Fig. 7

SNHG1 promotes USP8 mRNA Degradation. A and D Total RNA was extracted from UROtsa(SNHG1) versus UROtsa(Vector) cells, and U5637(shSNHG1#1) cells, U5637(shSNHG1#2) versus U5637(Nonsense) cells using Trizol reagent. Real-time PCR assays were conducted to measure USP8 mRNA expression levels, with GAPDH serving as an internal control. B and E A luciferase reporter driven by the human USP8 promoter was utilized to assess promoter transcription activity in the indicated transfectants. Results were normalized against internal TK activity. C and F The specified cells were seeded into 6-well plates and cultured until they reached 80% confluence. Post-synchronization, the cells were treated with Act D for designated durations. Total RNA was subsequently isolated and subjected to real-time PCR analysis to ascertain USP8 mRNA levels, again using GAPDH as an internal control. G Western blot analysis was performed on the chosen cell extracts to determine protein expression levels of NCL, AUF1, and HUR, with β-Actin acting as a protein loading control. H An NCL shRNA construct was stably transfected into U5637(shSNHG1#1) cells. The efficiency of NCL protein knockdown and the expression of USP8 were evaluated through Western blotting, utilizing β-Actin as a protein loading control