Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: Excessive cytoplasmic vacuolization results in cytoskeleton disruption in methuosis. ( A ) H9c2 cells were treated with either vehicle control (0.1% DMSO) or 1 µM maduramicin for durations ranging from 24 h to 48 h for further phalloidin staining (scale bars = 40 μm). ( B ) Similarly, U251 cells were exposed to vehicle control (0.1% DMSO) or 2.5 µM MOMIPP over the same time periods, followed by phalloidin staining assay (scale bars = 20 μm). ( C , D ) Cytoskeleton protein bands of filamin A, filamin B, and α-actinin-1 in H9c2 cells and U251 cells were determined by performing western blotting (WB) after the treatment with either vehicle control or maduramicin and MOMIPP at various time intervals (12 h, 24 h, 48 h, and 72 h), respectively. ( E ) Quantitative data of target proteins from triplicate WB assays, and the data are presented as mean ± standard deviation (SD), n = 3. Significance levels were denoted as * for P < 0.05 and ** for P < 0.01. ( F , G ) Immunofluorescence staining of α-tubulin and β-tubulin, along with phase-contrast microscopy, was conducted on H9c2 cells treated by 1 µM maduramicin for 48 h or vehicle control (0.1% DMSO), as well as on U251 cells treated by 2.5 µM MOMIPP for 24 h or vehicle control (0.1% DMSO) (scale bars = 40 μm–20 μm). ( H ) H9c2 cells and U251 cells were pretreated by maduramicin (1 µM, 48 h) or MOMIPP (2.5 µM) for 24 h, respectively, and followed by drug withdraw for different time intervals (4 h, 12 h, 24 h, and 48 h) and further phalloidin staining (scale bars = 20–40 μm)

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Disruption, Control, Staining, Western Blot, Standard Deviation, Immunofluorescence, Microscopy

Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: Cytoskeleton disruption in methuosis is reversed by blocking cytoplasmic vacuolization. ( A ) H9c2 cells were subjected to vehicle control (0.1% DMSO), bafilomycin A1 (Baf A1, 1 nM) for 1 h, maduramicin (Mad, 1 µM) for 48 h, or a sequential regimen of Baf A1 (1 nM) for 1 h followed by Mad (1 µM) for 48 h or cytochalasin D (Cyto D, 3 µM) for 15 min, after which cells were stained with phalloidin (scale bars = 20 μm). ( B ) U251 cells were exposed to vehicle control (0.1% DMSO), Baf A1 (100 nM) for 1 h, MOMIPP (2.5 µM) for 24 h, or a combination of Baf A1 (100 nM) for 1 h followed by MOMIPP (2.5 µM) for 24 h or cytochalasin D (Cyto D, 3 µM) for 15 min, and also followed by phalloidin staining (scale bars = 20 μm). ( C ) Western blotting was carried out to analyze the expression of filamin A and filamin B in H9c2 cells and U251 cells which were treated by vehicle control (0.1% DMSO), Baf A1 (1 nM or 100 nM) for 1 h, Mad (1 µM) for 48 h or MOMIPP (2.5 µM) for 48 h, and the combination of Baf A1 (1 nM or 100 nM) pretreatment for 1 h and Mad (1 µM) for 48 h or MOMIPP (2.5 µM) for 48 h. ( D ) Quantitative results of filamin A and filamin B expression of H9c2 cells and U251 cells in triplicate, data are shown as mean ± standard deviation (SD), * P < 0.05 and ** P < 0.01. ( E-H ) Immunofluorescence staining of filamin A and filamin B in H9c2 cells and U251 cells were performed with the same treatments above mentioned regimen in western blotting assay (scale bars = 40 μm)

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Disruption, Blocking Assay, Control, Staining, Western Blot, Expressing, Standard Deviation, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: RhoA-ROCK1 inhibition mediates cytoskeletal disruption in methuosis. ( A , B ) Western blotting was conducted to detect the expression of RhoA, ROCK1, and p-MLC in H9c2 and U251 cells at multiple time points (12 h, 24 h, 48 h, and 72 h) after treatment with either vehicle control (0.1% DMSO) and Mad (1 µM) or MOMIPP (2.5 µM), respectively. ( C ) Quantitative results of RhoA, ROCK1, and p-MLC expression of H9c2 cells and U251 cells, data in triplicate are shown as mean ± standard deviation (SD), * P < 0.05 and ** P < 0.01. ( D , E ) For fluorescence examination, H9c2 cells and U251 cells were treated by vehicle control (0.1% DMSO), calpeptin (100 µM) for 1 h, Mad (1 µM) for 48 h or MOMIPP (2.5 µM) for 24 h, and the combination of calpeptin (100 µM) pretreatment for 1 h and Mad (1 µM) for 48 h or MOMIPP (2.5 µM) for 24 h, respectively, followed by phalloidin staining (scale bars = 40 μm–20 μm)

