Journal: bioRxiv

Article Title: Germline Selection by Meiosis Defends the Transmission of defective Mitochondria with mtDNA variants

doi: 10.1101/2020.11.10.377390

Figure Lengend Snippet: A. Mitochondrial membrane potential was detected with TMRE and DilC 1 (5), Rhod2 and Fluo-3 in human PB1 and oocytes. B. The relative fluorescence intensity of human PB1 to its sister oocyte was quantified and the difference significance was evaluated using paired t test. C. Mitochondrial membrane potential was detected with TMRE and DilC 1 (5), Rhod2 and Fluo-3 in mouse PB1 and oocytes. D. The relative fluorescence intensity of mouse PB1 to its sister oocyte was quantified and the difference significance was evaluated using paired t test. In TMRE and DilC 1 (5), Rhod2 detection, red color represented TMRE and DilC 1 (5), Rhod2, green color marked mitochondria in PB1 and oocyte. In Fluo-3 detection, green color showed Fluo-3, red color stained mitochondria in PB1 and oocyte, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Error bars indicate SD, with the mean value, n=3 per group for human, n = 6 per group for mouse. Scale bar=40μm.

Article Snippet: For TMRE (ab113852, Abcam), DilC 1 (5) (M34151, life Technology) and Rhod-2 (M34151, life Technology) staining, MitoTracker (MitoTracker Green FM, M7514, Life Technology) were used to locate the mitochondria.

Techniques: Membrane, Fluorescence, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers.

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: Figure 2 NLRP3 inhibition attenuates ICI-induced cardiac injury. Mice were inoculated with B16F10 melanoma cells on day 0, and injected with either IgG+vehicle, or αPD-1 antibody+vehicle, or αPD-1 antibody+MCC950 every other day from day 7 to day 19. Hearts were collected on day 20. (A) Experimental design. (B) ELISA for plasma cTnT levels of indicated groups. (C) Echocardiographic analysis showing LVEF, LVFS, LVESV, LVEDV, LVIDs, and LVIDd, in mice of each group. (D, E and F) Representative immunofluorescence images for CD8 (D), F4/80 (E), TUNEL (F) and their statistical analysis of per high-power field (HPF, average of 3–6 different 200× visual fields) in the hearts of each group; scale bar, 50 µm. (G) Representative Masson staining images in mouse hearts of each group and their statistical analysis. Scale bar, 50 µm. (H) RT-qPCR analysis for the expression of Il1b, Il6, Tnf, Ifng, Ccl2, Ccl3, Ccl5. n=8 per group. Data are presented as the mean±SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc multicomparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with indicated group.cTnT, cardiac troponin t; ICI, immune checkpoint inhibitor; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; LVIDd, left ventricular internal dimension in diastole; LVIDs, left ventricular internal dimension in systole; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP nick-end labeling.

Article Snippet: TdT-mediated dUTP nick-end labeling (TUNEL) The TUNEL assay was performed using TMR (red) TUNEL Cell Apoptosis Detection Kit (Solarbio) according to the manufacturer’s instructions.

Techniques: Inhibition, Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunofluorescence, TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, End Labeling

Journal: Journal for immunotherapy of cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers.

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: Figure 6 NLRP3 inhibition ameliorates cardiotoxicity of combined ICI therapy. Mice were inoculated with B16F10 melanoma cells on day 0, and were administrated with IgG+vehicle, or anti-PD-1 + anti-CTLA-4 + vehicle, or anti-PD-1 + anti-CTLA-4 + MCC950 every other day from day 7 to day 19. Hearts were collected on day 20. (A) Experimental design. (B) ELISA for plasma cTnT levels of IgG group, dual ICI (D-ICI) group and dual ICI+MCC950 (D-ICI+MCC950) group. (C) Echocardiographic analysis showing cardiac function of each group. (D, E, and F) Representative immunofluorescence images for CD8 (D), F4/80 (E), TUNEL (F) and their statistical analysis of per high-power field (HPF, average of 3–6 different 200× visual fields) in the heart of each group; scale bar, 50 µm. (G) Representative Masson staining images in mouse hearts of each group and their statistical analysis. Scale bar, 50 µm. (H) RT-qPCR analysis for relative mRNA expression of Il1b, Il6, Tnf, Ifng, Ccl2, Ccl3, Ccl5 in the heart of each group. (I) Tumor volume measurements of the female B16F10 tumor-bearing mice of each group. (J) Tumor volume at day 19 of each group (left) and tumor weight at day 20 of each group (right). n=8 per group. Data are presented as mean±SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc multicomparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with indicated group. RT-qPCR, reverse transcription quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP nick-end labeling.

