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. Author manuscript; available in PMC: 2026 Jan 6.
Published in final edited form as: Cell Rep. 2025 Nov 10;44(11):116516. doi: 10.1016/j.celrep.2025.116516

Figure 5. miR-181d binds to the PNPT1 3′ UTR and regulates PNPT1 expression.

Figure 5.

(A and B) CMK3 cells were transfected with Myc-FLAG tagged PNPT1 cDNA construct with or without miR-181d MRE2, MRE3, mutated-MRE2, or mutated-MRE3 (disrupting miR-181d binding) as 3′ UTR. Total RNA and protein were extracted 24 h after transfection. PNPT1 mRNA expression was analyzed by RT-qPCR using primers specific to the endogenous (Endo)-PNPT1 or Myc-FLAG-PNPT1 (A). The expression of Myc-FLAG-PNPT1 was analyzed by western blotting. GAPDH used as loading control (B).

(C) CMK3 cells expressing Myc-FLAG-PNPT1 were transfected with Bi-miR181d or Bi-miR-NT. The lysate was affinity-purified with streptavidin-magnetic beads. Isolated RNA was analyzed for endo-PNPT1 or Myc-FLAG-PNPT1 mRNAs. Statistical significance ***p < 0.001.

(D and E) CMK3 cells expressing Myc-FLAG-PNPT1 cDNA construct containing MRE2, MRE3, mut-MRE2, or mut-MRE3 were transfected with Bi-miR-181d mimic. The lysate was affinity-purified with streptavidin-magnetic beads. Isolated RNA was analyzed for Myc-FLAG-PNPT1 transcripts. (D) MRE2 and mut-MRE2

(E) MRE3 and mut-MRE3. Statistical significance ***p < 0.001. Data are represented as mean ± SD.