Supplemental Fig. S1
Structure of green fluorescent protein (GFP) clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 plasmids. gRNA, guide RNA; NLS, nuclear localization signal; CBh, chicken β-actin hybrid promoter.
enm-34-203-s001.pdf
Fig. 1Neurofibromatosis 2 (NF2) overexpression suppresses cell growth in 8505C and KTC-1 cells. (A) Western blot analysis confirmed the differences in the Merlin protein expression between groups. (B) Proliferation assay to measure the cell growth rate in control and NF2 overexpressing 8505C cells. (C) Colony formation assay of 8505C cells in Day 7. (D) Proliferation assay to measure the cell growth rate in control and NF2 overexpressing KTC-1 cell. (E) Colony formation assay of KTC-1 cells in Day 7. Each data point represents mean±standard error of three independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Vec, vector. aP<0.05.
Fig. 2Neurofibromatosis 2 (NF2) overexpression inhibits cancer cell migration and invasion cancer in 8505C and KTC-1 cell. (A, B) In vitro wound healing assay in control and NF2 overexpressing thyroid cancer cells. Image was measured after 0, 24 hours after scratching (left) and quantification of wound closure was expressed in graph (right) (A) 8505C, (B) KTC-1. (C, D) In vitro transwells assay in control and NF2 overexpressing thyroid cancer cells. Representative images of invasive potential are shown and quantification was expressed in the bar graph (right) (C) 8505C, (D) KTC-1. The photographs were taken at 100×X and the bar size was 50 µm. Each data point represents mean±standard error of three independent experiments. Vec, vector. aP<0.05.
Fig. 3Neurofibromatosis 2 (NF2) loss fails to suppress growth in KTC-1 cells. (A) Proliferation assay to measure the cell growth rate in the control and small interfering NF2 (siNF2) KTC-1 cells. (B) Colony formation assay in the control and siNF2 KTC-1 cells. After the NF2/control clustered regularly interspaced short palindromic repeats (CRISPR) were conducted in the KTC-1 cell line, the NF2 gene knock out rate were measured using (C) confocal image, (D) fluorescence activated cell sorter sorting, (E) proliferation assay to measure the cell growth rate in the control KTC-1 cells and NF2 knock down KTC-1 cells using CRISPR/Cas9 system. (F) Colony formation assay in KTC-1 cells using CRISPR/Cas9 system. Each data point represents mean±standard error of three independent experiments. NS, no significant; FSC-A, forward Scatter-A; FITC-A, fluorescin isothiocyanate-A.
Fig. 4Neurofibromatosis 2 (NF2) mutagenesis enhances cell growth in 8505C and BHT101 cells. (A, B) Mutagenesis of the NF2 gene was performed on 8505C and BHT101. Cell proliferation was assessed in control and NF2 overexpressing thyroid cancer cells and also mutagenized thyroid cancer cells at 288 and 470 sites of NF2 gene. (A) 8505C: the control, 288, and 470 site of NF2 gene group showed significant decreased cell proliferation compared to NF2 group. (B) BHT101: the control, 288, and 470 site of NF2 gene group showed significant decreased cell proliferation compared to NF2 group. Each data point represents mean±standard error of three independent experiments. OD, optical density; pCMV, cytomegalovirus immediate early promoter. aP<0.001; bP<0.05.
Fig. 5Association between neurofibromatosis 2 (NF2) expression and cervical lymph node (LN) metastasis of papillary thyroid cancer. By The Cancer Genome Atlas data, mRNA expression of NF2 was significantly different according to cervical LN metastasis status. Especially, mRNA expression of NF2 was significantly decreased in papillary thyroid carcinomas (PTCs) with lateral cervical LN metastasis, compared to PTCs without LN metastasis in both (A) BRAF wild-type group and (B) BRAFV600E mutant group. Y-axis showed log2 transformed mRNA expression of NF2. In post hoc analysis, mRNA expression of NF2 was significantly different between N0 and N1b group. aP<0.05.