Journal: International Journal of Molecular Medicine
Article Title: Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG
doi: 10.3892/ijmm.2020.4487
Figure Lengend Snippet: UPF1 depletion-induced AREG disturbs keratinocyte homeostasis. (A) Cell counting kit-8 assays were performed to measure cell viability 48 h after UPF1 or AREG knockdown in HEKα and HaCaT cells. UPF1 and AREG depletion both decreased cell viability of keratinocytes. (B) The expression of Ki67 decreased in keratinocytes with UPF1 or AREG knockdown, while the expression of apoptosis-related gene BAD increased in both groups. (C) mRNA expression level of K5 , FLG and K10 was qualified by reverse transcription-quantitative PCR in keratinocytes. The expression levels of these differentiation makers decreased with UPF1 depletion and were rescued by supplementary AREG depletion. (D) Representative human keratinocyte scratch wounds with cells treated with the sh UPF1 construct, or sh UPF1 combined with si AREG . The wound images at 0 and 24 h are presented, and the epidermal sheet edge indicated by the yellow irregular curve was identified by ImageJ software automatically. UPF1 depletion induced the migration of keratinocytes by stabilization of AREG . (E) Quantitative analysis of the measured wound coverage at 0, 6, 12, 24 and 48 h. *** P<0.001 vs. NC group. (F) The motility markers SNAI1 , COX-2 and MMP1 and two chemokines associated with nuclear factor κB activation, CCL20 and CXCL1 , were identified at the mRNA level in keratinocyte cells. Expression levels of the migration makers and chemokines increased with UPF1 depletion and were rescued by supplementary AREG depletion. The results are presented as the mean ± SD from three independent experiments. *** P<0.001. UPF1 , up-frameshift suppressor 1 homolog; AREG , amphiregulin; BAD , BCL2 associated agonist of cell death; K5 , keratin-5; FLG , filaggrin; K10 , keratin-10; COX-2 , cyclooxygenase-2; MMP1 , matrix metalloproteinase 1; CCL20 , chemokine (C-C motif) ligand 20; CXCL1 , human recombinant GRO-alpha; NC, normal cells; Ki67 , proliferation marker protein Ki67; n.s., not significant.
Article Snippet: The prepared sections were incubated overnight at 4°C with anti-UPF1 primary antibodies (cat. no. sc-390096; 1:100; Santa Cruz Biotechnology, Inc.).
Techniques: Cell Counting, Expressing, Real-time Polymerase Chain Reaction, Construct, Software, Migration, Activation Assay, Recombinant, Marker