Journal: Febs Letters

Article Title: Hyperosmotic stress induces PARP1 ‐mediated HPF1 ‐dependent mono( ADP ‐ribosyl)ation

doi: 10.1002/1873-3468.70334

Figure Lengend Snippet: Time dependency of ADP‐ribosylation upon hyperosmotic stress. (A) U2OS wild‐type cells were subjected to 0.4 m D‐sorbitol treatment for 10, 30, or 60 min followed by recovery under normosmotic conditions. In total cell lysate, mono(ADP‐ribosyl)ation was visualized by HRP‐coupled AbD43647 . (B) U2OS wild‐type cells were pretreated with PARG inhibitor for 3 h, and then, the cells underwent 0.4 m D‐sorbitol treatment for 10, 30, or 60 min followed by recovery in normosmotic medium. Thereafter, poly(ADP‐ribosyl)ation was detected by the poly/mono‐ADP‐ribose antibody (D9P7Z) from Cell Signalling Technology. U2OS wild‐type cells treated with H 2 O 2 for 10 min in the presence or absence of PARGi served as a positive control of poly(ADP‐ribosyl)ation. (A, B) Ponceau S staining served as a loading control. Representative western blots of at least three independent experiments. [Correction added on 06 April 2026 after first online publication: The labels “10 min” and “30 min” in Figure 1A were inadvertently transposed and have been corrected in this version.]

Article Snippet: Untreated U2OS wide‐type cells or cells exposed to 0.4 m D‐sorbitol for 30 min were collected from two confluent 10 cm dishes by washing twice in 5–5 mL ice‐cold PBS supplemented with 1 μ m Olaparib and 1 μ m PARG inhibitor (HY‐108360; MedChem Express) then scraping the cells in 250 μL of the same inhibitor‐supplemented PBS.

Techniques: Positive Control, Staining, Control, Western Blot

Journal: Febs Letters

Article Title: Hyperosmotic stress induces PARP1 ‐mediated HPF1 ‐dependent mono( ADP ‐ribosyl)ation

doi: 10.1002/1873-3468.70334

Figure Lengend Snippet: MARylation of PARP1 during hyperosmotic stress. (A) U2OS wild‐type, PARP1 KO, and PARP2 KO cells were exposed to 30‐min‐long D‐sorbitol treatment followed by the collection of total cell lysates and detection of MAR by HRP‐coupled AbD43647 . PARP1 and PARP2 western blot verified the knockout status of the used cell lines. (B) U2OS wild‐type cells were pretreated with 5 μ m Olaparib, 5 n m Saruparib, or DMSO, followed by exposure to 0.4 m D‐sorbitol for 30 min together with inhibitors. MARylation was detected by HRP‐coupled AbD43647 . (C) U2OS wild‐type and GFP‐PARP1 expressing PARP1 KO cells were treated with D‐sorbitol for 30 min. Cell lysates were probed with HRP‐coupled AbD43647 and the poly/mono‐ADP‐ribose antibody (CSL, D9P7Z). (D) Streptavidin pulldown of biotinylated AbD43647 immunoprecipitates from untreated or D‐sorbitol‐treated wild‐type U2OS cells was performed. Input lysates and SDS eluates (IP) from streptavidin magnetic beads were analyzed by immunoblotting with anti‐PARP1 and anti‐PARP1 S499‐MAR ( AbD35251 )–HRP antibodies. (A, B, D) Ponceau S staining served as a loading control. Representative western blots of at least three independent experiments.

Article Snippet: Untreated U2OS wide‐type cells or cells exposed to 0.4 m D‐sorbitol for 30 min were collected from two confluent 10 cm dishes by washing twice in 5–5 mL ice‐cold PBS supplemented with 1 μ m Olaparib and 1 μ m PARG inhibitor (HY‐108360; MedChem Express) then scraping the cells in 250 μL of the same inhibitor‐supplemented PBS.

