Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis. K E Y W O R D Scell cycle, haspin, kinase, mitosis

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Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21 CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation.These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.

We present an efficient approach to slowing the progression of skin damage using a multi-mechanistic antioxidant blend suitable for cosmetic formulations and validate its performance in vitro. Three different mechanisms were targeted simultaneously: scavenging reactive oxygen species (ROS), mitigating intracellular ROS and reducing ROS-induced inflammaging markers. Using a Design of Experiment approach, a novel, non-phototoxic synergistic blend of antioxidants was identified and characterized in vitro for its ability to quench free radicals (DPPH assay) and intracellular ROS generated by UVA (DCFH assay). Further, we demonstrated that use of the blend results in a mitigation of markers correlated with inflammaging (photoaging and hyperpigmentation) caused by environmental oxidative stress (PGE 2 , IL-8, MMP-1). 3 These results support the notion that a multi-mechanistic antioxidant blend may effectively alleviate environmentally induced skin damage and be easily incorporated into skin care formulations providing an anti-inflammaging benefit. Brewer,

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