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Inhibition, Disruption, Western Blot, Expressing, Control, Standard Deviation, Fluorescence, Staining

Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: Excessive cytoplasmic vacuolization results in plasma membrane damage in methuosis. Lactate dehydrogenase (LDH) release was detected after Mad (1 µM) or MOMIPP (2.5 µM) exposed to H9c2 cells and U251 cells for 12 h, 24 h, 48 h and 72 h ( A ), intracellular ATP and extracellular ATP levels of H9c2 cells and U251 cells treated by Mad (1 µM) or MOMIPP (2.5 µM) for 24 h, 48 h and 72 h, respectively ( B , C ), compared with negative control (0.1% DMSO), data in triplicate or in quadruplicate are shown as mean ± standard deviation (SD), ** P < 0.01, compared to negative control. ( D , E ) H9c2 cells were treated with vehicle control (0.1% DMSO) or 1 µM maduramicin for 24 h to 72 h, and U251 cells were exposed to vehicle control (0.1% DMSO) or 2.5 µM MOMIPP for 24 h to 72 h, followed by Hoechst 33,342/PI staining. ( F-H ) The expression of p-MLKL, MLKL, GSDMD, GSDMD-N, and CRT was analyzed by performing western blotting in H9c2 cells and U251 cells treated by vehicle control (0.1% DMSO), Mad (1 µM) or MOMIPP (2.5 µM) at 12 h, 24 h, 48 h and 72 h. ( I ) Quantitative data of p-MLKL/MLKL, GSDMD-N/GSDMD and CRT expression of H9c2 cells and U251 cells, data in triplicate are shown as mean ± standard deviation (SD), * P < 0.05 and ** P < 0.01. ( J , K ) Immunofluorescence staining of CRT in H9c2 cells and U251 cells after treatment by Mad (1 µM) for 48 h and MOMIPP (2.5 µM) for 48 h, and DAPI staining for nucleus (scale bars = 20–40 μm)

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Clinical Proteomics, Membrane, Negative Control, Standard Deviation, Control, Staining, Expressing, Western Blot, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: Inhibition of cytoplasmic vacuolization prevents plasma membrane damage . H9c2 and U251 cells were treated with vehicle control (0.1% DMSO), Baf A1 (1 nM for H9c2 and 100 nM for U251), Mad (1 µM), MOMIPP (2.5 µM) or a pretreatment with Baf A1 followed by Mad (1 µM) for 48 h (H9c2) or MOMIPP (2.5 µM) for 24 h (U251). Thereafter, lactate dehydrogenase (LDH) release ( A ), intracellular ATP levels ( B ) and extracellular ATP levels ( C ) were determined accordingly at least in triplicate, data are shown as mean ± standard deviation (SD), ** P < 0.01, compared to negative control. ( D , E ) With the same drug exposure regimen, H9c2 and U251 cells were stained with Hoechst 33,342/PI to assess plasma membrane integrity. ( F , G , H ) Western blotting assay of CRT expression in H9c2 cells and U251 cells in H9c2 cells and U251 cells after treatment by negative control (0.1% DMSO), Baf A1 (1 nM for H9c2 cells, and 100 nM for U251 cells) for 1 h, Mad (1 µM) for 48 h, MOMIPP (2.5 µM) for 48 h, and Baf A1 pre-exposure for 1 h followed by the treatment of Mad (1 µM) for 48 h or MOMIPP (2.5 µM) for 48 h,, quantitative data of CRT expression are shown as bar graph, data from triplicate experiments are shown as mean ± standard deviation (SD), * P < 0.05, ** P < 0.01, compared to negative control or Mad/MOMIPP treatment. ( I , J ) Immunofluorescence staining of CRT in H9c2 cells and U251 cells after the same treatment regimen with above WB experiment, and DAPI staining for nucleus (scale bars = 20–40 μm)