Techniques: Western Blot, Knock-Out, Expressing, Magnetic Beads, Staining, Control

Journal: Febs Letters

Article Title: Hyperosmotic stress induces PARP1 ‐mediated HPF1 ‐dependent mono( ADP ‐ribosyl)ation

doi: 10.1002/1873-3468.70334

Figure Lengend Snippet: Hyperosmotic stress–induced HPF1‐dependent MARylation and HPF1‐independent PARylation. (A) U2OS wild‐type, HPF1 KO and ARH3 KO cells were subjected to 30 min 0.4 m D‐sorbitol treatment. Total cell lysate of sorbitol‐treated wild‐type U2OS was incubated in the presence of 1 m hydroxylamine (HA) for 2 h at RT, or it was heated at 95 °C for 5 min. As controls of HA treatment, U2OS wild‐type and HPF1 KO cells were exposed to 2 m m H 2 O 2 ‐induced oxidative stress followed by incubation with 1 m HA for 2 h at RT. MARylation was detected in western blot using HRP‐coupled Abd43647. (B) U2OS wild‐type or HPF1 KO cells were pretreated with 10 μ m PARG inhibitor for 3 h or left untreated, followed by 30 min 0.4 m D‐sorbitol or 10 min 2 m m H 2 O 2 treatment in the presence or absence of PARG inhibitor. MARylation was visualized by HRP‐coupled Abd43647 (C) U2OS wild‐type or HPF1 KO cells were pretreated with 10 μ m PARG inhibitor for 3 h or left untreated, followed by 30 min 0.4 m D‐sorbitol or 10 min 2 m m H 2 O 2 treatment in the presence or absence of PARG inhibitor. Thereafter, poly(ADP‐ribosyl)ation was detected by the poly/mono‐ADP‐ribose antibody (D9P7Z) from Cell Signalling Technology. (A–C) Ponceau S staining was used as a loading control. Representative western blot of at least three independent experiments.

Article Snippet: Untreated U2OS wide‐type cells or cells exposed to 0.4 m D‐sorbitol for 30 min were collected from two confluent 10 cm dishes by washing twice in 5–5 mL ice‐cold PBS supplemented with 1 μ m Olaparib and 1 μ m PARG inhibitor (HY‐108360; MedChem Express) then scraping the cells in 250 μL of the same inhibitor‐supplemented PBS.

Techniques: Incubation, Western Blot, Staining, Control

Journal: Febs Letters

Article Title: Hyperosmotic stress induces PARP1 ‐mediated HPF1 ‐dependent mono( ADP ‐ribosyl)ation

doi: 10.1002/1873-3468.70334

Figure Lengend Snippet: Effect of hyperosmotic stress on cell survival and CHK1 activation. (A) Immunofluorescent staining of phosphorylated histone H2AX (yH2AX) in U2OS wild‐type cells exposed to 10, 30, or 60 min hyperosmotic stress in 0.4 m D‐sorbitol followed by recovery in normosmotic medium. Cells were treated with 10 μ m camptothecin (CPT) for 1 h for positive control. Representative images of at least three independent experiments. Scale bar: 35 μm. (B) Representative western blot of at least three independent experiments to detect pCHK1(S317), CHK1, and PARP1 in U2OS wild‐type cells treated with 0.4 m D‐sorbitol for 10, 30, or 60 min followed by recovery in normosmotic media. GAPDH was used as a loading control. (C) Time course of cell survival in U2OS wild‐type, PARP1 KO and PARP2 KO cells during 0.4 m D‐sorbitol treatment. Wild‐type and knockout U2OS cells were treated with 0.4 m D‐sorbitol for the indicated time and subsequently cultured for 7 days before measuring the fluorescence of resorufin. Error bars are SD ( n = 3).

Article Snippet: Untreated U2OS wide‐type cells or cells exposed to 0.4 m D‐sorbitol for 30 min were collected from two confluent 10 cm dishes by washing twice in 5–5 mL ice‐cold PBS supplemented with 1 μ m Olaparib and 1 μ m PARG inhibitor (HY‐108360; MedChem Express) then scraping the cells in 250 μL of the same inhibitor‐supplemented PBS.

Techniques: Activation Assay, Staining, Positive Control, Western Blot, Control, Knock-Out, Cell Culture, Fluorescence