Fig 2: Expression of RGC-specific markers in 661W cells. 661W and RGC-5 cell lysates were subjected to western blotting for the expression of RBPMS (A), Brn3b (B), Brn3c (C) and Thy-1 (C,D and E). For Thy1 three different antibodies sourced from CST (C), R & D systems (D) and Santa Cruz (E) were used. Mouse retina and brain were used as positive controls. Immunofluorescence staining of 661W and RGC-5 cells for Brn3b (F), Brn3c (G) and ? Synuclein (H) is shown. Staining of nuclear DNA with DAPI is shown in blue. Blank indicates negative control where cells were processed similarly without addition of primary antibody. Panels show images captured using a fluorescence microscope at 40X magnification. Scale bar, 10 µm.

Fig 3: Comparison of the HP and HFL by multiplex immunohistochemistry with tyramide signal amplification for CD90, CD73 and CD105 with merged signals (A) or separate signals (B). Blue: Hoechst, Green: CD90, Yellow: CD73, Red: CD105. White arrows show the simultaneous presence of the 3 signals on the same location. C: cambial layer, F: fibrous layer, L: longitudinal layer, T: transverse layer. White scale bars: 100 µm.

Fig 4: Morphological analysis and differentiation of 661W and RGC-5 cells. 661W (A,C) and RGC-5 cells (B,D) were treated with 316 nM staurosporine (STSN) or vehicle (DMSO) for 24 hours, fixed and stained with specific antibody for acetylated tubulin (A,B). Staurosporine treatment induced neurite formation in 661W as well as RGC-5 cells. Scale bar: 10 µm. (C,D) Immunostaining of 661W and RGC-5 cells for RGC markers Brn3b, Brn3c and neuronal marker MAP-2 with antibodies. Scale bar, 10 µm. (E) Western blots showing the effect of differentiation with staurosporine on the level of Thy1, RBPMS, beta-III tubulin and NeuN proteins in 661W and RGC-5 cells. Numbers below the blots indicate quantitation of expression in differentiated cells relative to undifferentiated cells adjusted with actin loading control.

Fig 5: Analysis of the character of fibroblasts. The fibroblasts from stomach tissue (hGFCs) and BJ cells were collected, and the expressions of fibroblast markers calponin and CD90 and epithelium marker VE-cadherin were analyzed by (A) RT-PCR. In addition, the protein expressions of hGFCs, BJ cells, and skin fibroblasts were analyzed by Western blot. (B) The expression of fibroblast marker CD90 is presented, and none of epithelium marker VE-cadherin. (C) The wound healing assay was performed to investigate the migration of fibroblasts. (D) The data revealed that cell migration was slow in hGFCs compared with BJ cells and skin fibroblasts. Images were acquired at 40X. The hGFCs reveal less migration activity than skin fibroblasts and BJ cells. The experiment was performed in triplicate, and data are presented as mean ± SD. * p < 0.05, ** p < 0.01.