mrt68921  (Selleck Chemicals)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Ferrodoxin 1 (FDX1) drives paclitaxel resistance in ovarian cancer via copper metabolism and ULK1/ATG13-mediated autophagy: overcome by pH/ROS-responsive PPD/PDP@si-FDX1 nanomicelles

doi: 10.1186/s13046-025-03589-z

Figure Lengend Snippet: Mechanistic investigation of FDX1’s influence on autophagy and TAX resistance in A2780 cells. Note: A Western Blot analysis of P-ATG13, T-ATG13, and GST-ULK1 expression as CuCl 2 concentration increases from 0 µM to 10 µM; B Western Blot analysis of P-ATG13, T-ATG13, and GST-ULK1 expression with TTM concentration ranging from 0 to 50 µM or after treatment with 10 µM MRT68921; C IP analysis of P-ATG13, T-ATG13, and IP-ULK1 (ULK1 protein pulled down during IP); D Schematic diagram of the experimental procedures; E CCK-8 assay to assess cell viability across groups; F Western Blot analysis of ULK1, ATG13, LC3B-I, LC3B-II, and P62 protein expression in each group; G JC-1 assay to evaluate MMP levels in different groups; H Immunofluorescence staining to detect LC3-positive cells in each group; I TEM images showing mitochondrial morphology in A2780 cells, Scale bar = 1 μm (left), 500 nm (right), with red arrows indicating autophagosomes; J Colony formation assay to evaluate the proliferative capacity of each group; K Scratch assay to assess cell migration capability, Scale bar = 100 μm; L Transwell assay to evaluate cell invasion capability, Scale bar = 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. Experiments were repeated three times

Article Snippet: DMSO treatment was uniform across groups with cells treated with 10 nM BafA1 (Med Chem Express, Catalog no. HY-100558) for 24 h, 5 mM 3-MA (HY-19312, Med Chem Express, China) for 24 h, 10 μM Unc-51 Like Autophagy Activating Kinase 1 (ULK1) inhibitor MRT68921 (HY-100006, Med Chem Express, China) for 24 h, 5 μM ULK1 activator BL-918 (HY-124729, Med Chem Express, China) for 24 h, and 30 μM cuproptosis inhibitor TTM (HY-128530, Med Chem Express, China) for 24 h. (3) si-FDX1 group: Cells were not treated with BafA1 or BL-918. si-FDX1 + BafA1 group: Cells were treated with 10 nM BafA1 for 24 h prior to sample collection. si-FDX1 + BL-918 group: Cells were treated with 10 μM BL-918 for 24 h without BafA1. si-FDX1 + BL-918 + BafA1 group: Cells were first treated with 10 μM BL-918 for 24 h, followed by an additional 24-hour treatment with 10 nM BafA1.

Techniques: Western Blot, Expressing, Concentration Assay, CCK-8 Assay, Immunofluorescence, Staining, Colony Assay, Wound Healing Assay, Migration, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Ferrodoxin 1 (FDX1) drives paclitaxel resistance in ovarian cancer via copper metabolism and ULK1/ATG13-mediated autophagy: overcome by pH/ROS-responsive PPD/PDP@si-FDX1 nanomicelles

doi: 10.1186/s13046-025-03589-z

Figure Lengend Snippet: Effects of PPD/PDP@si-FDX1 on autophagy and TAX resistance in OC cells. Note: A Schematic of the cell experimental procedures; B Western Blot analysis of FDX1, ULK1, ATG13, LC3B-I, LC3B-II, and P62 protein expression in each group; C JC-1 assay to measure MMP levels across groups; D Immunofluorescence staining for LC3-positive expression in each group, Scale bar = 15 μm; E TEM images showing mitochondrial morphology in different groups, Scale bar = 1 μm (left) and 500 nm (right), with red arrows indicating autophagosomes; F CCK-8 assay to assess cell viability across groups. * p < 0.05, ** p < 0.01, *** p < 0.001. Experiments were repeated three times

Article Snippet: DMSO treatment was uniform across groups with cells treated with 10 nM BafA1 (Med Chem Express, Catalog no. HY-100558) for 24 h, 5 mM 3-MA (HY-19312, Med Chem Express, China) for 24 h, 10 μM Unc-51 Like Autophagy Activating Kinase 1 (ULK1) inhibitor MRT68921 (HY-100006, Med Chem Express, China) for 24 h, 5 μM ULK1 activator BL-918 (HY-124729, Med Chem Express, China) for 24 h, and 30 μM cuproptosis inhibitor TTM (HY-128530, Med Chem Express, China) for 24 h. (3) si-FDX1 group: Cells were not treated with BafA1 or BL-918. si-FDX1 + BafA1 group: Cells were treated with 10 nM BafA1 for 24 h prior to sample collection. si-FDX1 + BL-918 group: Cells were treated with 10 μM BL-918 for 24 h without BafA1. si-FDX1 + BL-918 + BafA1 group: Cells were first treated with 10 μM BL-918 for 24 h, followed by an additional 24-hour treatment with 10 nM BafA1.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, CCK-8 Assay

