Fig 2: Expression of FMRFaRRNAi and inhibition of CaMKII in dopaminergic neurons reduces calcium entry after a depolarizing stimulus.(A) Mean trace (±SEM) of normalized GCaMP6m responses observed upon KCl stimulation (green arrow) in 8 day adult neurons in the three indicated genotypes, THD1;TubGAL80ts>GCaMP6m, Control in black; THD1;TubGAL80ts>GCaMP6m;FMRFaRRNAi, in red and THD1;TubGAL80ts>GCaMP6m;Ala, in blue. (B) Area under the curve and (C) Peak change in fluorescence quantified from (A). Each bar is compared to control shown in black (**p<0.01; Mann-Whitney U-test). (D) Snap shots of GCaMP6m responses from the same genotypes shown in (A). In the FMRFaRRNAi knockdown condition (2nd row), the yellow arrow indicates a cell that responded whereas pink arrows indicate cells with no response or a minimal response to the depolarization stimulus. Scale bars represent 50 µm.

Fig 3: NOX4 knockdown prevents CLP-induced pulmonary hyperpermeability via CaMKII/ERK1/2/MLCK pathway inactivation. Sepsis was induced by CLP in mice transfected of control siRNA or NOX4 siRNA. Mouse lung tissues were harvested at 1, 2, 4, 6 h post CLP for Western blotting analyses. In parallel experiments, septic mice were pretreated with either KN93 (0.05 mg/day/mouse) or KN92 (0.05 mg/day/mouse) for 1 week before exposure to CLP for 16 h to examine effects of KN93 on pulmonary hyperpermeability in CLP-treated mice. (A): Representative Western blots and quantitative data of CaMKII phosphorylation in mouse lung tissues at 1, 2, 4, 6 h post CLP. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. 0 h group. (B): Representative Western blots and quantitative data of CaMKII, ERK1/2, and MLCK phosphorylation in sham mice or CLP treated mice transfected of control siRNA or NOX4 siRNA. Results indicate NOX4 knowdown markedly attenuated phosphorylation of CaMKII, ERK1/2 and MLCK. Data are presented as Mean±SEM; n = 3. **p < 0.01 vs. Control-CLP group. (C): Representative fluorescent images of DHE detection of ROS production in CLP mice with saline or KN92/KN93 treatment, indicating the upstream production of superoxide was not affected by CaMKII inhibition KN93. Data are presented as Mean±SEM; n = 3. (D): Representative Western blots and quantitative data of p-CaMKII expression in sham mice, or CLP treated mice pretreated with KN92 or KN93. Results indicate KN93 inhibition of CaMKII phosphorylation. Data are presented as Mean±SEM; n = 3. **p < 0.01. (E–F): Lung wet-to-dry weight ratio and Evans Blue index were determined in PBS or KN92/KN93 treated CLP mice, indicating attenuation of pulmonary hyperpermeability by KN93 treatment in vivo. Data are presented as Mean±SEM, n = 5. **p < 0.01 vs. CLP group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: CaMKII function is required in dopaminergic neurons to regulate flight.(A) Flight bout durations observed with overexpression of WT-CaMKII in the background of FMRFaR knockdown in THD1 neurons (THD1;TubGAL80ts>WT-CaMKII;FMRFaRRNAi). A significant rescue of the flight defect was observed by expression in the adults, but not in pupae (n=30, *p<0.05, **p<0.01, Mann-Whitney U-test). (B) Flight durations observed with expression of a genetically encoded CaMKII inhibitor peptide, Ala, in dopaminergic neurons as compared to Ala control (n=30, **p<0.01, Mann-Whitney U-test). (C) Flight time of flies with temporal expression of Ala in THD1 neurons using the TARGET system (THD1;TubGAL80ts>Ala). Significant deficits in flight were observed upon CaMKII inhibition in pupal and adult stages as compared to flies maintained at 18°C throughout (n=30, **p<0.01, Mann-Whitney U-test). (D) Mean traces (±SEM) of normalized GCaMP6m responses in control (THD1>GCaMP6m) and Ala expressing THD1 neurons (THD1>GCaMP6m;Ala) upon peptide stimulation (green arrow). (E) Area under the curve and (F) Peak ?F/F quantified from 0 s to 420 s in (D) were not significantly different between the two genotypes tested (p>0.05; ns—not significant; Mann-Whitney U-test).

