Journal: Scientific Reports

Article Title: Florfenicol-induced Mitochondrial Dysfunction Suppresses Cell Proliferation and Autophagy in Fibroblasts

doi: 10.1038/s41598-017-13860-9

Figure Lengend Snippet: FLO inhibits Parkin-mediated mitochondrial autophagy and promotes cell senescence. ( A ) Immunoblot analysis of autophagy-related proteins in L cells. Cells were treated with 0.1 mg/mL FLO for 12 h or 24 h, and combined treatment with Baf A1 (200 nM) as indicated. GAPDH served as a loading control. Densitometry was performed for quantification and the ratios of LC3B-II or p62 to GAPDH are presented at the bottom of the blots, respectively. Cropped blots are displayed and full-length blots are included in the Supplementary Information file. ( B ) Cox IV and Tim23 levels were detected by western blot with the whole cell lysis extracted form L cells. Cells were treated with FLO for 12 h or 24 h, or were pretreated with FLO for 8 h of 20 h and then co-treated with CCCP (100uM) for another 4 h as indicated. ( C ) Flow cytometric analysis of Mitotracker Deep Red fluorescence in L cells treated with FLO (0.1 mg/mL) for 24 h. The mean fluorescence of Mitotracker Deep Red represents the level of mitochondrial population. CCCP (100 uM for 4 h) was used as a positive control for the clearance of damaged mitochondria through mitophagy. ( D ) Representative images of SA β-gal staining in L cells in the presence of FLO (0.1 mg/mL) for eight days. The medium containing FLO was renewed every two days. SA β-gal-positive cells were quantified (n = 3). ( E ) Immunoblots show levels of the Pink1, recruited-Parkin and p62 on mitochondrial outer membrane in L cells. Cells were treated with FLO (0.1 mg/mL) only or co-treated with FLO and NAC (2 mM) for 24 h. CCCP (100 uM for 4 h) was used as a positive control for the induction of mitophagy through Pink1-Parkin pathway. Cox IV served as a loading control. ( F ) Flow cytometric analysis of Mitotracker Deep Red fluorescence in L cells that co-treated with FLO (0.1 mg/mL) and NAC (2 mM) for 24 h. Histograms in the figure represents the mean ± SD. **p < 0.01, as compared with the indicated group (LSD multiple comparison test after one-way ANOVA analysis).

Article Snippet: The apoptosis rates, cell cycle distribution, mitochondrial membrane potential, ROS levels and mitochondrial mass were assessed with the Annexin V-PE/7-AAD staining kit (KeyGen Biotech, Nanjing, China), cell cycle analysis kit (Beyotime, Shanghai, China), JC-1 detection kit (KeyGen Biotech, Nanjing, China), ROS assay kit (Beyotime, Shanghai, China), and MitoTracker Deep Red staining kit (KeyGen Biotech, Nanjing, China), respectively.

Techniques: Western Blot, Control, Lysis, Fluorescence, Positive Control, Staining, Membrane, Comparison

Journal: iScience

Article Title: Agent-based modeling of neuronal mitochondrial dynamics using intrinsic variables of individual mitochondria

doi: 10.1016/j.isci.2025.112390

Figure Lengend Snippet: Neuronal recordings of mitochondrial dynamics (A) Representative image of MitoTracker Deep Red-labeled neurons from live cell recordings. Scale bar: 10 μm. (B) Representative frames 60 s apart of MitoTracker Deep Red-labeled mitochondria performing fusion. Scale bar: 1 μm. (C) Quantification of MitoTracker Deep Red signal over time in neuron recordings. (D) Quantification of mitochondria undergoing fusion and fission events, as percentage of total mitochondrial objects, over time in MitoTracker Deep Red recordings. (E) Representative image of MitoTimer neuron from live cell recordings. Scale bar: 10 μm. (F) Representative frames 120 s apart of MitoTimer expression mitochondria performing fission. Scale bar: 1 μm. (G) Quantification of MitoTimer green signal, red signal (left y axis), and red/green ratio (right y axis) over time in MitoTimer neuron recordings. (H) Quantification of mitochondria undergoing fusion and fission events, as percentage of total mitochondrial objects, over time in MitoTimer recordings. n = 4–5 biological replicates.

Article Snippet: For MitoTracker Deep Red recordings, cells were washed once with sterile PBS and incubated with 30nM MitoTracker Deep Red FM (Fisher Scientific, M22426 ) in Earle’s Balanced Salts Solution (EBSS) without phenol red (Fisher Scientific, AAJ67559AP) during 30 minute acclimation.

