And differentiation; thus, affecting a myriad of biological processes in melanocytes [12,13]. Along with exerting an important physiological role, MITF is also a vital player in melanoma biology since it is didactically explained by a rheostat model: high, intermediate, and low levels of MITF lead to differentiated, proliferative, and invasive phenotypes, respectively, whereas MITF absence final results in senescence or cell death [147]. An essential feature of melanocytes is their sensitivity to UV and light stimulus responding with critical physiological processes, primarily pigmentation. A lot of the literature has focused on analyzing the endpoint of such response, i.e., pigmentation, N-Acetylcysteine amide Metabolic Enzyme/Protease proliferation, DNA damage, and others, CX-5461 manufacturer although just a handful of research have evaluated how melanocytes are essentially able to sense light and UV radiation photons. Inside this line of thought, opsins–light sensing molecules–known to become expressed in the eye, where they take part in visual and non-visual processes [182], have been 1st demonstrated within the skin in early 2000 in mice [23] and 2009 in humans [24]. Functional studies were only performed nearly a decade later by Oancea’s lab pioneering reports [257]. To the present day, the photosensitive system of your 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 growth 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 being light sensors was challenged by studies in murine melanocytes demonstrating that melanopsin can also detect thermal energy [37]. Moreover, it was shown that sperm cell thermotaxis is dependent on OPN2 and OPN4 presence [38,39]. Much more not too long ago, light- and thermo-independent roles of opsins have also been reported in human melanocytes, therefore, revealing an much more complex situation for opsin signaling. For example, OPN3 has been associated with unfavorable regulation on the MC1R pathway, leading to an inhibitory effect on melanogenesis [40] as well as Opn3 knockdown resulted in melanocyte apoptosis [41]. OPN5 has also been implicated as a adverse regulator of melanogenesis because its downregulation by gene silencing resulted in lowered expression of essential enzymes involved in melanin synthesis in a UV-independent manner [42]. Within 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. two. Material Methods two.1. Cell Culture Opn4KO Melan-a melanocytes were generated utilizing Clustered Regularly Interspaced Brief Palindromic Repeats (CRISPR) method. Cells underwent phenotypic characterization and Sanger sequencing revealed a disruption of one particular Opn4 allele that rendered these cells OPN4 impaired, as previously described in detail [28]. Opn4WT and Opn4KO cells were topic to Per1: Luc gene transfection as described previously [28] and have been also applied in this study. Cells were 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), ten fetal bovine serum (FBS, Atena, Campinas, So Paulo, Brazil), a 1 antibiotic/antimycotic option (10,000 U/mL penicillin.