Fig 5: NOX4 knockdown attenuates LPS-induced endothelial permeability via CaMKII/ERK1/2/MLCK pathway inactivation. Sepsis was mimicked in vitro by LPS (1 µg/ml) stimulation in HPMECs pre-transfected with control siRNA or NOX4 siRNA. (A): Representative Western blots and quantitative data of NOX4 protein expression in HPMECs transfected with control siRNA or NOX4 siRNA, indicating that NOX4 siRNA transfection effectively attenuated NOX4 protein abundance. Data are presented as Mean±SEM. n = 3. **p < 0.01. (B): Changes in TEER values in real time following LPS treatment up to 8 h. Data indicate that NOX4 knockdown substantially alleviated LPS induced decrease in TEER values in HPMECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. control siRNA transfected LPS group. (C): Representative images of DHE staining of HPMECs in control group or LPS treated groups transfected with control siRNA or NOX4 siRNA, indicating that NOX4 knockdown completely abrogated ROS production in response to LPS treatment. Cells were incubated with DHE (2 µM) in the dark for 30 min, and fluorescent images were captured using a Nikon A1R Confocal Microscope. Photographs were taken at ×200 magnification. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. control siRNA transfected LPS group. (D): Representative Western blots and qualitative data of CaMKII phosphorylation in HPMECs following LPS challenge, indicating a time-dependent increase. Data are presented as Mean±SEM. n = 3. **p < 0.01 vs. 0 h group. (E): Representative Western blots and quantitative data of CaMKII, ERK1/2, and MLCK phosphorylation in control or LPS (1 µg/ml) treated HPMECs transfected of control siRNA or NOX4 siRNA. Results indicate that NOX4 knockdown inactivated CaMKII/ERK1/2/MLCK pathway in LPS-treated HPMECs. Data are presented as Mean±SEM; n = 3. *p < 0.05, **p < 0.01 vs. control siRNA transfected LPS group. Results in F–H indicate that pharmacological inhibition of CaMKII is protective endothelial barrier dysfunction in LPS treated HPMECs. Cells were incubated with either KN93 (1 µmol/L) or KN92 (1 µmol/L) for 24 h and then exposed to LPS (1 µg/ml) for 2 h. (F): Representative fluorescent images of DHE detection of ROS production in LPS treated HPMECs with KN92 or KN93 treatment. Data indicate that the upstream production of superoxide was not affected by CaMKII inhibition KN93. Data are presented as Mean±SEM; n = 3. (G): Representative Western blots and quantitative data of p-CaMKII expression in control HPMECs, or LPS stimulated HPMECs pre-treated with KN92 or KN93. KN93 attenuated CaMKII phosphorylation while KN92 had no effects. Data are presented as Mean±SEM; n = 3. **p < 0.01 vs. LPS group. (H): Changes in TEER values in real time in LPS-stimulated HPMECs pre-treated with KN92 or KN93. Data indicate that KN93 treatment markedly preserved TEER values in LPS-treated HMPECs. Data are presented as Mean±SEM. n = 6–9, **p < 0.01 vs. LPS group. (I): Representative Western blots and quantitative data of Occludin and ZO-1 expression in control HPMECs or LPS (1 µg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. (J): Representative immunofluorescent analysis of ZO-1 expression in control HPMECs or LPS (1 µg/ml) stimulated HPMECs transfected with control siRNA or NOX4 siRNA. Cells were grown on a glass chamber slid to confluence and then challenged with LPS (1 µg/ml) for 6 h. Immunofluorescent staining of ZO-1 (red), and nuclei (blue) was imaged by a Nikon A1R Confocal microscope. Scale bars: 100 µm. Data are presented as Means±SEM. n = 3. **p < 0.01 vs. LPS group. Results in (I–J) indicate that NOX4 knockdown preserved protein abundance of Occudin and ZO-1 following LPS stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)