And differentiation; therefore, affecting a myriad of biological processes in Antiviral Compound Library site melanocytes [12,13]. As well as exerting an important physiological function, MITF can also be an essential player in melanoma biology as it is didactically explained by a rheostat model: higher, intermediate, and low levels of MITF cause differentiated, proliferative, and invasive phenotypes, respectively, whereas MITF absence benefits in senescence or cell death [147]. A vital function of melanocytes is their sensitivity to UV and light stimulus responding with critical physiological processes, mostly pigmentation. The majority of the literature has focused on analyzing the endpoint of such response, i.e., pigmentation, proliferation, DNA harm, and other individuals, when just a handful of research have evaluated how melanocytes are basically capable to sense light and UV radiation photons. Within this line of believed, opsins–light sensing molecules–known to be expressed in the eye, where they take part in visual and non-visual processes [182], were first demonstrated inside the skin in early 2000 in mice [23] and 2009 in humans [24]. Functional studies were only performed pretty much a decade later by Oancea’s lab pioneering reports [257]. For the present day, the photosensitive program in the skin has been shown to take part in murine and human: pigmentary responses [251], differentiation processes of keratinocytes [32,33], hair follicle growth [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 current years, the paradigm of opsins becoming light sensors was challenged by research in murine melanocytes demonstrating that melanopsin may also detect thermal power [37]. Additionally, it was shown that sperm cell thermotaxis is dependent on OPN2 and OPN4 presence [38,39]. Extra not too long ago, light- and thermo-independent roles of opsins have also been reported in human melanocytes, as a result, revealing an even more complex situation for opsin signaling. As an illustration, OPN3 has been related with damaging regulation in the MC1R pathway, major to an Fragment Library Purity & Documentation inhibitory impact on melanogenesis [40] as well as Opn3 knockdown resulted in melanocyte apoptosis [41]. OPN5 has also been implicated as a unfavorable regulator of melanogenesis given that its downregulation by gene silencing resulted in lowered expression of essential enzymes involved in melanin synthesis within a UV-independent manner [42]. In this study, we demonstrate a light- and thermo-independent function of OPN4 in murine melanocytes harboring a functional (Opn4WT ) and non-functional (Opn4KO ) OPN4 protein by evaluating cellular metabolism, proliferation, and cell cycle regulation. two. Material Strategies two.1. Cell Culture Opn4KO Melan-a melanocytes had been generated making use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. Cells underwent phenotypic characterization and Sanger sequencing revealed a disruption of one Opn4 allele that rendered these cells OPN4 impaired, as previously described in detail [28]. Opn4WT and Opn4KO cells were subject to Per1: Luc gene transfection as described previously [28] and have been also used within this study. Cells have been cultured in RPMI 1640 medium without the need of 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 answer (10,000 U/mL penicillin.