Fig 1: SCAI was a direct target of miR-424-5p. (A) Schematic of the miR-424-5p-binding sites within SCAI 3'UTR identified by the starBase v.2 software and mutated miR-424-5p-binding sites. (B and C) The luciferase activity in CAL-27 and SCC-15 cells cotransfected with SCAI 3'UTR WT or SCAI 3'UTR MUT and miR-424-5p mimic or miR-NC mimic. Blots were representative of n = 6. SCAI mRNA expression by qRT-PCR and protein level by Western blot in CAL-27 and SCC-15 cells transfected with anti-miR-NC or anti-miR-424-5p (D–G), OSCC tissues and matched normal tissues (H and I), HOK, CAL-27 and SCC-15 cells (J and K). Blots were representative of n = 3. (L) Correlation between SCAI mRNA level and miR-424-5p expression in OSCC tissues using the Spearman test. *P < 0.05.
Fig 2: Exosomal circGDI2 regulated SCAI expression through sponging miR-424-5p. (A) Correlation between circGDI2 expression and SCAI mRNA level in OSCC tissues using the Spearman test. CAL-27 and SCC-15 cells (Recipient cells) were transfected with miR-424-5p mimic or miR-NC mimic and then treated with 30 µg/mL of the exosomes from pcDNA-NC- or pcDNA-circGDI2-transfected Donor cells, followed by the measurement of SCAI mRNA expression by qRT-PCR (B and C), SCAI protein level by Western blot (D and E). Blots were representative of n = 6. *P < 0.05.Abbreviations: pcDNA-NC-exo, exosomes derived from OSCC cells transfected with pcDNA; pcDNA-circGDI2-exo, exosomes derived from OSCC cells transfected with pcDNA-circGDI2.
Fig 3: Control of caveolar proteins by megakaryoblastic leukemia 1 (MKL1). a. Western blots showing protein levels of caveolin-1/CAV1 and PTRF/CAVIN1 at different cell densities and at 3 and 5 days post-confluence in the absence (-) or presence (+) of 10 µM CCG-1423. Quantification of Western blot data from CCG-1423 treatment at 5 days post-confluence are shown in (b) (n = 4). c. Results of qPCR showing caveolin-1/CAV1 and PTRF/CAVIN1 mRNA levels after viral transduction with different multiplicities of infection (MOI) of Ad-CMV-MKL1. Panels d and e show qPCR for caveolin-2/CAV2 and SDPR/CAVIN2 at 100 MOI of Ad-CMV-null or Ad-CMV-MKL1. *(p < 0.05), **(p < 0.01), ***(p < 0.001). f. Western blots for the MKL1 coactivator FLNA and repressor SCAI, comparing levels in normal kidney cortical tissue lysates and cultured kidney epithelial cells obtained from the same kidneys 5 days post confluency. As caveolin-1/CAV1 and PTRF/CAVIN1 expression becomes evident during culture, a parallel de novo expression of FLNA is seen. Conversely, the pronounced cortical levels of SCAI are diminished during culture
Fig 4: Overexpression of exosomal circGDI2 hampered tumor growth in vivo. CAL-27 cells were subcutaneously injected into the nude mice (n= 6 each group). One week later, intratumor injection for the exosomes from lenti-NC- or lenti-circGDI2-transduced CAL-27 cells was performed. After 35 days cell implantation, all mice were euthanized. (A) Tumor volume measurement began on 7 days after implantation and was conducted every week. (B) Representative pictures and average weight of the xenograft tumors. (C–E) The levels of circGDI2, miR-424-5p and SCAI were assessed by qRT-PCR or Western blot in excised tumor tissues. A representative experiment was shown in triplicate. *P < 0.05.
Fig 5: The mitigative effects of miR-424-5p knockdown on OSCC cell malignant behaviors were mediated by SCAI. CAL-27 and SCC-15 cells were transfected with anti-miR-NC, anti-miR-424-5p, anti-miR-424-5p+si-NC or anti-miR-424-5p+si-SCAI. (A) SCAI expression by Western blot in transfected cells. (B and C) Cell proliferation by MTT assay. (D and E) Cell migration and invasion by transwell assay. (F and G) Glucose consumption and lactate production using a corresponding assay kit. (H and I) ECAR using a standard assay. (J and K) LDHA and GLUT1 levels by Western blot in transfected cells. A representative experiment was shown in triplicate. *P < 0.05.
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