Fig 2: CRYAB is the candidate to mediate its tumor-suppressive activity in PCa.a Workflow of the candidate screening. b Correlation analysis between MITF and CRYAB expression in primary tumor (PT) specimens of different PCa datasets. Sample sizes: Taylor, n = 131; Grasso, n = 49; Lapointe, n = 13; TCGA provisional data, n = 495; and Glinsky, n = 78. c CRYAB expression in normal prostate and primary tumor (PT) specimens in different PCa datasets9–13. Sample sizes: Taylor (N, n = 29; PT, n = 130); Grasso (N, n = 12; PT, n = 49); Varambally (N, n = 6; PT, n = 7); Lapointe et al. (N, n = 9; PT, n = 13), and Tomlins (N, n = 22; PT, n = 32). Data from Taylor dataset correspond to the mean signal of all isoforms of the transcripts. In (b) and (c), each dot corresponds to an individual specimen. d Western blot analysis of CRYAB expression in benign prostatic hyperplasia (BPH) and PCa specimens from Basurto University Hospital cohort (BPH n = 7 patient specimens; PCa n = 14 patient specimens). e Chromatin immunoprecipitation (ChIP) of exogenous MITF on CRYAB promoter in PC3 TRIPZ-MITFA cells after induction with 0.5 µg mL−1 doxycycline for 3 days (n = 4–5). Binding to ANGPT4 was used as a negative control. Final data were normalized to IgG (negative-immunoprecipitation control) and to No dox condition. No dox: MITFA noninduced conditions; Dox: MITFA-induced conditions. Statistic tests: Spearman correlation (b); Mann−Whitney test (c); one-sample t test (e); Error bars represent s.e.m. *p < 0.05, **p < 0.01

Fig 3: Identification of CRYAB p.E105K mutation.(A) Three Han Chinese pedigrees with optic nerve atrophy. Vision-impaired individuals are indicated by blackened symbols. Individuals harboring heterozygous (+/–) CRYAB (c.313G>A, p.Glu105Lys) mutation and WT (+/+) are indicated. (B) Fundus photographs from 8 (5 affected/3 control) members of 3 Chinese families. (C) Scheme for the structure of CRYAB and its products; multiple-sequence alignment of 7 homologs. (D) Western blot analysis of CRYAB in various lymphoblastoid cell lines. Representative of 3 independent experiments. (E) Subcellular localization of CRYAB by immunofluorescence in SH-SY5Y cells. CRYAB-GFP WT or MT (shown in green) and TOM20 (shown in red). Scale bar: 5 μm. (F) Subcellular localization of Cryab by Western blotting with Cryab, Tom20 (outer mitochondrial membrane), Atp5a (inner mitochondrial membrane), and Gapdh (cytosol). T, total cell lysate; D, debris; C, cytosol; Mito, mitochondria. Mitochondria isolated from C57 mice brain were treated with (+) or without (–) 1% Triton X-100 followed by proteinase K digestion.

Fig 4: In vitro analysis of structure and function of WT and MT CRYAB.(A) Schematic model for the α-crystallin domain (ACD) of CRYAB homodimer (PDB ID: 2klr) for WT (tawny) and MT (blue) contains chain A and chain B after MD simulations of 500 ns. The MT site was indicated. (B) Time evolution of the root mean square deviation (RMSD) values of all α-carbon (α-Cα) atoms for the WT (black line) and MT (red line) proteins. (C) Analysis for the oligomer formation of CRYAB. Both WT and MT proteins, labeled by Flag/His Tag, were expressed and purified from HeLa and BL21, electrophoresed through a blue native gel, and electroblotted and hybridized with antibodies specific for Flag-Tag, His-Tag, CRYAB, and GAPDH. (D) The chaperone activities of MT and WT CRYAB with DTT-induced denatured insulin. Insulin (0.4 mg/mL in 50 mM phosphate buffer [pH 7.4]) was reduced with 20 mM DTT, and the aggregation of the insulin CRYAB was monitored by measuring the apparent absorption at 360 nm. The aggregation rates were calculated. (E and F) Reduced interactions of CRYAB with cytochrome c (E) or VDAC (F). HEK293T cells, transiently expressing with WT, MT CRYAB-HA, or CRYAB-FLAG and empty vector, were solubilized with a lysis buffer. Lysate proteins and immunoprecipitates were immunoprecipitated with immunocapture buffer (IgG) (left), cytochrome c or VDAC antibody (right). Immunoprecipitates were then analyzed by SDS-PAGE and hybridized with anti-CRYAB and anti–cytochrome c or with anti-VDAC antibodies. Representative of 3 independent experiments.

Fig 5: Retinal deficiencies in CryabE105K mice.(A) Schema for the generation of CRYAB-knockin mice (C57BL/6J) using the CRISPR/Cas9 system. (B) Sequence chromatograms of Cryab gene. Sanger sequencing of WT and Cryab+/105K mice. (C) Levels of Cryab in the retinas from Cryab+/+, Cryab+/105K, and Cryab105K/105K mice at 8 weeks of age. (D) Cryab expression in the retinas of Cryab+/+, Cryab+/105K, and Cryab105K/105K mice by immunolabeling analysis. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar: 30 μm. (E) RGCs were stained with Brn-3a (green), β-III-tubulin (red), and DAPI (blue). Scale bar: 30 μm. (F) Ultrastructural analysis of RGC axons showing swelling and loss in Cryab+/105K and Cryab105K/105K mice. Swollen axons with thin myelin (asterisks) and degenerated axons with myelin clumping (arrow) were seen adjacent to normal caliber axons. Scale bar: 5 μm. (G) Representative optic coherence tomography of the retina in Cryab+/+, Cryab+/105K, and Cryab105K/105K mice. The circumpapillary retinal thickness was measured by OCT circle scan. (H) Fundus photograph eyes in Cryab+/+, Cryab+/105K, and Cryab105K/105K mice with OCT circle scan sites (black circles). (I) Fluorescence angiography of eyes in Cryab+/+, Cryab+/105K, and Cryab105K/105K mice. (J) Analysis of ffERG for Cryab+/+ (n = 6), Cryab+/105K (n = 6), and Cryab105K/105K (n = 6) mice. By dark adaptation for a night, mice were analyzed for scotopic response and then photopic response. Data are shown as mean ± SEM of triplicates. ***P < 0.001, by 1-way ANOVA followed by Bonferroni’s post hoc test.