Journal: Scientific Reports

Article Title: Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

doi: 10.1038/s41598-017-05145-y

Figure Lengend Snippet: Differential effects of exposure to hyperosmolarity on proinflammatory cytokine and AQP5 expression levels in immortalized HCECs. The immortalized HCECs were cultured in media with varied osmolarities (310, 400, 450, 500 or 550 mOsm) for 4 h and 24 h respectively. The real time semi quantitative PCR analysis (4 h) showed the mRNA levels of IL-1β, IL-6, IL-8, TNF-α, an inflammatory mediator caspase-1, and AQP5 increased when the medium osmolarity increased to either 450, 500 or 550 mOsm relative to those in a 310 mOsm isotonic medium ( P ≤ 0.034) ( A ). The protein expression levels measured by Western blot analysis of AQP5 ( P ≤ 0.019) ( B ) and p-JNK1/2 ( P ≤ 0.016) ( C ) rose after 24 h of exposure to hyperosmotic stress (450, 500 and 550 mOsm), while that of JNK remained unchanged in different groups. The cropped gels are displayed and the full-length gels are provided in a Supplementary Information file. Error bars represent the SD; * means P < 0.05 (Three independent experiments with 3 repeats each).

Article Snippet: Membranes were blocked with fat-free milk and then probed overnight with primary anti-AQP5 (sc-28628, Santa Cruz Biotechnology, Santa Cruz, CA, 1:500) primary anti-JNK (ab179461, Abcam, MA, USA, 1:500) and primary anti-p-JNK1/2 (ab124956, Abcam, MA, USA, 1:500), followed by incubating with horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG (Bioworld technology, Nanjing, China, 1:5000).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

doi: 10.1038/s41598-017-05145-y

Figure Lengend Snippet: p-JNK1/2 inhibitor suppressed hyperosmolarity-induced AQP5 protein expression. The immortalized HCEC cell viability was relatively unchanged in the 0.05%, 0.1%, 0.2%DMSO groups but decreased significantly in the 0.5% DMSO group, compared to the control group ( P = 0.018) ( A ). The cell viability in 10 μM, 20 μM, and 40 μM SP60012 dissolved in 0.1%DMSO was not different from that of the control group, while in the 80 μM SP600125 group cell viability significantly decreased ( P = 0.037) ( B ). Pre-treatment with 20 μM SP600125 decreased IL-1β, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels in 500 mOsm medium during 4 h ( P ≤ 0.012) ( C ), and the protein expression levels of p-JNK1//2 ( D ) and AQP5 ( E ) also declined by 33.3% ( P = 0.001) and 39.3% ( P = 0.008) respectively after 24 h. The cropped gels are displayed and the full-length gels are provided in a Supplementary Information file. Experiments were repeated three times each in triplicate.

Article Snippet: Membranes were blocked with fat-free milk and then probed overnight with primary anti-AQP5 (sc-28628, Santa Cruz Biotechnology, Santa Cruz, CA, 1:500) primary anti-JNK (ab179461, Abcam, MA, USA, 1:500) and primary anti-p-JNK1/2 (ab124956, Abcam, MA, USA, 1:500), followed by incubating with horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG (Bioworld technology, Nanjing, China, 1:5000).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells

doi: 10.1038/s41598-017-05145-y

Figure Lengend Snippet: Effect of SP600125 on AQP5 protein levels determined by immunofluorescent localization. After 24 h in 500 mOsm medium, the immortalized HCECs were collected for immunofluorescent assay. AQP5 staining intensity distinctly declined with the pretreatment of 20 μM SP600125, confirming that JNK1/2 inhibition reduced rises in AQP5 expression levels induced by exposure to hyperosmotic 500 mOsm medium. DAPI (blue, the left column) shows the nuclei; FITC secondary antibody staining (green, the middle column) documents AQP5 expression; and the right column is the merged image. Experiments were repeated in three independent experiments. The top row: HCECs cultured in 310 mOsm; The second row from the top: HCECs cultured in 500 mOsm; The third row from the top: HCECs cultured in 500 mOsm and DMSO (the solvent of SP600125); The bottom row: HCECs cultured in 500 mOsm and 20 μM SP600125, a JNK inhibitor.

Article Snippet: Membranes were blocked with fat-free milk and then probed overnight with primary anti-AQP5 (sc-28628, Santa Cruz Biotechnology, Santa Cruz, CA, 1:500) primary anti-JNK (ab179461, Abcam, MA, USA, 1:500) and primary anti-p-JNK1/2 (ab124956, Abcam, MA, USA, 1:500), followed by incubating with horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG (Bioworld technology, Nanjing, China, 1:5000).

Techniques: Staining, Inhibition, Expressing, Cell Culture