Fig 1: The subcellular distribution of the two splice isoforms of ANLN.A Immunofluorescence staining for ANLN-201 and ANLN-210 in SCC-9 cells. B The mRNA levels of ANLN-201 and ANLN-210 were examined in SCC-9 cells treated with 10 nM actinomycin D for 4, 8, and 12 h. **P < 0.05, *P < 0.01. C Sequence alignment of nucleotides between ANLN-210 and ANLN-201. D The predicted RNA-binding protein with ANLN-210 was listed in the table. E RNA co-immunoprecipitation was performed between ANLN-201/ANLN-210 and HNRNPC/SRSF10/U2AF2. *P < 0.05, **P < 0.01.
Fig 2: Expression pattern and functional effects of two ANLN splicing isoforms in exosomes.A The relative expression of ANLN-201 and ANLN-210 was examined in cells and exosomes by qRT-PCR. *P < 0.05, **P < 0.01. B The expression of ANLN-201 at mRNA level was examined in SCC-9 cells and exosomes when SCC-9 cells were transfected with si-HNRNPC. C The expression of ANLN-210 at mRNA level was examined in SCC-9 cells and exosomes when cells were transfected with si-HNRNPC and/or ANLN-210 overexpression. *P < 0.05, **P < 0.01. D The representative images of exosomes. Exosomes were extracted from SCC-9 cells transfected with ANLN-210 (210 O.E.) or exosomes were electrotransfected with ANLN-210 (210 E.T.). E Exosomal markers HSP70, CD63, and TSG101 were immunblotted in isolated exosomes. Calnexin which was expressing in cell lysates was used as the control. F The relative expression of ANLN-210 was examined in control, 210 O.E Exo. and 210 E.T. Exo groups. G Cell proliferation was performed in SCC-9 cell and HepG2 cells with the following treatment. PBS, exosomes derived from ANLN-sgRNA cells (Control Exo), exosomes derived from cells transfected with ANLN-210 (210 O.E. Exo), and exosomes electrotransfected with ANLN-210 (210 E.T. Exo). H Cell migration was analyzed in SCC-9 cells treated with the following treatment (PBS, Control Exo, 210 O.E. Exo, and 210 E.T. Exo).
Fig 3: ANLN-210 interacts with HNRNPC.A The knockdown efficiency of si-HNRNPC, si-SRSF10, and si-U2AF2 was assessed at protein level by western blot. B The RNA levels of ANLN-201 or ANLN-210 were measured in si-HNRNPC, si-SRSF10, and si-U2AF2 transfected cells by qRT-PCR. *P < 0.05, **P < 0.01. C The RNA stability of ANLN-210 was evaluated in si-HNRNPC or si-SRSF10 or si-U2AF2 treated with actinomycin D after 4, 8, and 12 h. *P < 0.05, **P < 0.01. D Schematic representation of the ANLN-210 WT and ANLN-210 MUT sequences used as probes in EMSA. MUT: The binding sequences between ANLN-210 and HNRNPC were mutant. ANLN-210 MUT could not interact with HNRNPC. E SCC-9 cells were transfected with HNRNPC in a dose-dependent manner. EMSA was performed using ANLN-210 WT probe (left) and ANLN-210 MUT probe (right).
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