Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A , B Western blot and quantification of PKM2 expression in neonatal rat cardiomyocytes (NRCMs) with saline or LPS administration for 24 h. β-Actin served as loading control, n = 6. C Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Pkm2 in NRCMs administrated with LPS. 18S was used as an internal reference gene, n = 6. D , E Western blot and quantification of cardiac PKM2 expression in AMCMs administrated with saline or LPS for 24 h. β-Actin served as loading control, n = 6. F qRT-PCR analysis of Pkm2 in AMCMs administrated with LPS. Gapdh was used as an internal reference gene, n = 8. G , H Western blot and quantification of cardiac PKM2 expression in myocardial tissues. β-Actin served as loading control, n = 6. I qRT-PCR analysis of Pkm2 in myocardial tissues. Gapdh was used as an internal reference gene, n = 6 to 7. Values represent the mean ± SEM of at least three independent experiments.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A – C Western blot and quantitation of apoptosis markers (cleaved-Caspase 3/Caspase 3 and Bax/Bcl-2 ratio) expression in NRCMs co-administered with Pkm2 siRNA and LPS. β-Actin served as loading control, n = 6. D , E Representative images and quantification of TUNEL staining from four groups (NC+Saline, n = 28; si- Pkm2 +Saline, n = 26; NC+LPS, n = 25; si- Pkm2 +LPS, n = 50; scale bar, 50 μm). White arrows point to TUNEL + cells. NC negative control. Values represent the mean ± SEM of at least three independent experiments.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Western Blot, Quantitation Assay, Expressing, TUNEL Assay, Staining, Negative Control

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A Representative M-mode echocardiography and heart cross-sections stained with hematoxylin-eosin (HE) (scale bar, 1 mm) of Pkm2 cKO and Pkm2 f/f mice 24 h after saline or LPS administration. B , C Quantitative analysis of ejection fraction (EF) and fractional shortening (FS) by echocardiography, n = 8 to 15. D , E Representative images of CD68 and statistical analysis, n = 7 to 9. Scale bar: 10 μm. F , G Representative images and quantification of TUNEL staining, n = 6. Scale bar: 50 µm. White arrows point to TUNEL positive cardiomyocytes. H , I Western blot and quantitation of apoptosis markers (cleaved-Caspase 3/Caspase 3 and Bax/Bcl-2 ratio) expression in hearts of Pkm2 cKO and Pkm2 f/f mice 24 h after saline or LPS administration. β-Actin served as loading control, n = 6. Values represent the mean ± SEM of at least three independent experiments.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Staining, TUNEL Assay, Western Blot, Quantitation Assay, Expressing

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A Immunoprecipitation assay to validate SERCA2a using anti-PKM2 antibody in NRCMs. B Representative immunofluorescence images showing the co-localization of PKM2 (green) and SERCA2a (red) in AMCMs and NRCMs by confocal immunofluorescence analysis (scale bars, 20 μm). Line profile analyses showing the distribution and intensity of fluorescence signals. The Pearson’s coefficient was measured from the images using ImageJ software ( n = 18). C Immunofluorescence analysis of the colocalization of exogenous-expressing SERCA2a (red) and PKM2 full-length and truncated forms. The right column indicates the line plot profiles of the region of interest ( n = 15). D Co-immunoprecipitation by anti-GFP antibody (PKM2 full-length and truncated forms) with Flag tagged SERCA2a in transiently transfected HEK-293T cells. E , F Western blot and quantitation of SERCA2a and PKM2 expression in NRCMs after Pkm2 knockdown. β-Actin served as loading control, n = 6. G , H Western blot and quantitation of SERCA2a expression in NRCMs co-administered with PKM2 overexpressing plasmid and LPS. β-Actin served as loading control, n = 6. Values represent the mean ± SEM of at least three independent experiments.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Immunoprecipitation, Immunofluorescence, Fluorescence, Software, Expressing, Transfection, Western Blot, Quantitation Assay, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A , B Western blot and quantitation of SERCA2a and PKM2 expression in NRCMs co-administered with Pkm2 siRNA and LPS. β-Actin served as loading control, n = 6. C – E Representative calcium transient curve and quantitation of calcium transient amplitudes and decay time in NRCMs, n = 24. F , G Assessment of the contractility of NRCMs co-administered with Pkm2 siRNA and LPS. Contractility waveform shows contraction amplitude of cardiomyocytes, n = 6. NC negative control. Values represent the mean ± SEM of at least three independent experiments.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Western Blot, Quantitation Assay, Expressing, Negative Control

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A – C Representative calcium transient curve and quantitation of calcium transient amplitudes and decay time in AMCMs from Pkm2 cKO and Pkm2 f/f mice after saline or LPS administration, n = 15. D , E Cardiomyocytes contraction depicting cell shortening in cardiomyocytes isolated from Pkm2 cKO and Pkm2 f/f mice after saline or LPS administration, n = 24. Values represent the mean ± SEM.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Quantitation Assay, Isolation

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A Representative M-mode echocardiography from littermate injected AAV9- null or AAV9- Pkm2 virus after CLP surgery. B – D Quantitative analysis of stroke volume (SV), cardiac output (CO), and heart rate by echocardiography, n = 8. E , F Representative images and quantification of TUNEL staining, n = 7. Scale bar: 50 µm. White arrows point to TUNEL positive cardiomyocytes. G – I Western blot and quantitation of apoptosis markers (cleaved-Caspase 3/Caspase3 and Bax/Bcl-2 ratio) expression in hearts of littermate injected AAV9- null or AAV9- Pkm2 virus after CLP surgery. β-Actin served as the loading control, n = 6.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Injection, TUNEL Assay, Staining, Western Blot, Quantitation Assay, Expressing

Journal: Cell Death Discovery

Article Title: PKM2 deficiency exacerbates gram-negative sepsis-induced cardiomyopathy via disrupting cardiac calcium homeostasis

doi: 10.1038/s41420-022-01287-9

Figure Lengend Snippet: A – C Representative calcium transient curve and quantitation of calcium transient amplitudes and decay time in AMCMs from littermates injected AAV9- null or AAV9- Pkm2 virus after CLP surgery, n = 17. D , E Cardiomyocytes contraction depicting cell shortening in cardiomyocytes isolated from littermate injected AAV9- null or AAV9- Pkm2 virus after CLP surgery, n = 22–25. Values represent the mean ± SEM.

Article Snippet: Plasmid homo-GFP-PKM2 (Sino Biological, HG11430-ACG) was used as template to generate exon 10 and Pkm2 -Δexon 10 by ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and Phanta Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), based on homologous recombination method.

Techniques: Quantitation Assay, Injection, Isolation