ROS generation in human PBMCs induced by PR-GO.

<p>ROS generation in human PBMCs induced by PR-GO.</p

Flow cytometry and confocal microscopy of human PBMC treated with different concentrations of PR-GO.

<p>Flow cytometry and confocal microscopy of human PBMC treated with different concentrations of PR-GO.</p

DLS elucidation of the synthesized PR-GO.

<p>DLS elucidation of the synthesized PR-GO.</p

Cytotoxicity of PR-GO in terms of MTT, Resazurin and neutral red uptakeassays.

<p>Cytotoxicity of PR-GO in terms of MTT, Resazurin and neutral red uptakeassays.</p

From left, dispersed and exfoliated graphene oxide, aqueous solution of the polysaccharide, polysaccharide-reduced graphene oxide.

<p>From left, dispersed and exfoliated graphene oxide, aqueous solution of the polysaccharide, polysaccharide-reduced graphene oxide.</p

Green conversion of graphene oxide to graphene nanosheets and its biosafety study - Fig 8

<p>Raman Spectrum of PRGO (Curve A) and GO (Curve B).</p

XRD spectrum of PR-GO nanosheets.

<p>XRD spectrum of PR-GO nanosheets.</p

Characteristics of adherent cells and spheroids.

<p>(<b>A1</b>) Phase contrast image showing adherent stromal cells in serum-based growth media. (<b>B1</b>) Phase contrast image of spheroids in ultra-low attachment plates with serum-free media. (<b>A2–4</b>) On immunofluorescence staining, adherent cells stain weakly with KERA (<b>A2</b>, red) and ALDH3A1 (<b>A3</b>, red), and are strongly positive for α-SMA (<b>A4</b>, red). (<b>B2–4</b>) In contrast, spheroids stain strongly positive for KERA (<b>B2,</b> red), ALDH3A1 (<b>B3,</b> red), as well as α-SMA (<b>B4,</b> red). Cell nuclei are stained with DAPI (blue). (<b>C</b>) Western blot results for protein abundance in adherent cells and spheroids. Representative blot image (<b>C1</b>) and graph (<b>C2</b>) show that KERA and ALDH3A1 are significantly more abundant in spheroids, while α-SMA is more abundant in adherent cells. β-Actin was used as a loading control. (<b>D</b>) RT-qPCR confirms KERA and ALDH3A1 gene expressions to be higher in spheroids than in adherent cells, while α-SMA is expressed more in adherent cells than in spheroids. KERA, keratocan; ALDH3A1, aldehyde dehydrogenase 3 family member A1; α-SMA, alpha-smooth muscle actin. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars: 50µm; Error bars = Standard error of mean; *p≤0.05.</p

Expression of stemness transcription factor genes in spheroids and adherent cells.

<p>(<b>A</b>) RT-qPCR analyses shows that Oct4, and Nanog are expressed significantly more in spheroids than in adherent cells. (<b>B and C</b>) Immunofluorescence stained images show that adherent cells do not stain with Oct4 (<b>B1</b>, red) and Nanog (<b>B2</b>, red), whereas spheroids show positive staining with both (<b>C1, C2</b>). Nuclei stain with DAPI (blue). (<b>B3, C3</b>) Negative controls. Oct4, octamer-binding transcription factor 4; Nanog, Nanog homeobox; DAPI, 4′,6-diamidino-2-phenylindole. Error bars = Standard error of mean; Scale bars: 50 µm; *p≤0.05.</p

Spheroid-derived cells in KIM resemble keratocytes and are more viable than adherent culture-derived cells.

<p>(<b>A1–4</b>) Wide field fluorescence images of Calcein AM stained cells show very few dendritic processes in adherent culture-derived cells in KIM. (<b>B1–4</b>) In contrast, spheroid-derived cells show typical keratocyte morphology with dendritic processes and stellate shape, and this is maintained over 2 weeks of culture. (<b>C</b>) Cell viability analysis shows that keratocytes derived from spheroids were more viable than those derived from adherent cells over 2 weeks. Error bars = Standard error of mean; Scale bars: 100 µm; *p≤0.05.</p