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. 2020 Feb 18;9(2):26. doi: 10.1038/s41389-020-0205-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Expressions of FIR family and related proteins were examined by western blotting in five paired-tumor (T) and adjacent non-tumor (N) tissue samples from human gastric cancer tissues. a, bottom Four splicing variants of the FIR/PUF60 gene PUF60, FIR, PUF60Δexon2, and FIRΔexon2 were detected in gastric cancer tissues in five paired tumors (T) and adjacent non-tumors (N). tub1: well-differentiated tubular adenocarcinoma, tub2: moderately differentiated tubular adenocarcinoma; por1: poorly differentiated adenocarcinoma, solid type; muc: mucinous adenocarcinoma. b The ratio of mRNA expression of FIRΔexon2/FIR was significantly higher in gastric cancer tissues (T) that in corresponding non-cancer tissues (N). N = 14, P = 0.007 by Student’s t-test. c Clones of stably transfected pcDNA3.1-FIRΔexon2 plasmids (clones 1,2,3,7,9,10,12,14, and 15 among 30 clones). At least 30 clones were screened by immunoblotting and immunostaining with anti-FLAG and anti-FIR antibodies (6B4) to find FIR-FLAG-expressing clones for FIR-FLAG stably expressing cells, or with anti-c-Myc antibody to examine c-Myc expression for FIRΔexon2 stably expressing cells. Soft-agar colony formation assay of clone12. The cells of clone12 (2 × 10³) were inoculated in 0.3% low-melting-temperature agarose (FMC Bio Products, Rockland, ME, USA) in DMEM supplemented with 10% FCS, and colonies were scored after incubating for 2 weeks. 1 A cells was a positive control cells provided by Dr Ariga²⁷. d Comparison of protein expressions from five paired tumors determined by statistical analysis in gastric cancer tissues in 14 paired tumors (T) and adjacent non-tumors (N). N = 5, P < 0.01, R < 1.0 was obtained by Student’s t-test.