Fig 2: Overexpressing HAS3 in the N2a cells significantly promotes cell differentiation. (A) HAS3 overexpression plasmid was transfected into the N2a cells. The protein level of HAS3 and differentiation markers (TUBB3 and GFAP) were detected by Western blot assay (left panel). Differentiated N2a cells were calculated based on 1000 random cells in the vector control group and the HAS3 overexpression group. The morphological change after HAS3 overexpression was captured by microscopy. Magnification, 100×; scale bar, 10 µm. (B) Neurite length was measured in six random microscopic fields for both the vector control group and the HAS3 overexpression group at 24, 48 and 72 h. (C) Differentiated N2a cells were identified by immunofluorescence staining after transfection with an HAS3 expression plasmid. TUBB3-rhodamine was used as a marker for differentiated N2a cells. Endogenous and overexpressed HAS3 were identified via HAS3-FITC staining. Magnification, 630×; scale bar, 50 µm. The data are presented as the mean ± SD; *P < 0.05 and **P < 0.01

Fig 3: Glial activation in retinas of Vps13b?Ex3/?Ex3 eyes with and without cataract. (A–D) Representative images of retinal sections (n = 3 per group) from Vps13b+/+ eyes (left panels) and Vps13b?Ex3/?Ex3 eyes with hypermature cataract (right panels) at 3 months of age stained with H&E (A) or antibodies against GFAP (B) or Iba1 (C). Cataractous eyes (HC) showed large and multiple regions with retinal folds and edemas in which astroglial and microglial proliferations were prominent along with activated Müller cells. The arrow in A (lower panel) highlights a fibrotic area in the vitreous humor near an edema. VH, vitreous humor; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ED, edema. Scale bars: 10 µm.

Fig 4: Inflammatory and proliferative profiles of Vps13b?Ex3/?Ex3 retinas based on protein expression levels. (A) Western blot analysis of Iba1 and GFAP expression levels, as well as Stat3 and Erk1/2 phosphorylation levels, on protein extracts from Vps13b+/+, cataract-free Vps13b?Ex3/?Ex3, and nuclear sclerotic Vps13b?Ex3/?Ex3 eyes, as well as Vps13b?Ex3/?Ex3 eyes with HC, at 2 months of age. GAPDH was used as loading control. (B–F) Relative protein levels of Iba1, GFAP, Stat3, Phospho-Stat3/Stat3, and Phospho-Erk2/Erk2 from western blot analysis presented in (A). Protein levels were normalized to GAPDH levels and the average Vps13b+/+ value. The data are presented as a boxplot showing minimum, maximum, and quartile values for each group (n = 3). Although some Vps13b?Ex3/?Ex3 retinas of eyes without HC presented with increased Iba1 and GFAP levels representative of glial activation and proliferation, this increase became systematic in eyes with HC. Iba1 was increased by 11-fold (B) and GFAP by 5-fold (C) compared to controls. In addition, the pro-inflammatory protein Stat3 was also more prominent in retinas of eyes with HC. Comparison of the Phospho-Stat3/Stat3 ratio (D) and Stat3/GAPDH ratio (E) shows that increased levels of Phospho-Stat3 in cases of HC were mostly due to the overexpression of Stat3. Interestingly, Vps13b?Ex3/?Ex3 retinas of eyes without HC also displayed a moderately elevated expression of Stat3 that did not translate into increased phosphorylation of the protein. Last, the key regulator of cell proliferation Erk was only over-activated in eyes with HC (F).

Fig 5: The growth and differentiation of NE-4C and SH-SY5Y cells after cocultured with PVA, GO/PVA, MoS2/PVA, and MoS2/GO/PVA hydrogels in vitro. Confocal microscopic images of (a) NE-4C cells and b SH-SY5Y cells. Tuj1-positive cells (green) and GFAP-positive cells (red) in different groups (Control, GO/PVA, MoS2/PVA, and MoS2/GO/PVA hydrogels) at 3 and 7 days. All nuclei were stained with DAPI (blue). Scale bar = 50 µm. c Fluorescence intensity values corresponding to confocal photos of each group. d Quantitative of relative gene expression levels of Tuj1, GFAP, ChAT, Islet-1, and HB9 in NE-4C cells in different groups (Control, GO/PVA, MoS2/PVA, and MoS2/GO/PVA hydrogels) at 3 and 7 days. Error bars indicate the SD from four different experiments; *p < 0.05, **p < 0.01, all groups were compared with the control group