Techniques: Labeling, Expressing

Journal: iScience

Article Title: Agent-based modeling of neuronal mitochondrial dynamics using intrinsic variables of individual mitochondria

doi: 10.1016/j.isci.2025.112390

Figure Lengend Snippet: Dynamic mitochondria have unique intrinsic properties (A) Histogram of log-transformed mitochondrial area (μm 2 ) from individual mitochondrial objects during MTDR recordings. Curves are fitted Gaussian distributions. (B) Histogram of log-transformed aspect ratio (AR) from individual mitochondrial objects during MTDR recordings. Curves are fitted Gaussian distributions. (C) Histogram of log-transformed relative MitoTracker Deep Red (MTDR) mean intensity from individual mitochondrial objects during MTDR recordings. Curves are fitted Gaussian distributions. (D) Correlation matrix of intrinsic variables collected from individual mitochondrial objects during MTDR recordings. (E) Histogram of log-transformed MitoTimer red/green ratio from individual mitochondrial objects during MitoTimer recordings. Curves are sum of two Gaussian distributions. (F) Correlation matrix of intrinsic variables collected from individual mitochondrial objects during MitoTimer recordings.

Article Snippet: For MitoTracker Deep Red recordings, cells were washed once with sterile PBS and incubated with 30nM MitoTracker Deep Red FM (Fisher Scientific, M22426 ) in Earle’s Balanced Salts Solution (EBSS) without phenol red (Fisher Scientific, AAJ67559AP) during 30 minute acclimation.

Techniques: Transformation Assay

Journal: iScience

Article Title: Agent-based modeling of neuronal mitochondrial dynamics using intrinsic variables of individual mitochondria

doi: 10.1016/j.isci.2025.112390

Figure Lengend Snippet: Intrinsic mitochondrial properties predict fission and fusion events (A) ROC curve of multiple logistic regression classifying stable and fission mitochondria from MTDR recordings. (B) Violin plot of predicted probability for fission of MTDR-labeled mitochondria by multiple logistic regression. (C) ROC curve of multiple logistic regression classifying stable and fusion mitochondria from MTDR recordings. (D) Violin plot of predicted probability for fusion of MTDR-labeled mitochondria by multiple logistic regression. (E) ROC curve of multiple logistic regression classifying stable and fission mitochondria from MitoTimer recordings. (F) Violin plot of predicted probability for fission of MitoTimer mitochondria by multiple logistic regression. (G) ROC curve of multiple logistic regression classifying stable and fusion mitochondria from MitoTimer recordings. (H) Violin plot of predicted probability for fusion of MitoTimer mitochondria by multiple logistic regression.

Article Snippet: For MitoTracker Deep Red recordings, cells were washed once with sterile PBS and incubated with 30nM MitoTracker Deep Red FM (Fisher Scientific, M22426 ) in Earle’s Balanced Salts Solution (EBSS) without phenol red (Fisher Scientific, AAJ67559AP) during 30 minute acclimation.

Techniques: Labeling

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . TNT structure. T24 and RT4 cells were co-cultured at a 1:1 ratio for 24 h, and images of TNTs, thin microtubular connections, were captured under white-light visual (a) and fluorescence (b) microscopy. (c) is the merged of visual image (a) and fluorescence image (b). Bar = 50 μm. B . F-actin based structure. T24 and RT4 cells were co-cultured for 24 h, and stained with Actin-Tracker Green and DAPI. (a) Actin-Tracker (Green); (b) nuclei-DAPI (Blue); (c) the merged image of (a) and (b). TNTs were observed and marked by Actin-Tracker Green (White arrows), which indicated TNTs had an F-actin based structure. Images were captured under fluorescence microscopy. Bar = 50 μm.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Cell Culture, Fluorescence, Microscopy, Staining

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . Open-ended filopodia-like cell protrusions of TNTs between T24 and RT4 cells. TNTs (a, black arrows) were observed between T24 cells and RT4 cells (b, c, d). Continuous, membranous, micro-tubular connection between T24 (b) and RT4 (d) cells were featured. The caliber of the membranous tubes ranged from 100-200 nm, and the lengths of TNTs between T24 and RT4 cells spanned a large range from 20 μm to 1 mm. B . Blindly ending filopodia-like cell protrusions of TNTs between T24 and RT4 cells. TNTs (a, black arrows) were extended from T24 cells (b), indicating TNTs were originally formed by T24 cells.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques:

