Fig 2: Proximity ligation assay quantification for C8orf37 and FAM161A in the marmoset retina. Confocal images of the retina were divided into layers (example is shown in (A)). PLA puncta in each layer were counted from images of 5 stained sections from one marmoset eye, showing: (B,C) the numbers of PLA puncta in each layer of the retina, as well as the total number of PLA puncta across all layers of the retina. Error bars represent standard error of the mean (SEM). (D) Table of p-values from ANOVA and Tukey HSD post-hoc tests. The number of PLA puncta was significantly increased when PLA was performed with both probes compared with when anti-C8orf37-PLUS or anti-FAM161A-MINUS probes were used alone. Scale bar in (A): 20 μm.

Fig 3: C8orf37 and FAM161A were widely expressed in the marmoset retina. Marmoset retinal sections were double-immunostained with anti-C8orf37 (red) and anti-acetylated α-tubulin (green) (A–F) or anti-FAM161A and acetylated α-tubulin (green) (G–L). The sections were counterstained with DAPI (blue). Images (A–F) show C8orf37 and acetylated α-tubulin double staining. C8orf37 immunoreactivity was observed in the inner segment layer, outer nuclear layer, inner nuclear layer, and ganglion cell layer. C8orf37 immunoreactivity puncta was also observed at the base of acetylated α-tubulin-labeled cilia (arrows in F), as well as along the cilia (arrowheads). Images (G–L) show FAM161A and acetylated α-tubulin double staining. FAM161A immunoreactivity was widely distributed in the retina. Its puncta were also enriched at the base of cilia (arrows in L) and along the cilia. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment. Scale bar: 20 μm for (A–C) and (G–I); 6 μm for (D–F) and (J–L); 3 μm for insets in (F) and (L).

Fig 4: N-terminus of C8orf37 interacts with the UPF0564 domain of FAM161A. Y2H assays were performed to identify the region of interaction on C8orf37 and FAM161A. Transformants were plated at different dilutions on selective medium lacking leucine and tryptophan (-L -W); on medium lacking leucine, tryptophan, and histidine (-L -W -H); on stringent selective medium lacking leucine, tryptophan, and histidine treated with 1mM 3-amino-1,2,4-triazole (-L -W -H +1 mM 3AT); and on medium lacking leucine, tryptophan, and histidine treated with 5mM 3AT (-L -W -H +5 mM 3AT). Schematic (A) shows full-length C8orf37, mutant C8orf37, and C8orf37 fragments used in the Y2H experiments. Images in (B) show that positive interaction, indicated by colony growth, could be observed between full length C8orf37 (C8orf37–FL) and FAM161A (aa 317–549), C8orf37-R177W and FAM161A (aa 317–549), and C8orf37-Q182R and FAM161A (aa 317–549). No growth was observed between C8orf37 (aa 69–207) and FAM161A. Images in (C) show that positive interaction could be observed between the N-terminal fragment of C8orf37 (aa 1–75) and FAM161A (aa 317–549) on -L -W -H plates treated with 1 mM 3AT and 5 mM 3AT, respectively. Abbreviations: EV, empty vector; BD, binding domain; AD, activation domain.

Fig 5: Overexpressed C8orf37 and FAM161A were co-localized. HEK293 cells were transfected with pCMV-C8orf37-tGFP and pCMV-FAM161A-c-Myc expression constructs. The cells were immunostained with anti-c-Myc (red) (A–C) or processed for proximity ligation assay using anti-tGFP and anti-c-Myc antibodies (D–G). Images (A–C) show pCMV-C8orf37-tGFP and pCMV-FAM161A-c-Myc double transfected cells. HEK293 cells were efficiently transfected with both plasmids. Image (D) shows that PLA puncta were frequently observed in cells double transfected with pCMV-C8orf37-tGFP and pCMV-FAM161A-c-Myc. Images (E–G) show that PLA puncta were significantly reduced when the cells were single-transfected with pCMV-C8orf37-tGFP (E), or pCMV-FAM161A-c-Myc (F), or non-transfected (G). Image (H) shows quantification of PLA puncta three-repeat transfections. ANOVA followed by post-hoc Student’s t-test with Bonferroni correction. Error bars represent SEM. Scale bar in (G): 20 μm.