Hoechst nucblue

Skin samples were obtained from healthy donors undergoing surgery under ethical approval from the UK National Research Ethics Service (14.NW.1153). Epidermal sheets were separated from dermal tissues by overnight incubation in dispase (5 U/ml; Sigma Aldrich) and cultured up to 48 h in KGM-2 keratinocyte medium (Lonza) adjusted with CaCl₂ to a 1.5 mM final calcium concentration. The Ni(NO₃)₂ solution was added at the 1 mM final concentration. Experiments were repeated on skin explants from 10 donors. For fluorescent antibody staining epidermal sheets were fixed with 4% formaldehyde (Sigma), followed by 0.1% Triton X-100 (Sigma Aldrich) and incubated in a blocking buffer (5% FCS, 2% BSA in PBS) for 1 h. Anti-FLG goat G20 (Santa Cruz), and the secondary anti-goat Alexa488 (Life Technologies) antibody staining was carried out in PBS for 1 h. The nuclei were visualized by Hoechst (NucBlue, Life Technologies). The sheets were mounted on microscope cover-slides with Mowiol 4-88 (Sigma) for imaging. Data acquisition was carried out on the Zeiss 780 inverted confocal microscope by recording z-stacks of 2D images (at 0.38 µM intervals) and images taken using inverted confocal microscope (Zeiss 780) by recording 2D images in a 3D z-stack.

Normal human epidermal keratinocytes (NHEKs) (purchased from Lonza) were cultured in monolayers in a dedicated medium (Lonza, KBM-2) at the Ca²⁺ level of 0.06 mM. To stimulate differentiation and FLG expression, a calcium switch was conducted over a period of 24 h by replacing the culture media with fresh media adjusted to a 1.5 mM final calcium concentration. A Ni(NO₃)₂ (Sigma) solution was added to achieve various final concentrations (10 μM, 100 μM and 1 mM). Doses were chosen based on MTT test results (Supplementary Figure S1). After 24 h of incubation, the cells were fixed, permeabilized and immunostained with anti-FLG antibodies (Anti-FLG goat G20 (Santa Cruz), and secondary anti-goat Alexa-488 and anti-rabbit Alexa-568 (Life Technologies) antibodies were used. Staining was carried out in PBS and nuclei were visualized by Hoechst (NucBlue, Life Technologies). The slides were coversliped with Mowiol 4-88 (Sigma). Data acquisition was carried out on the Zeiss 780 inverted confocal microscope. Images from three separate experiments were analysed; KHG diameter and integrated intensity from the signal were measured using Fiji: ImageJ program (Abramoff, 2007 (link)). For the statistical analysis the Mann–Whitney U test was used.