Fig 2: PARP16 knockdown suppresses ER stress in APP/PS1 mice and Aß-treated neuron cells. (A) Knockdown of PARP16 attenuated ER stress by mediating the PERK and IRE1a signaling pathways in APP/PS1 mice. APP/PS1 and WT mice were injected intracerebroventricularly (i.c.v.) with an AAV-expressing shRNA targeting PARP16 (shRNA-Parp16) or AAV-scramble control shRNA (scRNA, used as a control). p-PERK and p-IRE1a signaling was determined by western blots in the hippocampi and cortices. Data represent the mean ± S.D. ***p < 0.001, n = 6. (B) PARP16 knockdown attenuated ER stress in Aß1-42-treated HT22 cells. HT22 cells transfected with negative control (siNC) mimics or siParp16 were treated with or without Aß1-42, and p-PERK and p-IRE1a signaling were measured by western blotting. Data represent the mean ± S.D, **p < 0.01. At least three experiments were repeated.

Fig 3: PARP16 binds and stabilizes APP mRNA. (A) PARP16 expression was increased in HEK-APP cells. Data are presented as the mean ± SD, ***p < 0.001. At least three experiments were repeated. (B) Knockdown of PARP16 attenuated APP expression. HEK and HEK-APP cells were transfected with negative control (siNC) mimics or siRNA PARP16, and PARP16 and APP were measured by western blotting. Data represent the mean ± S.D, ***p < 0.001. At least three experiments were repeated. (C) PARP16 knocked down-HEK-APP cells were treated with MG132, 3-MA, or NH4Cl, and APP levels were measured by western blotting. (D) Knockdown of PARP16 attenuated APP mRNA levels. HEK-APP cells were transfected with negative control (siNC) mimics or siPARP16, and APP mRNA was measured by qPCR. Data are presented as mean ± S.D, *p < 0.05, n = 4. (E) The nuclear run-on assay was used to determine PARP16 knockdown on APP transcription efficiency. Data are presented as the mean ± S.D, n = 3. (F) Control HEK-APP cells, and PARP16 knocked down-HEK-APP cells were treated with the transcription inhibitor Actinomycin D (5 µg/ml). APP mRNA levels were quantified using RT-qPCR. Data are presented as the mean ± S.D, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3. (G) Control HEK-APP cells, PARP16 knockdown cells, and knockdown HEK-APP cells with PARP16 re-expressed were treated with the transcription inhibitor Actinomycin D (5 µg/ml). APP mRNA levels were quantified using RT-qPCR. Data are presented as the mean ± S.D, *p < 0.05, **p < 0.01, n = 3. (H) The 5-Ethynyluridine (EU) labeling assay was used to determine APP mRNA stability under PARP16 knockdown. Cells were first treated with EU (0.1 mM) for 24 h, and the incorporation of EU was determined. Then, EU was removed, and cells were cultured in fresh media for another 0, 2, 4, 6, or 8 h. The stability of APP mRNA was determined as the percentage of labeled mRNA after the removal of EU divided by the percentage of labeled mRNA before removal. Data are presented as the mean ± S.D, *p < 0.05, ***p < 0.001, n = 3. (I) RIP assay in HEK-APP cells demonstrating significant enrichment of APP mRNA using PARP16 antibodies compared to the negative control IgG. Data are presented as the percentage of input (± S.D, n = 3), ***p < 0.001. (J) The RNA pull-down assay in Aß1-42-treated HT22 cells lysate using either the APP or mutated-APP RNA. GFP RNA was used as a negative control.