Fig 2: miR-7 directly targets TGFBR2 and Smad3 binds to the CD44 promoter region. (A) Lenti-miR-7 and lentivector MDA-MB-231 cells were respectively incubated with TGF-β1 and TGF-β1 + SB431542, and then CD44 mRNA relative expression levels were measured by RT-qPCR. (B) The relative expression levels of Smad2, Smad3, and Smad4 were measured by RT-qPCR in lenti-miR-7 and lentivector MDA-MB-231 cells. (C) TGFBR2 mRNA relative expression levels were measured by RT-qPCR. (D) TGFBR2 3'UTR with the miR-7 binding site was predicted, and complementary sequences of miR-7 to TGFBR2 3'UTR mutated are presented in red. (E) Cells were harvested and luciferase activities were measured after 48-h transfection. (F) The expression levels of TGFBR2, Smad3, and CD44 were analyzed by western blotting. (G) TGFBR2 mRNA relative expression levels were measured by RT-qPCR in BCSC-siALDH1A3, BCSC-lenti-miR-7, and control cells. (H) ChIP-PCR assay in MDA-MB-231 cells. *P<0.05, **P<0.01 and ***P<0.001. miR-7, microRNA-7; TGFBR2, transforming growth factor-β receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BCSC, breast cancer stem cell; si, small interfering.

Fig 3: ALG10 knockdown suppressed the activity of TGF-β signaling by reducing TGFBR2 glycosylation. (A and B) The activity of Luc-TCF-1 and Luc-smad was evaluated in HCT-116 and HT-29 cells with or without ALG10 knockdown. (C) The status of TGFBR1/2 glycosylation was detected in HCT-116 and HT-29 cells with or without ALG10 knockdown. (D) The status of TGFBR2 glycosylation and p-smad2 expression were evaluated in HCT-116 cells with or without ALG10 knockdown or Tunicamycin treatment. (E) The nucleus expression of p-smad2 was examined in HCT-116 and HT-29 cells with or without ALG10 knockdown or Tunicamycin treatment. (F) The TGFBR1-TGFBR2 and TGFBR1-p-smad2 interaction was measured in HCT-116 and HT-29 cells with or without ALG10 knockdown. (G) The mRNA expression of FOXD1, SPHK1, CHST1, and EST2 was determined in HCT-116 and HT-29 cells with or without ALG10 knockdown or Tunicamycin treatment. n = 3, **P < 0.01 vs. Control.

Fig 4: TGFBR2 is necessary for ALG10-induced stemness of CRC cells in vitro. (A and B) CRC cells with or without ALG10 knockdown were treated with rTGFBR2 or not and then subjected to detect the mRNA levels of stemness markers. (C) The protein levels of stemness markers were examined in the CRC cells described in (A). (D) ALDH activity was measured in the CRC cells depicted in (A). (E and F) The sphere formation ability was evaluated in the CRC cells described in (A) by detecting sphere size (E) and number (F). (G) CD133+ cell sub-population was determined in the CRC cells depicted in (A). n = 3, **P < 0.01 vs. Control, ##P < 0.01 vs. ALG10-kd & rTGFBR2.

Fig 5: Detection of the molecular expression in SK-BR-3, MCF-7, and LD breast cancer cell lines. (A-C) TGFBR2, Smad3, and CD44 expression levels in SK-BR-3, MCF-7, and LD cells were measured by RT-qPCR analysis after miR-7 mimic transfection. (D-F) TGFBR2, Smad3, and CD44 relative expression levels in SK-BR-3, MCF-7, and LD cells were detected after the inhibitor transfection by RT-qPCR. *P<0.05, **P<0.01 and ***P<0.001. TGFBR2, transforming growth factor-ß receptor 2; RT-qPCR, reverse transcription-quantitative polymerase chainreaction; miR-7, microRNA-7.