Fig 1: Galectin-7 (Gal-7) release from normal human epidermal keratinocytes (NHEKs) via interleukin (IL)-4/IL-13–induced signal transducers and activator of transcription (Stat)6 activation. (A) NHEKs were treated with or without IL-4/IL-13, interferon (IFN)-? and IL-17/IL-22 for 15 minutes at a concentration of 50 ng/mL, respectively. Western blot analyses for Stat1, phosphorylated (p)-Stat1 (tyrosine [Tyr]701), Stat3, p-Stat3 (Tyr705), Stat5, p-Stat5 (Tyr694), Stat6 and p-Stat6 (Tyr641) were performed. (B) NHEKs were transiently transfected with 10 nM small interfering (si) RNA targeting Stat3 (siStat3), Stat6 (siStat6) or non-targeting siRNA (siControl; siCtr) for 48 hours, followed by 48-hour stimulation with or without IL-4/IL-13 (50 ng/mL). Stat6, p-Stat6, Stat3, p-Stat3 and Gal-7 were analysed by Western blot. (C, D) The expression values of Stat3 and Stat6 in each siRNA-treated NHEKs were normalized to the siCtr-treated control group (set as 1) (n = 3). (E) Gal-7 protein levels in culture supernatants obtained from each siRNA-treated NHEKs with or without IL-4/IL-13 (50 ng/mL) were measured by ELISA. Data were shown as means ± SD. *P < .05, **P < .01; ***P < .005. n.s., not significant. NT, no treatment
Fig 2: Methylation-specific polymerase chain reaction analysis of the Galectin-7 gene promoter in 6 vulvar squamous cell carcinoma samples (C1–6) and 6 normal vulvar tissues (N1–6). The methylated (M) and unmethylated (U) DNA was amplified using primers specific for each methylation status. Universal methylated DNA was used as a positive loading control and distilled water was used as a negative control.
Fig 3: Representative immunohistochemical staining images of Galectin-7 in (A) normal vulvar tissue and (B) vulvar squamous cell carcinoma (magnification, ×200).
Fig 4: Interleukin (IL)-4/IL-13–mediated Galectin-7 (Gal-7) release from normal human epidermal keratinocytes (NHEKs). (A) NHEKs were treated with or without IL-4/IL-13, interferon (IFN)-? and IL-17/IL-22 for 48 h at a concentration of 50 ng/mL Gal-7 mRNA levels were assessed by real-time reverse transcriptase-PCR. (n = 3). (B) Gal-7 protein levels in culture supernatants were measured by ELISA (n = 3). (C) IL-4/IL-13–induced cell death was analysed by flow cytometry (n = 6). (D) Confocal images of immunostaining for Gal-7 (green). Nucleus, actin filaments and tight junctions were counterstained with DAPI (blue), phalloidin (red) and ZO-1 (yellow), respectively. Scale bar = 30 µM. (E) Representative images of haematoxylin and eosin (HE) and immunohistochemical staining for Gal-7 in 3-dimensional (D)–reconstructed epidermis treated with or without IL-4/IL-13 for 48 hours at a concentration of 50 ng/mL. Scale bars = 50 µm (upper and middle) and 10 µm (bottom). Black arrows indicate intercellular deposits of Gal-7. Data were shown as means ± SD of three independent experiments performed in one (A, B) or duplicate (C). *P < .05, **P < .01; n.s., not significant
Fig 5: Galectin-7 (Gal-7) is required to avoid interleukin (IL)-4/IL-13–induced disruption of cell-to-cell and/or cell-to-extracellular matrix (ECM) adhesion junction of the epidermis. (A) Three-dimensional (D)–reconstructed epidermis transduced with short hairpin control (shCtr) or shGal-7 was treated with or without IL-4/IL-13 for 48 hours at a concentration of 50 ng/mL Gal-7, E-cadherin, Desmocollin-1 (DSC1), Claudin-1 (CLDN1) and Desmoglein-1 (DSG1) mRNA levels were assessed by real-time reverse transcriptase-PCR. (B) Representative images of haematoxylin and eosin (HE) and immunohistochemical (IHC) staining for Gal-7 in shCtr or shGal-7–transduced 3D-reconstructed epidermis treated with or without IL-4/IL-13 for 48 hours at a concentration of 50 ng/mL. The boxed areas were enlarged in the right panels. Black arrows indicate acantholysis. Scale bars = 50 µm. Data were shown as means ± SD of two or three independent experiments. *P < .05. NT, no treatment
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