Article Snippet: TdT-mediated dUTP nick-end labeling (TUNEL) The TUNEL assay was performed using TMR (red) TUNEL Cell Apoptosis Detection Kit (Solarbio) according to the manufacturer’s instructions.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Immunofluorescence, TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, End Labeling

Journal: Carbohydrate polymers

Article Title: An oxidized dextran-composite self-healing coated magnesium scaffold reduces apoptosis to induce bone regeneration.

doi: 10.1016/j.carbpol.2023.121666

Figure Lengend Snippet: Fig. 6. Exploration of the osteogenic mechanism of scaffolds in vitro. (A) Reactive oxygen detected by cellular reactive oxygen species detection assay Kit. (B) The mitochondrial membrane potential was used to evaluate the mitochondrial function. (C) The apoptosis detection was performed using an apoptosis detection kit. (D) The apoptosis related genes were tested by qPCR. The apoptosis related proteins (E) tested by Western blot. (F) Tested by immunofluorescence.

Article Snippet: To evaluate mitochondrial function by assessing the mitochondrial membrane potential using a Mitochondrial Membrane Potential Assay Kit (TMRE, Solarbio, China).

Techniques: In Vitro, Detection Assay, Membrane, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Inhibition of autophagy and MEK promotes ferroptosis in Lkb1-deficient Kras-driven lung tumors

doi: 10.1038/s41419-023-05592-8

Figure Lengend Snippet: A Clonogenic survival assay of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with HCQ or Trametinib individually at indicated concentrations. B Cell growth inhibition curve of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with HCQ or Trametinib individually at indicated concentrations in Table . C Clonogenic survival assay of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with the combination of HCQ and Trametinib at indicated concentrations. D Relative proliferation of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. E Western blot for LC3, pERK, total ERK, pS6, total S6 and β-actin of KL and KP TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h. F Scheme of the KL or KP TDCLs for measuring oxygen consumption rate (OCR) using Seahorse XFe24 analyzer. G Basal respiration and ATP production of KL TDCLs (clone 2126 3-2 and clone 2126 5-5 (with black squares)) after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. H Basal respiration and ATP production of KP TDCLs (clone 2871-1 and clone 2871-8 (with black squares)) after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. I Scheme of the metabolomics analysis via LC-MS of KL TDCLs after 6 h’ treatment. J The levels of metabolites of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. K Left: Overlapping images of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with MitoTracker Red CMXRos for mitochondrial membrane potential and MitoTracker Green FM for mitochondrial mass. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KL TDCLs quantified by the ratio of red fluorescence intensity and green fluorescence intensity. L Left: Overlapping images of KP TDCLs (clone 2871-7 and clone 2871-8) treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with MitoTracker Red CMXRos for mitochondrial membrane potential and MitoTracker Green FM for mitochondria mass. Blue: Hoechst 33342 for nuclear staining. Right: graph of relative mitochondrial membrane potential of KP TDCLs quantified by the ratio of red fluorescence intensity and green fluorescence intensity. M Left: Overlapping images of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with TMRM (red fluorescence) for mitochondrial membrane potential. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KL TDCLs quantified by the ratio of red fluorescence intensity and total cell numbers. N Left: Overlapping images of KP (clone 2871-1 and clone 2871-8) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with TMRM (red fluorescence) for mitochondrial membrane potential. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KP TDCLs quantified by the ratio of red fluorescence intensity and total cell numbers. Data are mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TMRM Assay Kit (Mitochondrial Membrane Potential) (Cat#: ab228569) was purchased from Abcam.

Techniques: Clonogenic Cell Survival Assay, Inhibition, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Staining, Membrane, Fluorescence