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Fig. 7 | Molecular Cancer

Fig. 7

From: The circROBO1/KLF5/FUS feedback loop regulates the liver metastasis of breast cancer by inhibiting the selective autophagy of afadin

Fig. 7

circROBO1 inhibits selective autophagy of afadin via KLF5. a RT-qPCR was performed to detect the mRNA change of genes associated with autophagy. b According to STARBASE, BECN1 was negatively correlated with KLF5. c After knockdown or overexpression of circROBO1, the change of BECN1 in mRNA level was analyzed by RT-qPCR. d Schematic diagram showed that two binding sites of KLF5 in BECN1 promotors were predicted by JASPAR. ChIP, qPCR and nucleic acid electrophoresis results indicated that the first one is the most enriched. e Dual-luciferase report assays were conducted to show that the luciferase activity was disappear in E1 mutant group. f Autophagosomes in mCherryGFP-LC3 labeled BT-549 cells after transiently transfected with siRNAs of circROBO1, KLF5, BECN1 respectively and rescued si-circROBO1 group with KLF5 overexpression vector (Scale bar=20μm). Quantification of the number of autolysosomes (red LC-3 puncta) and autophagosomes (yellow LC-3 puncta) per cell. The rescue group “si-circROBO1+KLF5” was compared to si-circROBO1 group and the others were compared to the NC control group. g The alteration of protein associated with autophagy and afadin was detected by western blot after transiently transfected with indicated siRNAs or vectors. h The F-actin was detected by IF in BT-549 cells transfected with siRNA or circROBO1 overexpression vector respectively (Scale bar=20μm). i After coimmunoprecipitation (CoIP) by antibody of afadin in BT-549 cell lysates, immunoblots with NBR1, p62, NDP52, TAX1BP1, Tollip were performed. j CoIP and immunoassay of lysates of 293T cells transfected with Flag-tagged wild-type NBR1 or mutant-type with UBA domain deletion, together with HA-tagged afadin, were performed. k After transfected with NBR1 siRNA for 12h, HA-tagged afadin was co-transfected for another 48h. Then EBSS was used to treat the cells for different hours and the results were determined by western blot

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