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (A) Schematic illustrating the time course of lysosomal membrane damage-induced changes in ESCRTs, lysophagy, PI(4)P, PI(3)P, canonical mTORC1 signalling, and protein translation. Curves were modelled according to experimental data shown in [PI(3)P], (mTORC1), and (Lysophagy), [PI(4)P], (translation), (ESCRT). PI(3)P, mTORC1, and translations curves were normalized between 0 and 1 to allow for comparison of the kinetics. The ESCRT curve represents an average of CHMP4B, IST1, and Alix data . (b) Volcano plot illustrating the enrichment of proteasome subunits and ubiquitylation machinery on lysosomes immunoprecipitated from control or 1 h 1mM LLOMe-treated HEK293-TMEM192-3xHA cells determined by quantitative proteomics. (c) Representative confocal images of HMC3 and U2OS cells co-stained with DAPI and PSMB5 at indicated time points after 1mM LLOMe treatment. Scale bar, 10 µm. (d) Representative confocal images of 3 h 1mM LLOMe-treated HMC3 cells co-stained for the proteasome subunit PSBM5, Lamp1, and DAPI. Scale bar, 2 µm. (e) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 1 h 1mM LLOMe, or 5 h 1mM LLOMe. Right: densitometric quantification of mScarlet-MTMR14 relative to GAPDH in control, 1 h and 5 h LLOMe treatment from immunoblots as shown in the left panel. one-way ANOVA (n = 3 independent experiments). (f) Immunoblot of global ubiquitinated protein probed by FK2 antibodies in immunoprecipitation fractions collected from control or 2 h 1mM LLOMe-treated U2OS-mScarlet-MTMR14 KI cells with RFP-trap beads to enrich for mScarlet-MTMR14. (g) Representative confocal images of 2 h LLOMe-treated U2OS cells co-expressing mCherry-MTMR14 and eGFP-Ubiqutin. Scale bar, 10 µm. (h) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with control (DMSO) or 5 h LLOMe with or without 3 µM TAK-243 co-incubation. Right: densitometric quantification of mScarlet-MTMR14 intensity/ ß-actin from immunoblots as shown in the left panel. t test (n = 4 independent experiments). Dotted line denotes mScarlet-MTMR14 level in controls set to 1. (i) Left: representative confocal live cell images of U2OS cells expressing GFP-2xFYVE imaged after treatment with 5 h LLOMe with or without 3 µM TAK-243. Scale bar, 10 µm. Right: number of PI(3)P puncta/cell area quantified in cells as shown in the left panel, fold change over control. t test (n = 3 independent experiments, total number of cells is 108 in LLOMe and 115 in LLOMe + TAK-243 conditions. (j) Quantification of mean LysoTracker intensity/field of view in U2OS cells treated with LLOMe for 5 h with or without 3 µM TAK-243 co-incubation, fold change over control. t test (n = 3 independent experiments, each data point represents 30 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (k) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 5 h LLOMe, or 5 h LLOMe with 200 nM Baf A1 and 2 µM Bortezomib. Right: densitometric quantification of mScarlet-MTMR14 relative to GAPDH from immunoblots as shown on the left panel. one-way ANOVA (n = 3 independent experiments). (l) Schematic illustration of MTMR14 recruitment to damaged lysosomes via calcium and sphingomyelin leading to local hydrolysis of PI(3)P during early lysosome damage. Upon prolonged lysosome damage, MTMR14 becomes ubiquitinated, and PI(3)P levels are restored. Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05. See also .

