Fig 2: Nrf2/ARE signaling pathway was activated by CKIP-1 in high-glucose treated HRECs. a, b Co-IP was used to investigate the protein–protein interactions of CKIP-1 and Nrf2 (n = 3). c Luciferase reporter gene system was used to detect the transcriptional activity of Nrf2 (n = 3). d Western Blot was used to detect nuclear Nrf2 and cytoplasm Nrf2 expressions (n = 3). e Immunofluorescence assay was performed to detect the expression levels and cellular localization of Nrf2 (Scale bar is 50 μm). f The downstream targets of Nrf2 (including HO-1, NQO-1, γGCS and SOD) were detected by Western Blot (n = 3). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001

Fig 3: The effects of CKIP-1 knock-down on high-glucose treated HRECs in terms of cell viability, oxidative stress, inflammation and apoptosis. a Relative CKIP-1 mRNA levels were detected by Real-Time qPCR (n = 3). b Relative CKIP-1 protein levels were detected by Western Blot (n = 3). c CCK-8 assay kit was utilized to detect cell viability. d MDA levels, SOD activity and GSH-PX activity were detected by MDA assay kit, SOD kit abd GSH-PX kit respectively (n = 3). Inflammation associated cytokines (TNF-α, IL-6 and IL-1β) were detected by ELISA (n = 3). e Calcein-AM/PI double stain kit was employed to detect cell apoptosis (Scale bar is 200 μm). f Apoptosis associated proteins (Bcl-2, Bax and Cleaved Caspase 3) were detected by Western Blot (n = 3). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001

Fig 4: CKIP-1 was downregulated in DR tissues and high-glucose treated HRECs. a Real-Time qPCR was used to detect CKIP-1 mRNA levels in DR tissues (N = 20) and normal tissues (N = 10). b Western Blot Was performed to detect CKIP-1 protein levels in DR tissues (The “1#, 2# and 3#” represented 3 individual clinical specimens). c MDA assay kit was used to detect MDA levels, SOD kit was used to detect SOD activity and GSH-Px kit was used to detect GSH-Px activity in DR tissues respectively (n = 3). d The morphology of HRECs treated with low-glucose (5.5 mM) and high-glucose (25 mM) (Scale bar is 200 μm). e Relative CKIP-1 mRNA levels in HRECs treated with low-glucose (5.5 mM) and high-glucose (25 mM) was detected by Real-Time qPCR (n = 3). f Relative CKIP-1 protein levels in HRECs treated with low-glucose (5.5 mM) and high-glucose (25 mM) was detected by Western Blot (The “1#, 2# and 3#” represented 3 individual repetitions). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001

Fig 5: The effects of overexpressed CKIP-1 on high-glucose treated HRECs in terms of cell viability, oxidative stress, inflammation and apoptosis. a Relative CKIP-1 mRNA levels were detected by Real-Time qPCR (n = 3). b Relative CKIP-1 protein levels were detected by Western Blot (n = 3). c CCK-8 assay kit was utilized to detect cell viability (n = 3). d MDA levels, SOD activity and GSH-PX activity were detected by MDA assay kit, SOD kit abd GSH-PX kit respectively (n = 3). Inflammation associated cytokines (TNF-α, IL-6 and IL-1β) were detected by ELISA (n = 3). e Calcein-AM/PI double stain kit was employed to detect cell apoptosis (Scale bar is 200 μm). f Apoptosis associated proteins (Bcl-2, Bax and Cleaved Caspase 3) were detected by Western Blot (n = 3). The data above in one experiments were repeated at least 3 times and performed as mean ± standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001