Figure 1.

Effect of IL-4 on the expression of PER2. HaCaT cells were treated with vehicle (PBS) or 20 ng/mL IL-4 for various periods (0–36 h). (A,B) Total RNA was isolated, and the levels of PER2 mRNA were measured by RT-PCR (A) and qPCR (B). The mRNA level of GAPDH was used as an internal control. ns, not significant; ** p < 0.01; *** p < 0.001 compared to vehicle (PBS)-treated control group (n = 3) by Sidak’s multiple comparisons test. (C) Whole-cell lysates were prepared and immunoblotted using anti-IL31 antibodies. GAPDH was used as the loading control. (D) HaCaT cells cultured on coverslips were treated with vehicle (PBS) or 20 ng/mL IL-4 for 24 h, followed by fixing and permeabilization. Immunofluorescence staining was performed using anti-PER2 and Alexa Fluor 555-conjugated secondary antibodies (red staining). α/β-tubulin was counterstained with anti-α/β tubulin and Alexa Fluor 488-conjugated secondary antibodies (green staining). Nuclear DNA was stained with 1 μg/mL Hoechst 33258 (blue staining). Fluorescent cells were captured with an EVOSf1 fluorescence microscope. The last panel on the right is a zoomed-in view of the dotted box. Scale bars, 50 μm. IL-4, interleukin 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline; PER2, Period2; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative real-time PCR.