Fig 2: Immunoblot analysis of tissue biomarkers. (a) Immunoblots of Kallikrein-1, LMW kininogen-1, Bradykinin RB2, and beta-Actin as a loading control. (b) Chemiluminescent analysis of band intensities of Kallikrein-1 in (a). (c) Chemiluminescent analysis of band intensities of LMW kininogen-1 in (a). (d) Chemiluminescent analysis of band intensities of Bradykinin RB2 in (a). (e) Heatmap depicting trend of changes in tissue biomarkers analyzed by immunoblots in (a). Not significant (ns); * p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001, (n = 8).

Fig 3: (A) shows western blots of nuclear extracts of mCCD cells treated with 100 µmol/L CoCl2. Treatment with CoCl2 increased stabilization of HIF1a in the nucleus of the cells. GAPDH levels are shown as loading controls. (B) shows western blots of whole cell extracts of mCCD cells treated with 100 µmol/L CoCl2 for 4 h. As shown in the images, treatment with CoCl2 increased the EPO production by cells. Beta actin levels are shown as loading controls. (C) shows western blots of whole cell extracts of mCCD cells treated with 100 µmol/L CoCl2 for 4 h and chronic fluid flow. As shown in the images, treatments decreased ZO-1 production by cells independently of fluid flow stimulation. Beta actin levels are shown as loading controls. (D) shows western blots of whole cell extracts of mCCD cells treated with 100 µmol/L CoCl2 for 4 h and chronic fluid flow. As shown in the images, treatments decreased NaKATPase production by cells independently of fluid flow stimulation. Beta actin levels are shown as loading controls. (E) Densitometric analysis of Western blots presented in (A–D). HIF1a level: P = 0.004 (N = 3), t-test, showing significant nuclear stabilization of HIF1a. EPO level: increases slightly though not statistically significant (N = 3, P > 0.05, One-way ANOVA). ZO-1 level: P < 0.0001 (N = 4), One-way ANOVA, showing significant decrease in ZO-1 levels, independent of fluid flow. NaKATPase a1 subunit level: P = 0.001 (N = 3), One-way ANOVA, showing significant decrease in NaKATPase a1 subunit levels, largely independent of fluid flow.

Fig 4: Lon expression is induced by a variety of stressorsA. The normal culture medium (10% FBS) of D-54 and U-251 cells were replaced by serum-free medium for 3 hours. The cells were then allowed to recover in normal medium for the amount of time indicated. B. D-54 cells were treated with TMZ (500 µM). C. D-54 cells were exposed to 4 or 6 Gy of irradiation. D. D-54 and U-251 cells were cultured in low-oxygen concentrations (1%) or chemically-induced hypoxia (200 µM cobalt chloride) for 24 hours. Cells were collected at the indicated time points and RNA was extracted. qRT-PCR was then performed to measure the Lon mRNA levels. The relative expression levels were normalized by ACTB. E. D-54 cells were treated with 100µM or 200µM of CoCl2 for 24 hours. Western blot was used to detect HIF-1a, LONP1 and COX IV. Actin was used as the internal control. F. Treatment with four different siRNA targeted against the HIF-1a sequence leads to 80-90% decrease of HIF-1a mRNA levels as compared with siControl-treated cells, 72 hours after transfection. Treatment of cells with siRNA targeting HIF-10a reduced Lon mRNA levels (~80% in D-54 and ~50% in U-251), representative data showed. *p < 0.05, **p < 0.01, ***p < 0.001.