Fig 2: Effect of TQ on PI3K/Akt/mTOR signaling expression in MV4-11 and K562 cells. RT-qPCR analyses were performed. The graph demonstrates the downregulation of PI3K, Akt, and mTOR genes and upregulation of PTEN in (A) MV4-11 cells and (B) K562 cells after treatment with TQ. The data represent three separate experiments and are presented as the median and interquartile range (error bars). Wilcoxon signed ranks test was conducted. Where * p < 0.05 and *** p < 0.001 are significant versus untreated control cells.

Fig 3: Schematic of the mechanisms underlying the therapeutic effects of HMW HA on injured human lung microvascular endothelial cells. Cytomix, a mixture of IL-1ß, TNFa, and IFN? often used as a surrogate for acute respiratory distress syndrome pulmonary edema fluid, caused an increase in protein permeability across HLMVEC which was associated with an increase in apoptosis/LDH release and actin stress fiber and gap formations between cells. Cytomix was also capable of breaking down HMW HA into LMW HA further exacerbating the injury. In response to cytomix, AKT phosphorylation and HAS2 expression were increased in HLMVECs as an inherent reparative effect although there was no restoration of intracellular HMW HA levels or protein permeability. Surprisingly, exogenous HMW HA administration increased HAS2 expression further after cytomix through CD44, leading to an increase in intracellular HMW HA synthesis and restoration of protein permeability. This finding was unexpected given normal feedback inhibition seen with receptors and end-products and may result from an increase in AKT phosphorylation from HMW HA leading to increased HAS2 expression. Although exogenous LMW HA administration suppressed Caspase-3 activation in the current study, LMW HA is known to increase inflammation, apoptosis, and permeability in endothelial cells whether through a direct effect or through increased release of cytokines or through inhibition of HMW HA binding to CD44 as a competitive antagonist

Fig 4: Effect of 4-MU, a HAS inhibitor, with or without hyaluronic acid on HLMVEC Injured With an Inflammatory Insult. (A) The addition of 4-MU significantly suppressed the expression of HAS2 mRNA in HLMVEC in a dose-dependent manner over 24 h. Only 4-MU at a concentration of 1.0 mM led to an increase in protein permeability, whereas there were no significant differences with a 4-MU concentration of 0.1–0.5 mM on permeability compared with vehicle control. Data are presented as mean ± SD. ****p < 0.0001; N = 3–6 for PCR and N = 9 for permeability experiments. (B) Addition of 4-MU 0.5 mM with cytomix further decreased HAS2 mRNA expression and increased protein permeability across HLMVEC. There was no restorative effect of HMW HA on the decrease in HAS2 mRNA or increase in protein permeability. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; N = 3–6 for PCR and N = 9 for Permeability experiments. (C–E) However, by fluorescence microscopy and Western blot analyses, the addition of HMW HA partially decreased F-actin stress fiber and increased both ZO-1 and VE-Cadherin total protein levels and distribution to cell–cell junctions in HLMVEC injured with both cytomix and 4-MU 0.5 mM. Yellow Arrows = Gap formations between cells. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; N = 3. (F) 4-MU further suppressed CD44 protein expression and the phosphorylation of AKT after the addition of cytomix in HLMVEC. Only the addition of HMW HA partially increased the expression of CD44 at 24 h but not the phosphorylation of AKT. Data are presented as mean ± SD. *p < 0.05, **p < 0.01; N = 3. Individual p values are the statistical comparison between two groups by Student’s t test. Graphs were created using GraphPad Prism 8.4.3 for OS X (GraphPad Software, www.graphpad.com)

Fig 5: Dock7-mediated AKT phosphorylation in serum-deprived conditions requires Rictor, but not Raptor, and mTORC1-like activity is partially responsible for survival.(A) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. (B) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 µl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 µM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. (C) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. (D) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. (E) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. (F) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (B and F) represent means ± SD; ***p < 0.001, and **p < 0.01.