Journal: Cell Death & Disease

Article Title: Targeting mitochondrial phosphatase PGAM5 alleviates ferroptosis and acute pancreatitis by upregulating NRF2-mediated FSP1 expression

doi: 10.1038/s41419-026-08484-9

Figure Lengend Snippet: a PGAM5 inhibitor, LFHP-1c treatment suppresses erastin-induced ferroptosis and lipid peroxidation. AC16 cells were treated with erastin (20 μM) with or without LFHP-1c (2 μM) for 16 h for cell death measurement or 12 h for lipid ROS measurement. Cells were stained with Propidium Iodide (PI) and cell death was analyzed by flow cytometry. Cells treated as indicated were stained with BODIPY 581/591 C11, and lipid ROS levels were measured by flow cytometry. b Knockdown of PGAM5 blocked erastin-induced ferroptosis and lipid peroxidation. Western blot images confirm the expression of the indicated proteins. Cells, as indicated, were treated with erastin (20 μM) for 16 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in ( a ). c DRP1 was dramatically phosphorylated at serine 616 and de-phosphorylated at serine 637 in a time-dependent manner upon erastin treatment. AC16 cells were treated with erastin (20 μM) as indicated. Western blot images confirm the expression of the indicated proteins. d PGAM5 inhibitor LFPH-1c treatment suppresses erastin induced de-phosphorylation of DRP1 at serine 637. AC16 cells were treated with erastin (20 μM) with or without LFPH-1c (2 μM) for the indicated time. Western blot images confirm the expression of the indicated proteins. e Knockdown of PGAM5 blocks erastin-induced de-phosphorylation of DRP1 at serine 637. AC16 cells, as indicated, were treated with erastin (20 μM) for the indicated time. Western blot images confirm the expression of the indicated proteins. f Knockdown of PGAM5 blocks ferroptosis-associated mitochondrial fragmentation. SU9-GFP labeled AC16 cells were treated with erastin (20 μM) as indicated. SU9-GFP labeled mitochondria were imaged by using a confocal laser scanning microscope (scale bars in images of all figures were 10 μM). Quantification of mitochondria with different morphologies (fragmented, short, long). “Short” represents cells with a majority of mitochondria shorter than 10 µm, “Long” represents cells with a majority of mitochondria longer than 10 µm, “Fragmented” represents cells with a majority of granular mitochondria. At least 100 cells were counted for each sample. g Overexpression of PGAM5 suppresses erastin-induced ferroptosis and lipid peroxidation. Western blot images confirm the expression of the indicated proteins. AC16 cells, as indicated, were treated with erastin (20 μM) for 16 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in ( a ). h The level of PGAM5 influences erastin-induced ferroptosis. Different dose PGAM5-flag plasmids were transfected into AC16 cells, and AC16 cells were cultured for 48 h, and then treated with erastin (20 μM) for 16 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in ( a ). Western blot images confirmed the expression of the indicated proteins. Data are derived from three independent experiments, and each value represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, t test ( n = 3).

Article Snippet: Chemical reagents used were erastin (Selleck, Cat#S7242), ML385 (APE×BIO, Cat#B8300), LFHP-1c (MCE, Cat# HY139598 ), L -arginine (Sigma, Cat#74-79-3), and iFSP1 (MCE, Cat#HY-136057).

Techniques: Staining, Flow Cytometry, Knockdown, Western Blot, Expressing, De-Phosphorylation Assay, Labeling, Laser-Scanning Microscopy, Over Expression, Transfection, Cell Culture, Derivative Assay

Journal: Cell Death & Disease

Article Title: Targeting mitochondrial phosphatase PGAM5 alleviates ferroptosis and acute pancreatitis by upregulating NRF2-mediated FSP1 expression

