Fig 2: RasGRP1 expression in human cSCC and normal skin and effects on human keratinocyte morphology. (A) Analysis of RasGRP1 mRNA expression in the Riker-87 dataset shows that RasGRP1 is significantly higher expressed in cSCC (11 samples) than in normal skin (4 samples). The results shown are for a rank-based Kruskal-Wallis test to prevent undue influence by very high or very low mRNA expression values. RasGRP1 also shows significant overexpression using 2log (3,21 ± 0,14 > 2,61 ± 0,21; P = 0,040) and normal t-tests (10,50 ± 0,84 > 6,95 ± 1,02; P = 0,039). (B) Immunoblot of endogenous RasGRP1 expression was determined for 1 keratinocyte and 1 human cSCC cell line. (C) Immunoblot analysis confirming the exogenous expression of mouse RasGRP1 (mRG1, using M199 antibody) and Ras in human keratinocytes (HKn) transduced with adenovirus at the indicated times post transduction. Expression of endogenous human RasGRP1 (hRG1) was also determined. (D) HKn were transduced with adenovirus encoding GFP, GFP-RasGRP1, or HRasQ61L. Images taken 72 h post transduction at 200× magnification (Olympus Microscope). Insets show transduction efficiency for GFP, RasGRP1, and HRasQ61L via fluorescence and staining. Scale bar = 50 µm. Percent of GFP positive transduced cells is indicated at the bottom.

Fig 3: RasGRP1 expression led to defective stratification in skin tissue reconstructs. (A) Left: Diagram illustrating the architecture of normal human skin including basal and suprabasal layers. Right: Diagram of the skin reconstruct procedure for generation of a 3D organotypic culture. Keratinocytes transduced with adenoviral vectors encoding GFP, RasGRP1 or HRasQ61L were seeded onto the dermal layer of a 3D skin reconstruction culture. (B) Micrographs of HKn 48 h post transduction prior to harvesting for preparation of the 3D cultures. Insets shown are fluorescence images for GFP, RasGRP1 or HRasQ61L-expressing HKn. (C) HKn were seeded onto the dermal layer of a 3D skin reconstruction culture 48 h post transduction. Tissue reconstructs were harvested after 10 days and processed by H&E staining for histological assessment.

Fig 4: Rasgrp1 is selectively expressed in ipRGCs. A, Whole-mount retina immunostained for Opn4, Rasgrp1, and the pan-RGC marker Rbpms (gray scale). Focal plane is at the GCL. We quantified colocalization of the three markers in confocal images of 49 regions that were topographically dispersed across three whole-mount adult retinas. Colocalization of Rasgrp1 (green), Rbpms (red), and Opn4 (magenta). Rasgrp1 is expressed in a subpopulation of amacrine cells and RGCs (Rbpms-negative and -positive, respectively). Scale bar, 20 µm. B, Rasgrp1 immunolabeling (antibody sc-8430) of cell bodies in GCL of Rasgrp1± heterozygous mice (left, yellow arrows). Absence of cell body immunolabeling in Rasgrp1-/- knock-out mice (right) suggests a lack of cellular off-target antibody staining. C, Quantification of Rasgrp1-expression across Opn4-immunopositive ipRGC subtypes. 70% of M1 and displaced M1 (dM1) cells were Rasgrp1-immunopositive, whereas only 20–30% of either M2, M5, or M6 cells were Rasgrp1-immunopositive. None of the identified M4 cells were Rasgrp1-immunopositive. M1 and M3 types were combined during the process of coexpression analysis (designated M1/M3). Error bars represent SEM. D, Distribution of all Rasgrp1-expressing RGCs (Rasgrp1+;Rbpms+) that belong to specific RGC types, to the extent that could be determined, including Opn4-immunoreactive ipRGC subtypes. No examples of M4 cells were observed to express Rasgrp1. The vast majority (96%) of Rasgrp1-RGCs are Opn4-immunopositive and therefore ipRGCs. The remaining “unknown” RGC types expressing Rasgrp1 (Rasgrp1+; Rbpms+; Opn4neg.) could be a low-expressing ipRGC type or conventional RGCs. Error bars represent SEM.

Fig 5: The ipRGCs projecting to the SCN have a molecularly diverse pattern of Rasgrp1 and Tbx20 expression. A, Experimental design of fluorescent bead injection to SCN, followed by examination of Rasgrp1 and Tbx20 expression in retrograde labeled RGCs. B, Neuro-anatomical study to verify that retrograde injection is within the SCN, but not the optic nerve. C, Triple immunofluorescence of Opn4, Rasgrp1, and Tbx20 in combination with fluorescent Retrobeads. Retrobeads were mostly observed in Opn4-immunopositive RGCs (M1–M3 ipRGCs). Quantification of Rasgrp1 and Tbx20 in retrolabeled cells. D, SCN-projecting ipRGCs in the ipsilateral and contralateral retina are molecularly diverse for Tbx20 and Rasgrp1 expression.