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Inhibition, Clinical Proteomics, Membrane, Control, Standard Deviation, Negative Control, Staining, Western Blot, Expressing, Immunofluorescence

Journal: Cell & Bioscience

Article Title: Cytoskeleton disruption and plasma membrane damage determine methuosis of normal and malignant cells

doi: 10.1186/s13578-025-01441-7

Figure Lengend Snippet: ESCRT-III membrane repair system negatively control methuosis. H9c2 cells ( A ) and U251 cells ( B ) were treated by negative control (0.1% DMSO), Mad (1 µM) or MOMIPP (2.5 µM) for 12 h, 24 h, 48 h and 72 h, respectively, followed by western blotting analysis of ESCRT-III subunits CHMP2B, CHMP3, CHMP4B, and CHMP5. ( C ) Quantitative data of western blotting bands from triplicate assays, data are shown as mean ± standard deviation (SD), * P < 0.05, ** P < 0.01, compared with negative control. ( D ) siRNA targeting the ESCRT-III subunits CHMP3 and CHMP5 was transfected into H9c2 cells, and siRNA targeting CHMP3 was transfected into U251 cells using the transfection reagent jetPRIME, followed by western blotting assay of CHMP3 and CHMP5. ( E-G ) CCK-8 assay was conducted to assess the effect of siRNA-mediated knockdown of CHMP3 and CHMP5 on cell viability upon drugs exposure for 24–48 h ( n = 6), * P < 0.05, ** P < 0.01 compared to negative control or Mad/MOMIPP alone treatment. ( H, I ) LDH release assay was performed in H9c2 cells and U251 cells treated by Mad or MOMIPP after siRNA knockdown of CHMP3 or/and CHMP5 ( n = 3). ** P < 0.01, compared to negative control or Mad/MOMIPP alone treatment. ( J-L ) Hoechst 33,342/PI staining was carried out to determine the effect of siRNA knockdown of CHMP3 or CHMP5 on membrane integrity after Mad (48 h) or MOMIPP (24 h) exposure

Article Snippet: To determine the effects of maduramicin or MOMIPP on the F-actin arrangement, H9c2 cells were treated by 0.1% dimethyl sulfoxide (DMSO, D8418, Sigma-Aldrich, negative control group) or 1 μM maduramicin (Mad, obtained from China Institute of Veterinary Drug Control) for 24 h and 48 h, whereas U251 cells were exposed to 0.1% DMSO (negative control group) or 2.5 μM MOMIPP ( T33467 , TargetMol) for 12 h and 24 h. Similarly, to explore whether drugs-induced alteration of cytoskeleton was reversible, H9c2 cells were exposed to 0.1% DMSO, maduramicin (1 μM, 48 h), and maduramicin (1 μM, 48 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h, U251 cells were treated by 0.1% DMSO, MOMIPP (2.5 μM, 24 h) and MOMIPP (2.5 μM, 24 h) followed by drug withdraw for 4 h, 12 h, 24 h and 48 h. In addition, to understand the effect of bafilomycin A1 on the microfilament change, H9c2 cells were pretreated with 1 nM bafilomycin A1 (Baf A1, S1413, Selleck) for 1 h prior to the treatment with 0.1% DMSO or 1 μM maduramicin for 48 h. U251 cells underwent a pretreatment with 100 nM bafilomycin A1 for 1 h, followed by the treatment with 0.1% DMSO (negative control group) or 2.5 μM MOMIPP for additional 24 h. To further elucidate the functional role of the RhoA-ROCK1 signaling pathway in microfilament disruption, H9c2 cells were pretreated with the RhoA-ROCK1 pathway agonist Calpeptin (Cal, T6432, TargetMol, 100 μM) for 1 h prior to exposure to either 0.1% DMSO (vehicle control) or 1 μM maduramicin for 48 h. In parallel, U251 glioblastoma cells were subjected to an identical Calpeptin pretreatment protocol (100 μM, 1 h), followed by treatment with 0.1% DMSO or 2.5 μM MOMIPP for an additional 24 h. The obtained H9c2 and U251 cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) in phosphate-buffered saline (PBS) for 15 min, and were washed three times with PBS, and permeabilized with 0.2% Triton X-100 (P0096, Beyotime) in PBS for 5 min at room temperature.

Techniques: Membrane, Control, Negative Control, Western Blot, Standard Deviation, Transfection, CCK-8 Assay, Knockdown, Lactate Dehydrogenase Assay, Staining