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Membrane, Comparison, Immunoprecipitation, Control, Quantitative Proteomics, Staining, Western Blot, Expressing, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (a) Volcano plot illustrating the enrichment of proteasome subunits and ubiquitylation machinery on lysosomes immunoprecipitated from control or 30min 1mM LLOMe-treated HEK293-TMEM192-3xHA cells determined by quantitative proteomics. (b) Immunoblot of U2OS cells treated with DMSO control, or 1mM LLOMe for 30 minutes, 1 h, 2 h, or 5 h. (c) Representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 5 h 1mM LLOMe, and 5 h LLOMe with 200 nM Baf A1. (d) Representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 5 h 1mM LLOMe, and 5 h LLOMe with 2 µM Bortezomib. Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Immunoprecipitation, Control, Quantitative Proteomics, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (a) Time course of LLOMe-induced lysophagy. Quantification of acidic TMEM192-mKeima puncta/cell area in HeLa cells after LLOMe treatment, each datapoint represents 24-50 cells. Data are mean ± s.e.m. Representative confocal images are shown in . (b) Schematic illustrating the time course of lysosomal membrane damage-induced changes in lysophagy, PI(3)P, and canonical mTORC1 signalling. Curves were modelled according to experimental data shown in [PI(3)P], (mTORC1), and (Lysophagy). PI(3)P and mTORC1 curves were normalized between 0 and 1 to allow for comparison of the kinetics. (c) Defective lysotracker recovery of U2OS cells treated with ULK1 inhibitor (2µM MRT68921). Left: cells were imaged before and 30min and 5h after LLOMe treatment. Scale bar, 20µm. Right: quantification of representative data shown in left. Mean LysoTracker intensity/field of view, fold change over control. Data are from n = 3 independent experiments (t test), each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm. (d) Quantification of cytotoxicity by LDH assay of 5 h 4mM LLOMe-treated U2OS cells in absence or presence of ULK1 inhibitor 2µM MRT68921. t test (n = 4 independent experiments, each datapoint represents 3 replicate measurements). (e) Representative confocal live cell images of LLOMe-treated WT or MTMR14 KO U2OS cells expressing TMEM192-mKeima. Cell outlines are shown in yellow. Quantification is shown in . Scale bar, 10 µm. (f) Quantification of acidic TMEM192-mKeima puncta per cell area in WT or MTMR14 KO U2OS cells treated with LLOMe in the absence or presence of the mTOR inhibitor AZD8055 (200 nM). one-way ANOVA (n = 5 independent experiments, total number of cells is 106 for WT, 106 for, MTMR14 KO and 83 for MTMR14 KO + AZD8055). (g) Schematic illustration showing that VPS34-IN1 inhibits VPS34 leading to PI(3)P depletion. (h) Left: effects of ULK1 and VPS34 inhibition on LLOMe-induced lysophagy in U2OS cells analyzed by confocal live cell imaging of TMEM192-mKeima. Cell outlines are shown in yellow. Scale bar, 10 µm. Right: quantification of acidic mKeima puncta /cell area. One-way ANOVA (n = 3 independent experiments, total number of cells is 76 for LLOMe, 77 for LLOMe+MRT68921, and 78 for LLOMe +VPS34-IN1). (i) Lysotracker recovery measured as fold change over control of 5 h 1mM LLOMe-treated U2OS cells in the absence or presence of 5µM VPS34-IN1. t test (n = 3 independent experiments, each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (j) Quantification of cytotoxicity of 5 h 4mM LLOMe-treated U2OS cells in absence or presence of 5µM VPS34-IN1 inhibitor. t test (n = 3 independent experiments, each datapoint represents triplicate measurements). Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05. See also .

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Membrane, Comparison, Control, Lactate Dehydrogenase Assay, Expressing, Inhibition, Live Cell Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation

doi: 10.64898/2026.04.04.716461

Figure Lengend Snippet: (a) Representative confocal live cell images of TMEM192-mKeima expressing HeLa cells treated with LLOMe for the indicated time points. Quantification is shown in . (b) Quantification of acidic TMEM192-mKeima puncta/cell area in U2OS cells after different durations of LLOMe treatment. n = 28-37 cells per datapoint. Data are mean ± s.e.m. (c) Quantification of acidic TMEM192-mKeima puncta/cell area after 5 h LLOMe treatment in U2OS WT and MTMR14 KO cells. t test (n = 4 independent experiments, total number of cells is 116 for WT and 113 for MTMR14 KO). Representative confocal images in . (d) Quantification of mean LysoTracker intensity/field of view fold change over control in BV2 cells after 5 h LLOMe plus control (DMSO), 5 µM VPS34-IN1, or 2µM MRT68921 treatment. one-way ANOVA (total number of fields of view is 30 each group, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (e) Quantification of LDH cytotoxicity assay in BV2 cells treated for 5 h with 4mM LLOMe plus DMSO control or 5 µM VPS34-IN1. t test (n = 3 independent experiments, each datapoint represents triplicate measurements). (f) Quantification of mean LysoTracker intensity/field of view fold change over control in HeLa cells after 5 h LLOMe plus control (DMSO), 5 µM VPS34-IN1, or 200 nM Apilimod treatment. one-way ANOVA (total number of fields of view is 30 for LLOMe, 29 for LLOMe + VPS34-IN1, and 28 for LLOMe + Apilimod, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).

Techniques: Expressing, Control, LDH Cytotoxicity Assay, Two Tailed Test