/− MEF cells were treated with 10 μM SB202190 for 2 h and analyzed for P-S6K1 and P-S6 by immunoblot. B. CRC cells were transfected with two different TSC1 or TSC2 siRNAs, and then treated with 10 μM SB202190 for 2 h. TSC1, TSC2 and P-S6K1(T389) were analyzed by immunoblot. C. CRC cells were transfected with two different TSC1 or TSC2 siRNAs, and then treated with 10 μM SB202190 for 48 h. Cell growth was measured by SRB assay and expressed as percentage of SB202190/control (DMSO). Data represent means ± s.d. from three independent triplicate experiments. *P < 0.01 versus NC. D. Drug-sensitive CACO2, SW480 and RKO cells, and drug-resistant HCT116, SW620 and SW1116 cells were treated with 10 μM SB202190 for 2 h and analyzed for phosphorylation of TSC2 at S664, S1254, T1462 and S1798 by immunoblot. Boxed area highlights the different effects of SB202190 on TSC2 phosphorylation in drug-sensitive versus -resistant CRC cells. SE, short exposure, LE, long exposure. E. HCT116 and SW620 cells were treated with 10 μM SB202190 with or without Mek inhibitors 30 μM PD98059 or 10 μM U0126 for 2 h, and then analyzed for phosphorylation of TSC2, ERK, RSK, S6K1 and S6 by immunoblot.