doi: 10.1038/s41419-026-08484-9

Figure Lengend Snippet: a Schematic illustration of the experimental design of the mouse arginine-induced acute pancreatitis model. b LFHP-1c treatment protects against arginine-induced higher levels of serum α-AMS. n = 6. c LFHP-1c treatment protects against arginine-induced higher levels of serum LDH and ALT. n = 6. d LFHP-1c treatment blocks arginine-induced upregulation of pro-inflammatory cytokines (miL-1β, miL-6, and TNF-α) in pancreatic tissue. RT-qPCR analyzes the expression of indicated pro-inflammatory cytokines. n = 3. e Representative H&E sections obtained from the mouse pancreas in response to different treatments after 72 h. f LFHP-1c treatment suppresses arginine-induced upregulation of 4-HNE and activates AMPK and promotes the expression of NRF2 and FSP1 in pancreatic tissue. g LFHP-1c treatment suppresses the arginine-induced higher level of pancreatic tissue MDA. n = 6. h LFHP-1c treatment promotes the mRNA level of FSP1 in pancreatic tissue. RT-qPCR analyzes the expression of FSP1. i LFHP-1c treatment suppresses the elevated mRNA level of PTGS2 induced by arginine in pancreatic tissue. RT-qPCR analyzes the expression of PTGS2. n = 3. Data are derived from independent experiments, and each value represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, t test.

Article Snippet: Chemical reagents used were erastin (Selleck, Cat#S7242), ML385 (APE×BIO, Cat#B8300), LFHP-1c (MCE, Cat# HY139598 ), L -arginine (Sigma, Cat#74-79-3), and iFSP1 (MCE, Cat#HY-136057).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5

doi: 10.7150/ijbs.95707

Figure Lengend Snippet: OTUD1 directly binds to PGAM5. (A) Schematic illustration of mass spectrometry analysis to identify proteins binding to OTUD1. (B) PGAM5 peptides derived from the mass spectrometric analysis of OTUD1. (C-D) Co-IP assays of the interaction between OTUD1 and PGAM5 in 293T cells transfected with the indicated plasmids. (E-F) Co-IP assays of Otud1 and Pgam5 in NRCMs. (G-H) Co-IP assays of Otud1 and Pgam5 in heart tissues. (I) Schematic illustration of the OTUD1 domain deletion mutants. (J) Co-IP analysis of the binding region of OTUD1 and PGAM5.

Article Snippet: To test the role of PGAM5 in pathological cardiac hypertrophy, we used PGAM5 inhibitor LFHP-1c (HY-139598, MCE) in TAC mice.

Techniques: Mass Spectrometry, Binding Assay, Derivative Assay, Co-Immunoprecipitation Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: METTL3-mediated m6A modification of OTUD1 aggravates press overload induced myocardial hypertrophy by deubiquitinating PGAM5

doi: 10.7150/ijbs.95707

Figure Lengend Snippet: OTUD1 deubiquitinates PGAM5. (A) Representative western blot of Pgam5 protein in heart of Flox and Otud1-CKO mice subjected to Sham or TAC (n=3). (B) Representative western blotting result of Pgam5 in NRCMs infected with Ad-GFP and Ad-Otud1 and treated with Ang II (10 μM) for 48h and cycloheximide (CHX, 50 μM) for the indicated time points (n=3). (C) Results of ubiquitination assays confirming the ubiquitination of PGAM5 after overexpression of OTUD1 for 48 h and treated with MG132 (50 μM) for 6 h in 293T. (D) Results of ubiquitination assays confirming the K48 ubiquitination of PGAM5 after co-transfected with Flag-OTUD1, HA-Ub-WT, HA-Ub-K48, HA-Ub-K63 for 24 h and treatment with MG132 (50 μM) for 6 h in 293T. (E) Results of ubiquitination assays confirming the expression and ubiquitination of Pgam5 in NRCMs infected with Ad-GFP and Ad-Otud1 and followed by Ang Ⅱ treatment. (F) Results of ubiquitination assays confirming the expression and ubiquitination of Pgam5 in heart tissue from Flox and CKO mice subjected to sham or TAC. (G) Results of ubiquitination assays confirming the ubiquitination of Pham5 after overexpression of OTUD1, OTUD1(C320A) and Pgam5 for 24 h and treated with MG132 (50 μM) for 6 h in 293T cells. (H) Immunoblotting analysis of Pgam5, Ad-Otud1 and Ad-Otud1 (C294S) in NRCMs.

Article Snippet: To test the role of PGAM5 in pathological cardiac hypertrophy, we used PGAM5 inhibitor LFHP-1c (HY-139598, MCE) in TAC mice.

Techniques: Western Blot, Infection, Over Expression, Transfection, Expressing