And differentiation; as a result, affecting a myriad of biological processes in melanocytes [12,13]. In addition to exerting a vital physiological part, MITF is also an essential player in melanoma biology since it is didactically explained by a rheostat model: higher, intermediate, and low levels of MITF lead to differentiated, proliferative, and invasive phenotypes, respectively, whereas MITF absence benefits in senescence or cell death [147]. An important function of melanocytes is their sensitivity to UV and light stimulus responding with essential physiological processes, mostly pigmentation. The majority of the literature has focused on analyzing the endpoint of such response, i.e., pigmentation, proliferation, DNA damage, and others, though just a handful of studies have evaluated how melanocytes are actually capable to sense light and UV radiation photons. Within this line of thought, opsins–light sensing molecules–known to be expressed in the eye, exactly where they take part in visual and non-visual processes [182], were very first demonstrated inside the skin in early 2000 in mice [23] and 2009 in humans [24]. Functional studies had been only performed nearly a decade later by Oancea’s lab pioneering reports [257]. For the present day, the photosensitive system from the skin has been shown to participate in murine and human: pigmentary responses [251], differentiation processes of keratinocytes [32,33], hair follicle development [34], UVA-induced photoaging [35], cellular development and apoptosis in response to UVA radiation [28], and UV- and blue light-induced calcium influx [25,27,36]. In recent years, the paradigm of opsins being light sensors was challenged by research in murine melanocytes demonstrating that melanopsin can also detect thermal power [37]. Moreover, it was shown that sperm cell thermotaxis is Hesperadin Cancer dependent on OPN2 and OPN4 presence [38,39]. Far more not too long ago, light- and thermo-independent roles of opsins have also been reported in human melanocytes, thus, revealing an even more complicated situation for opsin signaling. As an example, OPN3 has been associated with adverse regulation in the MC1R pathway, major to an inhibitory effect on melanogenesis [40] also as Opn3 knockdown resulted in melanocyte apoptosis [41]. OPN5 has also been implicated as a adverse regulator of melanogenesis considering the fact that its downregulation by gene silencing resulted in lowered expression of important enzymes involved in melanin synthesis in a UV-independent manner [42]. In this study, we demonstrate a light- and thermo-independent role of OPN4 in murine melanocytes harboring a functional (Opn4WT ) and non-functional (Opn4KO ) OPN4 protein by evaluating cellular metabolism, proliferation, and cell cycle regulation. 2. Material Methods two.1. Cell Culture Opn4KO Melan-a melanocytes were generated employing Clustered Frequently Interspaced Quick Palindromic Repeats (CRISPR) technique. Cells underwent phenotypic characterization and Sanger sequencing revealed a disruption of 1 Opn4 allele that rendered these cells OPN4 impaired, as previously described in detail [28]. Opn4WT and Opn4KO cells were subject to Per1: Luc gene Biotin-azide Chemical transfection as described previously [28] and had been also utilized within this study. Cells had been cultured in RPMI 1640 medium without having phenol red (Atena, Brazil), supplemented with 25 mM NaHCO3 (Sigma-Aldrich, St. Louis, MO, USA), 20 mM HEPES (Santa Cruz, Dallas, TX, USA), 10 fetal bovine serum (FBS, Atena, Campinas, So Paulo, Brazil), a 1 antibiotic/antimycotic remedy (10,000 U/mL penicillin.