Fig 1: USP18 was upregulated in LUAD tissues and cells. (A-B) USP18 expression in LUAD in Timer dataset (A) and Ualcan dataset (B). (C) USP18 expression analysis in LUAD was conducted by IHC. According to the positive cells (brown), IHC quantification analysis was carried out by normalization of normal group to 1. (D-E) RT-qPCR (D) and western blot (E) were employed for mRNA and protein analysis in LUAD tissues. (F-G) RT-qPCR (F) and western blot (G) for USP18 expression detection in LUAD (H1299, A549) and normal 16HBE cells. mP < 0.001
Fig 2: USP18 stabilized POU4F1 protein expression through inducing de-ubiquitination. (A) POU4F1 was predicted as a USP18 substrate in Ubibrowser. (B-C) Analysis of USP18 and POU4F1 interaction by IP assay. (D) The degree of POU4F1 ubiquitination was examined using western blot after USP18 knockdown. (E-F) Half-life of POU4F1 with CHX treatment under knockdown of USP18 was detected by western blot. (G) POU4F1 protein in LUAD tissues was measured by IHC. According to the positive cells (brown), IHC quantification analysis was performed by normalization of normal group to 1. (H-I) RT-qPCR (H) and western blot (I) were administrated for POU4F1 detection in LUAD samples. (J-K) POU4F1 mRNA and protein detection by RT-qPCR and western blot in LUAD cells. (L) POU4F1 protein analysis by western blot after co-transfection with sh-USP18 and POU4F1, and control groups. mP < 0.001
Fig 3: Knockdown of USP18 promoted apoptosis and ferroptosis of LUAD cells. Transfection with sh-NC or sh-USP18 was performed in H1299 and A549 cells. (A) Analysis of USP18 protein level by western blot. (B-C) Cell proliferation (B) and migration (C) were assessed via colony formation assay and wound healing assay. (D) Apoptosis detection by flow cytometric assay. (E-G) ROS (E), MDA (F), and GSH (G) examination using kits. (H) Fe2+ level was determined via iron level assay. nP < 0.01, mP < 0.001
Fig 4: USP18 regulated proliferation, migration, apoptosis, and ferroptosis by mediating deubiquitination of POU4F1 to affect PRKAA2
Fig 5: Silencing USP18 reduced tumor growth in vivo by mediating POU4F1/PRKAA2 axis. (A) Establishment of xenograft model. (B-C) Tumor volume and weight of sh-NC, sh-USP18, sh-USP18 + POU4F1, sh-POU4F1, or sh-POU4F1 + PRKAA2 group. (D) Western blot was used to measure protein expression of PRKAA2 in tumor tissues. (E) PRKAA2 protein detection was carried out using IHC in tumor tissues. According to the positive cells (brown), IHC quantification analysis was completed by normalization of sh-NC group to 1. nP < 0.01, mP < 0.001
Supplier Page from Abcam for Recombinant Human UBP43/USP18 protein (GST tag N-Terminus)