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . MitoTracker Deep Red labeled T24 (a, red) and CFSE Green labeled RT4 cells (b, green) were co-cultured for 24 h, and nuclei were marked by DAPI (c, blue); (d) is the merged images of (a), (b), and (c). Spontaneous mitochondria trafficking (white arrows) from T24 cells to RT4 cells were obtained by capturing “double positive” (red and green) RT4 cells under fluorescence microscopy (d). Bar = 25 μm. B . Mitochondria in T24 cells were labeled by MitoTracker Deep Red, and could be observed as a thin red line in the tube-like structures between T24 and RT4 cells (white arrows) (a). Then F-actin was labeled by Actin-Tracker Green (b), and nuclei were labeled by DAPI (c). Mitochondria could be observed migrating from T24 cells to RT4 cells via F-actin based TNTs (d, merged images of a, b, and c). Bar = 10 μm. C . Latrunculin B was used to inhibit TNT formation and mitochondria transfer between T24 cells and RT4 cells. (a) T24 cells were labeled by MitoTracker Deep Red and co-cultured with RT4 cells and Latrunculin B (1.25 μmol/L) for 24 h. No mitochondria transportation from T24 cells to RT4 cells can be observed. (b) T24 and RT4 cells were labeled by Actin-Tracker Green. (c) The nuclei in T24 and RT4 cells was marked by DAPI. (d) is the merged images of a, b, and c. Bar = 10 μm. D . MitoTracker Deep Red labeled T24 cells were co-cultured with MitoTracker Green labeled RT4 cells. Double labeled RT4-Mito-T24 cells were sorted out and obtained by FACS. (a) In RT4-Mito-T24 cells, mitochondria originally migrated from T24 cells, and was labeled by MitoTracker Deep Red. Image was captured under a red fluorescent filter. (b) In RT4-Mito-T24 cells, mitochondria originally in RT4 cells were labeled by MitoTracker Green. Image was captured under a green fluorescent filter. After nuclei were marked by DAPI (c), mitochondria distribution in RT4-Mito-T24 cells was observed by LCM. The immigrated mitochondria (a) from T24 cells and original mitochondria (b) from RT4 cells had a high concordance in sub-cellular distribution (d, white arrows, d is the merged images of a, b, and c), which implied mitochondria fusion. Bar = 10 μm.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Labeling, Cell Culture, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . The invasive ability of RT4, T24, and RT4-Mito-T24 cells were detected by Transwell assay. After the incubation, images of cells migrating through the Matrigel-coated filter were captured respectively. Bar = 50 μm. B . Cells invading the Matrigel and reaching the lower surface of the filter were counted. The invasive ability in RT4-Mito-T24 cells was up-regulated compared to parental RT4 cells.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Transwell Assay, Incubation

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . The invasive ability of RT4, T24, and RT4-Mito-T24 cells were detected by wound healing assays. The images of the cells along the wound were captured at 0 h and 24 h, and marked by lines under an inverted microscope. Bar = 50 μm. B . Then the healing area was analyzed. The closure of the wounded area was accelerated in RT4-Mito-T24 cells relative to parental RT4 cells.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Inverted Microscopy

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . RT4, T24, and RT4-Mito-T24 cells were inoculated in the forelimb of athymic mice (A Left) respectively. After 30 days, mice were sacrificed, and the volume of the xenografts were measured (A Right). B . Tumor growth curves indicated that the average size of the tumors in the RT4 group was smaller than that in the other two groups.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques:

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A . After tumor formation, blood flow circumference around xenografts (green square area, shown in A lower panel) were detected and measured by ultrasound in vivo . B . T24 group obtained a higher relative vascular index than the RT4 group. RT4-Mito-T24 group had a higher relative vascular index than parental RT4 cells, but the difference was not statistically significant.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: In Vivo

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: RT4, T24, and RT4-Mito-T24 cells were cultured separately for 24 h and labeled by MitoTracker Deep Red, F-actin (green), and DAPI (blue). The images of F-actin and mitochondria distribution (white arrows) were captured by LCM. F-actin staining was restricted to the inner membrane of RT4 cells, and diffused along the filopodia-like cell protrusions. The mitochondria were restricted around the R24 nuclei, but diffusely distributed in the cytoplasm of T24 and RT4-Mito-T24 cells. Bar = 10 μm.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Cell Culture, Labeling, Staining, Membrane

Journal: Oncotarget

Article Title: Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

doi: 10.18632/oncotarget.14695

Figure Lengend Snippet: A, B . Akt expression was increased in RT4-Mito-T24 cells compared to parental RT4 cells. However, the p-Akt levels between the three types of cells had no significant difference. Both mTOR and p-mTOR were increased in RT4-Mito-T24 cells compared to RT4 cells. As two main downstream regulators of mTOR, the expression of 4EBP1 was higher in RT4-Mito-T24 cells, while no difference was noted in p70S6K levels.

Article Snippet: MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA).

Techniques: Expressing

Journal: iScience

Article Title: mtDNA extramitochondrial replication mediates mitochondrial defect effects

doi: 10.1016/j.isci.2024.108970

Figure Lengend Snippet:

Article Snippet: MitoTracker Deep Red , YEASEN , Cat40734ES50.

Techniques: ATP Assay, Isolation, Software