Fig 1: Whole-genome expression sequencing and RIP assay. (A) Two ECA-109 groups were transfected with negative control (NC) and shFSCN1 separately. Relative mRNA level of FSCN1 was detected. n = 3 per group, ***p < 0.001. (B) ECA-109 cells were transfected with NC (shCtrl) and shFSCN1 separately. The sample correlation among the control groups (shCtrl) and FSCN1 knock down groups (shFSCN1) is shown. (C, D) The differentially expressed genes (DEG) were analyzed and shown. (E) RNA immunoprecipitation (RIP) was performed by using FSCN1 monoclonal-antibody in ECA-109 cell lysate. The immunoprecipitation was detected by western blotting. (F) The sample correlation is shown. (G) Gene Ontology enrichment is shown in the sequencing analysis of RIP in (F). (H) The peak reads of the sequence of PTK6 pulled down by FSCN1 in RIP analysis. (I) ECA-109 lysates were immunoprecipitated by FSCN1 antibody or IgG. Relative pre-mRNA level of PTK6 was detected. n = 3 per group, ***p < 0.001.
Fig 2: FSCN1 downregulates PTK6 expression in ESCC cell lines. (A–D) Lentivirus containing FSCN1 shRNA sequence (FSCN1-KD) and over-expressed sequence (FSCN1-OE) were added into ECA-109 and KYSE-150 mediums separately. Lentivirus expressing GFP only was used as a negative control (NC). The mRNA (A) and protein level (B–D) of FSCN1 in the 2 cell lines were detected. n = 3 per group, **p < 0.01, ***p < 0.001. (E–H) ECA-109 and KYSE-150 infected by NC, FSCN1-KD, and FSCN1-OE as indicated were harvested, and the mRNA (E) and protein level (F–H) of PTK6 in the 2 cell lines were detected. n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: PTK-T2 increases ECA-109 apoptosis. (A) ECA-109 cells expressing NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 as indicated were stained by TUNEL. (B) Statistical analysis of A. n = 3 per group, *p < 0.05, ***p < 0.001. (C) ECA-109 cells expressing NC, FSCN1-KD, and FSCN1-KD + PTK6-KD as indicated were stained by TUNEL. (D) Statistical analysis of C. n = 3 per group, ***p < 0.001. (E) ECA-109 cells expressing NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 as indicated were harvested. BCL2 and BAX were detected by western blotting. (F) Statistical analysis of E. n = 3 per group, ***p < 0.001. (G) ECA-109 cells expressing NC, FSCN1-KD, and FSCN1-KD + PTK6-KD as indicated were harvested. BCL2 and BAX were detected by western blotting. (H) Statistical analysis of G. n = 3 per group, *p < 0.05, ***p < 0.001.
Fig 4: PTK-T2 inhibits ESCC tumor progression ex vivo. (A) The mRNA level of PTK6 in tumor tissues of NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 groups was detected. n = 5 per group, ***p < 0.001. (B) PTK6 in tumor tissues of NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 groups was detected by immunohistochemical staining (C) Statistical analysis of B. n = 5 per group, ***p < 0.001. (D) TUNEL assay was performed in tumor tissues of NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 groups. (E) Statistical analysis of D. n = 5 per group, *p < 0.05, ***p < 0.001. (F) EdU assay was performed in tumor tissues of NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 groups. (G) Statistical analysis of F. n = 5 per group, *p < 0.05, ***p < 0.001.
Fig 5: The binding region of the pre-mRNA of PTK6 with FSCN1 is PTK-T2 (A) Schematic diagram of the full length and truncated mutant of PTK6 intron located in chromosome 20. (B) Plasmids expressing PTK6-FL and five truncated mutants (?T5, ?T4, ?T3 and ?T2) were transfected into ECA-109 for 48 h. RIP was performed using the FSCN1 antibody. The pull-down RNA level was detected. n = 3 per group, ***p < 0.001. (C) Cultured ECA-109 cells were infected with NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 as indicated. The protein level of PTK6 was detected by western blotting (D) Statistical analysis of C. n = 3 per group, ***p < 0.001. (E) CCK-8 assay was performed in cultured ECA-109 cells infected with NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 as indicated. n = 3 per group, ***p < 0.001. (F) CCK-8 assay was performed in cultured ECA-109 cells infected with NC, FSCN1-KD, and FSCN1-KD + PTK6-KD as indicated. n = 3 per group, ***p < 0.001. (G) ECA-109 cells expressing NC, FSCN1-OE, and FSCN1-OE + PTK6-T2 as indicated were stained by EdU. (H) Statistical analysis of G. n = 3 per group, *p < 0.05. (I) ECA-109 cells expressing NC, FSCN1-KD, and FSCN1-KD + PTK6-KD as indicated were stained by EdU. (J) Statistical analysis of I. n = 3 per group, *p < 0.05.
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