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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 2. PNPT1 is required for TMZ-induced degradation of miR-181d.

Figure 2.

(A) CMK3 cells were treated with 500 μM TMZ for up to 24 h. RNA was isolated, and the precursor (pre) or mature (mat) miR-181d expression was analyzed using RT-qPCR.

(B) CMK3 cells were pretreated with actinomycin D1 (100, 250, and 500 nM) and exposed to TMZ (500 μM) for 24 h. Mature (mat) miR-181d expression was analyzed by RT-qPCR.

(C) CMK3 cells transfected with siExoSC4, siPNPT1, or siXRN1 and miR-181d expression were analyzed by RT-qPCR. Statistical significance, *p < 0.05.

(D and E) Exogenous expression of PNPT1 restored TMZ-induced miR-181d degradation. CMK3 WT or PNPT1-KO cells were transfected with pCMV6-AC-GFP-PNPT1 and exposed to TMZ or DMSO. miR-181d (D) or MGMT (E) expression was analyzed by RT-qPCR.

(F) CMK3 cells transfected with biotinylated (Bi)-miR-181d or miR-NT control mimic. Bi-miRNA was affinity pull-down (AP) with streptavidin-magnetic beads. The associated protein complex was eluted and analyzed by western blotting. Total cell lysate was used as input.

(G and H) PNPT1 protein was immunoprecipitated and incubated with miR-181d or miR-21 mimic (1 × 105 copies). RNA was extracted and quantified by RT-qPCR for miR-181d (G) and miR-21 (H).

(I) Affinity-purified PNPT1 was isolated from CMK3 cells with and without TMZ treatment and co-incubated with miR-181d. Statistical significance: ***p < 0.001 between the same treatment groups.

(J and K) CMK3 cells were treated with 500 μM TMZ or DMSO and fractionated into cytoplasmic and mitochondrial fractions. These fractions were analyzed for PNPT1 localization by western blotting. Alpha (α)-tubulin was used as the cytoplasmic, and succinate dehydrogenase (SDH) was used as the mitochondrial control (J, left). Densitometry quantitation of PNPT1 in cytoplasmic and mitochondrial fractions (J, right). (K) Total miRNA was isolated from these fractions and analyzed for miR-181d by RT-qPCR. Statistical significance: ***p < 0.001 between the same treatment groups. Data are represented as mean ± SD.