Fig 1: Overexpression of PDCD6IP counteracts the miR-363-3p-mediated protective effects against OGD/R-induced injury. SH-SY5Y cells were divided into the following groups according to different transfections: pcDNA3.1, pcDNA3.1-PDCD6IP, empty vector, empty + miR-363-3p mimics, and miR-363-3p mimics + PDCD6IP, followed by OGD/R treatment. (A and B) The protein expression level of PDCD6IP was detected in SH-SY5Y cells from different groups. (C) Cell viability, (D) LDH release and (E) apoptosis were evaluated by CCK-8 assay, LDH assay and flow cytometric analysis, respectively. (F-H) ELISA assays were applied to determine the levels of IL-1β, IL-6 and TNF-α. Data are presented as the mean ± SD. **P<0.01 and ***P<0.001 compared with empty; and ##P<0.01 and ###P<0.001 compared with mimics + empty. PDCD6IP, programmed cell death 6-interacting protein; miR, microRNA; OGD/R, oxygen and glucose deprivation/re-oxygenation; LDH, lactate dehydrogenase; CCK-8, Cell Counting Kit-8; IL, interleukin; TNF, tumor necrosis factor.
Fig 2: MiR-363-3p directly targets the 3′-UTR of PDCD6IP. (A) The potential interaction between miR-363-3p and PDCD6IP was predicted by TargetScan. (B) Investigation of the regulatory effects of miR-363-3p on PDCD6IP expression using luciferase reporter assay in SH-SY5Y cells. **P<0.01 compared with miR-NC; SH-SY5Y cells were transfected with miR-363-3p mimics or miR-NC, followed by OGD/R treatment. (C) The expression level of PDCD6IP mRNA was detected by RT-qPCR. Data are presented as the mean ± SD. (D and E) Western blot analysis was performed to assess the protein level of PDCD6IP in (D) the normoxic and OGD/R groups, as well as that in (E) the miR-363-3p mimics/miR-NC-transfected OGD/R groups of SH-SY5Y cells. ***P<0.001 compared with normoxia; and ##P<0.01 and ###P<0.001 compared with OGD/R + miR-NC. miR, microRNA; 3′-UTR, 3′-untranslated region; PDCD6IP, programmed cell death 6-interacting protein; NC, negative control; OGD/R, oxygen and glucose deprivation/re-oxygenation; RT-qPCR, reverse transcription-quantitative PCR.
Fig 3: PDCD6IP knockdown suppresses OGD/R-induced apoptosis and inflammation. SH-SY5Y cells were transfected with si-PDCD6IP, followed by OGD/R treatment. (A) The protein expression level of PDCD6IP in SH-SY5Y cells was detected. (B) The viability of SH-SY5Y cells was evaluated using the CCK-8 assay. (C) Cell cytotoxicity was analyzed using LDH assay. (D) Flow cytometric analysis was conducted to detect apoptosis of SH-SY5Y cells. ELISA assay was applied to determine the levels of (E) IL-1β, (F) IL-6 and (G) TNF-α. Data are presented as the mean ± SD. **P<0.01 and ***P<0.001 compared with si-NC. PDCD6IP, programmed cell death 6-interacting protein; OGD/R, oxygen and glucose deprivation/re-oxygenation; si-, small interfering; CCK-8, Cell Counting Kit-8; LDH, lactate dehydrogenase; IL, interleukin; TNF, tumor necrosis factor; NC, negative control.
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