Fig 1: SHANK1 overexpression reduces the protein expression of KL in a dose-dependent manner.A A549 cells were transfected with SHANK1, and relative protein levels of KL were detected by western blotting assay. Data shown are the mean ± SEM of three separate experiments (*p < 0.05, compared with the control group (0 ng), one-way ANOVA). B A549 cells were transfected with SHANK1, and relative mRNA levels of KL were detected by qRT-PCR. C, D Protein expression of SHANK1 and KL in NSCLC samples, as measured by WB, relative to that of Tubulin. T tumor. N normal. E, F A549 cells were transfected with empty vector or SHANK1 for 48 h and then serum-starved for the indicated times at 37 °C in the presence of cycloheximide (CHX, 20 µg/ml). KL was detected by anti-KL immunoblotting. Data shown are the mean ± SEM of three separate experiments (*p < 0.05, analysis of variance followed by post hoc tests). G, H A549 cells were transfected with SHANK1 vector for 48 h and then serum-starved for the indicated times at 37 °C in the presence of DMSO(1/1000), cycloheximide (CHX, 20 µg/ml) and MG132 (10 µM). KL was detected by anti-KL immunoblotting. Data shown are the mean ± SEM of three separate experiments (*p < 0.05, analysis of variance followed by post hoc tests).
Fig 2: Acute knockdown of Shank family proteins via lentivirus-mediated shRNA expression. A, Schematics of Shank proteins indicating shRNA target sites. B, Schematics of lentiviral constructs used to introduce shRNAs into cells. Constructs contained one of four shRNAs targeting Shank1 (shShank1), Shank2 (shShank2), Shank3 (shShank3), or Shank1 and Shank3 simultaneously (shShank13), as well as one of two fluorescent proteins (GFP, green fluorescent protein; tdT, tdTomato). pCMV, cytomegalovirus promoter; LTR, long terminal repeats; HIV-flap, a nuclear import sequence; pH1, constitutive H1 promoter; pUb, constitutive ubiquitin promoter; WRE, woodchuck hepatitis virus post-transcriptional regulatory element. C, D, Example (C) and quantification (D) of Western blotting for Shank protein levels in dissociated cortical neuron culture. Actin was used as a loading control. GFP and tdT refer to cultures infected with virus constructs containing the fluorescent protein and no shRNA. shShank13 + 2, knockdown of all three Shank members by superinfection of shShank13 and shShank2. E, A Western blotting for Shank3 levels in HEK cells cotransfected with a rat Shank3 expression vector and a GFP or shRNA expressing construct as indicated. One-way ANOVA was used for each quantification, followed by Tukey’s test, *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3: Knockdown of SHANK1 inhibits the migration and invasion, and it increases the apoptosis of human lung cancer cell lines.A A549 and H1299 cells were transfected with SHANK1 shRNAs, followed by qRT-PCR assays to detect knockdown efficiency of siRNAs. B A549 cells were transfected with SHANK1 shRNAs, followed by WB assays to detect knockdown efficiency of siRNAs. C, D CCK8 assay showed that SHANK1 knockdown inhibited cell proliferation in A549 and H1299. E, F Clone formation assay showed that SHANK1 knockdown inhibited cell growth in A549, H1299, and SK-MES-1. G Transwell was performed to detect the migration and invasion of A549 cells. The number of migratory and invasive A549 cells was counted. H Transwell was performed to detect the migration and invasion of H1299 cells. The number of migratory and invasive H1299 cells was counted. *p < 0.05. I The apoptosis of A549 and H1299 cells was detected by flow cytometry. The column diagram shows the apoptosis cell number. J The apoptosis-related protein levels were detected by western blot. The column diagram shows the related expression levels of target genes. K The PI3K-AKT-related protein levels were detected by western blot. The column diagram shows the related expression levels of target genes. *p < 0.05.
Fig 4: Enhancing excitatory drive does not rescue the decrease in AMPAR eEPSC caused by Shank knockdown. A, B, Comparison of uninfected (control) and infected [shShank1 (A); shShank2 (B)] neuronal responses measured by evoked AMPAR eEPSCs (? individual data point, ? mean) with bicuculline (20 µM). C, Summary of effects on AMPAR eEPSCs of knockdown of Shank1 or Shank2 under control (light gray, data from Fig. 2, the same as in C as a comparison), with bicuculline (dark gray), mean ± SEM.
Fig 5: SHANK1 regulates ubiquitination of KL by interacting with KL.A A549 cells were transfected with SHANK1-myc and Flag-KL constructs and lysed by TNE buffer, followed by immunoprecipitation with anti-FLAG antibody. Western blotting analysis was performed. B Endogenous association of KL and SHANK1 in adult rat lung tissue was detected by co-immunoprecipitation. C Subcellular co-localization of endogenous KL and SHANK1 in A549 cells visualized by confocal microscopy. Staining was carried out with rabbit anti-SHANK1 (green) and mouse anti-KL (red) antibodies. D The ubiquitination of KL was detected by IP and western blotting with Ub antibody. E The relative Ub levels in (D) are shown (relative to vector expression level, the error bars indicate SEM for three experiments, **p < 0.01, determined by student’s t test).
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