Fig 2: Kinase Assay validates FTS hits as inhibitors.MAP2K4 kinase assays were performed as described in Methods using either p38 MAPK (K53A) or JNK1 (K55M) as substrates, and was done using a range of concentrations of either staurosporine A or daidzein B. Representative phosphorylated JNK1 and phosphorylated p38 MAPK and total JNK and p38 MAPK Western blots are depicted. C. A plot of densitometric measurement of bands from separate experiments utilizing Staurosporine (2X each on p38 and JNK1 substrates) is depicted with intensity measurements shown as mean ± S.D along with the curve fit used for determining the IC50 values; Red – JNK1 and Black – p38MAPK. D. 3 indicated concentrations of either MAP2K4-EE or MAP2K4-AC were employed in the kinase assay and the western blot performed as before.

Fig 3: The levels of p38 MAPK, Cyclins and cell cycle inhibitors changed after REX1 knockdown and p38 MAPK inhibitor treatment.(A) Significant phosphorylation of p38 MAPK (Pp38) was seen after REX1 knockdown in hUCB-MSCs. (B) hUCB-MSCs, hAD-MSCs and hBM-MSCs express all four types of p38 isoforms. (C) After p38 MAPK inhibitor treatment (1 µM BIRB796), the phosphorylation of p38 MAPK was similar in REX1 knocked-down hUCB-MSCs compared to vehicle control-infected hUCB-MSCs. (D) Cell proliferation was measured with CCK-8 for four days. The cell proliferation of REX1 knocked-down hUCB-MSCs was similar with vehicle control-infected hUCB-MSCs after BIRB796 (1 µM) treatment. Without BIRB796, the proliferation of REX1 knocked-down hUCB-MSCs significantly decreased compared with vehicle control-infected hUCB-MSCs. **, p<0.01. (E) The p38 MAPK of REX1 knocked-down hUCB-MSCs was not activated after 1 µM or 10 µM BIRB796 treatment compared to those of vehicle control-infected hUCB-MSCs. (F) Changes in the expression of CDK and cell cycle inhibitors. The decreased expression of CDK2 and CCND1 recovered after p38 MAPK inhibitor treatment. (G) The expression levels of ERK1/2, MEK, phospho-MEK (pMEK1/2) and NF-?B did not significantly change after REX1 knockdown in hUCB-MSCs. (H) The expression changes of STAT3 and STAT5. The expression levels of STAT3 and phospho-STAT3 were significantly increased after REX1 knockdown without regard to p38 MAPK inhibitor treatment. STAT5 expression did not change after the knockdown of REX1. (I) p38a and p38ß were up-regulated but p38? was down-regulated after REX1 knockdown in hUCB-MSCs. (J) REX1 expression of hUCB-MSCs did not change after BIRB796 (p38 MAPK inhibitor) treatment. Error bars represent the standard deviation from three independent experiments.

Fig 4: Locomotor activity rhythms of p38b knockdown flies with respective controls.Flies were recorded in LD 12:12 for 6 days and subsequently in DD for at least 14 days. A daily average activity profile for day 2–7 in LD was calculated for each genotype and is shown above the double-plot of a representative actogram (left panels in A and B). In addition, for each genotype the onset of evening activity in LD (upper right panel in A and B) as well as the average free-running period in DD (lower right panel in A and B) was determined and is depicted as boxplot in the right panel. While average activity profiles of dicer2;UAS-p38bRNAi/tim(UAS)-Gal4;+ (A) displayed wildtype-like behavior in LD with activity bouts around lights-on and lights-off, evening activity onset of dicer2;UAS-p38bRNAi/Pdf-Gal4;+ flies (B) was significantly delayed compared to respective controls. When transferred to DD, dicer2;UAS-p38bRNAi/tim(UAS)-Gal4;+ mainly became arrhythmic (see Table 1 and lower left panel in A), just 7% remained rhythmic displaying a significant longer free-running period than UAS- and Gal4-controls (lower right panel in A). p38b knockdown in PDF-expressing neurons also significantly lengthened the free-running period in DD as compared to respective controls (lower panels in B), and 58% of the flies remained rhythmic (see Table 1). Bars above the daily average activity profiles and actograms depict the light regime of the LD 12:12 cycle and black arrowhead indicate the shift to constant DD. Black lines in daily average activity profiles represent mean relative activity, gray lines SEM and dotted grey lines the calculated evening activity onset. Gray lines in boxplots illustrate the median, boxes 25–75%, and whiskers 10–90% of the data. UAS refers to respective UAS-control, GAL4 to respective Gal4-control and UAS>GAL4 to the experimental line. Significant differences (p<0.05) are indicated by *, highly significant differences (p<0.001) by **. Numbers in brackets indicate n.

Fig 5: The schematic of the proposed VEGF and PlGF signaling in HG-induced hRECs injuries. Hyperpermeability and increased cell death was detected in HG-treated hRECs. Following HG treatment, expression of VEGF and PlGF was upregulated, phosphorylation of VEGFR1 and VEGFR2 as well as PKC activity and NOS1 abundance were increased, and activation of Erk1/2, p38MAPK and STAT1 were increased. Inhibition of PKC activity predominantly prevented activation of Erk1/2 and decreased NOS1 level and permeability, while did not affect apoptotic cell death. Inhibition of p38MAPK dramatically inhibited activation of STAT1 and decreased cell death, while did not show significant effect on permeability. Knockdown of VEGF mainly decreased phosphorylation of VEGFR2 and p38MAPK as well as cell death; knockdown of PlGF mainly decreased phosphorylation of VEGFR1 as well as PKC activity and permeability. Go 6976: PKC inhibitor, SB203580: p38MAPK inhibitor.