Clump formation in mouse pituitary-derived non-endocrine cell line Tpit/F1 promotes differentiation into growth-hormone-producing cells
Cell and Tissue Research
Masashi Higuchi, Saishu Yoshida, Naoko Kanno, Hideo Mitsuishi, Hiroki Ueharu, Mo Chen, Naoto Nishimura, Takako Kato & Yukio Kato*
*Department of Life Science, School of Agriculture, Meiji University, Kanagawa214-8571, Japan (E-mail: )
Fig.S1Specificity of guinea pig anti-human GH antisera.a-dImmunohistochemical(a, b, adult mouse pituitary) and immunocytochemical analyses (c, d,Tpit/F1 cells cultured for 20 d in clump-forming medium; D/F with bFGF and B27 supplement)were performed using guinea pig anti-human GH antisera (a, c) and antisera pre-absorbed with recombinant human GH protein (b, d), which was provided by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) courtesy of Dr. A. F. Parlow.GH were visualised with Cy3 (red).Bars 100 μm.
Fig.S2Expression of Gh in Tpit/F1 cells cultured in clump-forming medium. Quantitative real-time PCR was performed to estimate the mRNA levels ofGrowth hormone (Gh)using total RNAs prepared from all Tpit/F1 cells cultured in clump-forming medium (D/F with bFGF and B27 supplement) for 4, 10 and 20 d. Expression levels were calculated by the comparative Ct method to estimate the amount of transcripts relative to that of the TATA-box binding protein (Tbp) used as an internal standard. Data are presented as means ± SD (n = 4 in each duplicated PCR). A part of samples (2 samples in 4 samples) is below detectable limit in Tpit/F1 cells cultured for 4 and 10 d, respectively.
Fig.S3Expression of receptors and chromograninin Tpit/F1 cells cultured in clump-forming medium.a-f Quantitative real-time PCR was performed to estimate the mRNA levels ofGlucocorticoid receptor (Gr), Growth hormone releasing hormone receptor (Ghrhr), Growth hormone secretogogue receptor (Ghsr), Somatostatin receptor 5 (Sstr5) and 2 (Sstr2), and chromogranin A (Chga) using total RNAs prepared from all Tpit/F1 cells cultured in clump-forming medium (D/F with bFGF and B27 supplement) for 4, 10 and 20 d, Tpit/F1 cells cultured in a conventional medium (conv.), and mouse adult pituitary (mPituitary).Nucleotide sequences of primers used were as follows: mouse Gr (NM_008173.3, forward primer 5’-cagtggaaggacagcacaattac-3’ and reverse primer 5’-agtgtcttgtgagactcctgc-3’), Ghrhr (NM_001003685, forward primer 5’-acccgtatcctctgcttgct-3’ and reverse primer 5’-aggtgttgttggtcccctct-3’), Ghsr (NM_177330.4, forward primer 5’-tatgggtgtcgagcgtcttc-3’ and reverse primer 5’-gcagccagcagaggatgaaa-3’), Sstr5 (NM_011425, forward primer 5’-ttccacctactgagctccaag-3’ and reverse primer 5’-ccagttatggctactgctgga-3’), Sstr2 (NM_001042606, forward primer 5’-agagaacacagggaagcgag-3’ and reverse primer 5’-ggctcccattcaactgctca-3’), and Chga (NM_007693.2, forward primer 5’-agactacagacccactcccg-3’ and reverse primer 5’-ggagatgacttccaggacgc-3’). Expression levels were calculated by the comparative Ct method to estimate the amount of transcripts relative to that of the TATA-box binding protein (Tbp)used as an internal standard. Data are presented as means ± SD (n = 4 in each duplicated PCR). The DNA sequences of the PCR products were confirmed by nucleotide sequencing (data not shown).
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