nmol l mitophagy dye  (Dojindo Labs)

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Iron accumulation impairs mitophagy, promotes senescence, and suppresses osteogenic differentiation in BMSCs. (a) Schematic diagram of extraction of BMSCs from human femur. (b) Western blot analysis of osteogenic marker proteins (RUNX2, ALP) in BMSCs from normal controls and postmenopausal osteoporosis patients and osteoporosis patients with iron accumulation. (c) Alizarin Red S (ARS) staining of BMSCs treated with increasing concentrations of FAC (0, 50, 100, 200 μM) for 21 days and alkaline phosphatase (ALP) staining of BMSCs treated with increasing concentrations of FAC (0, 50, 100, 200 μM) for 14 days. Scale bar: 50 μm. (d) Western blot analysis of osteogenic markers (RUNX2, ALP) in FAC-treated BMSCs for 5 days. (e) RT-qPCR analysis of osteogenic genes ( Runx2, Alpl, Bglap, Sp7 ) in FAC-treated BMSCs for 72h. (f) KEGG pathway enrichment analysis of differentially expressed genes from RNA sequencing of control and 200 μM FAC-treated BMSCs for 72h. (g, h) Immunofluorescence staining of senescence markers (γ-H2AX, H3K9me3) in FAC-treated BMSCs for 72h. Scale bar: 20 μm. (i) Senescence-associated β-galactosidase (SA-β-gal) staining of FAC-treated BMSCs for 72h. Scale bar: 50 μm. (j) Flow cytometric quantification of SA-β-gal activity in FAC-treated BMSCs for 72h. (k) Western blot analysis of senescence-related proteins (P53, P21, P16) in FAC-treated BMSCs for 72h. (l) Mitophagy assessment by immunofluorescence co-staining with Mitophagy Dye (red) and MitoTracker (green) in FAC-treated BMSCs for 72h. Scale bar: 20 μm. (m) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in FAC-treated BMSCs for 72h. (n) Mitochondrial membrane potential (MMP) detection by MT-1 staining in FAC-treated BMSCs for 72h. Scale bar: 30 μm. Data are presented as mean ± SEM; One-way ANOVA (Dunnett's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Extraction, Western Blot, Marker, Staining, Quantitative RT-PCR, RNA Sequencing, Control, Immunofluorescence, Activity Assay, Membrane, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Mitophagy activation rescues iron accumulation-induced mitochondrial dysfunction, cellular senescence, and impaired osteogenic differentiation in BMSCs. BMSCs were isolated from normal mice and treated with 200 μM FAC with or without CCCP co-treatment for the same duration in each assay. The time points for the indicated assays were the same as those in . (a) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3). (b, c) Flow cytometric analysis of (b) intracellular ROS and (c) mitochondrial superoxide levels. (d) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining. Scale bar: 30 μm. (e) Cellular ATP content measurement. (f – i) Immunofluorescence analysis of senescence markers (f, h) γ-H2AX and (g, i) H3K9me3. Scale bar: 40 μm. (j) Western blot analysis of senescence-related proteins (P53, P21, P16). (k) Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining. Scale bar: 50 μm. (l) Western blot analysis of osteogenic marker proteins (RUNX2, ALP). Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Activation Assay, Isolation, Western Blot, Membrane, Immunofluorescence, Staining, Marker, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Mitophagy activation alleviates BMSC senescence and restores bone mass in iron-accumulating mice. (a) Representative micro-CT images of distal femoral trabecular bone. (b) Quantitative micro-CT analysis of trabecular bone parameters: Tb.BMD (trabecular bone mineral density), BV/TV (bone volume fraction), BS/TV (bone surface density), and Tb.N (trabecular number). (c) Detection of the serum OCN and P1NP levels from the mice in each group. (d) Histological analysis of tibial sections via H&E staining, toluidine blue staining, and DAPI immunofluorescence from the mice in each group. Scale bar: 250 μm. (e) Detection of the bone formation rate by calcein double labeling from the mice in each group. Scale bar: 20 μm. (f – i) Immunofluorescence analysis of senescence markers (γ-H2AX and H3K9me3) in BMSCs isolated from different treatment groups. Scale bar: 50 μm. (j) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs. (k) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in BMSCs. (l) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining in BMSCs. Scale bar: 50 μm. (m) Cellular ATP content measurement in BMSCs. (n – o) Flow cytometric analysis of (n) intracellular ROS and (o) mitochondrial superoxide levels in BMSCs. Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Activation Assay, Micro-CT, Staining, Immunofluorescence, Labeling, Isolation, Western Blot, Membrane, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: PINK1 overexpression rescues iron accumulation-induced mitochondrial dysfunction, senescence, and osteogenic impairment in BMSCs. The time points for the indicated assays were the same as those in . (a) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, PARKIN, P62, LC3) in BMSCs transduced with control or PINK1-overexpressing lentivirus followed by FAC treatment. (b) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining. Scale bar: 50 μm. (c) Cellular ATP content measurement. (d, e) Flow cytometric analysis of (d) intracellular ROS and (e) mitochondrial superoxide levels. (f) Western blot analysis of senescence-related proteins (P53, P21, P16). (g – j) Immunofluorescence analysis of senescence markers (γ-H2AX and H3K9me3). Scale bar: 50 μm. (k, l) Alizarin Red S (ARS) staining and Alkaline phosphatase (ALP) staining. Scale bar: 50 μm. (m) Western blot analysis of osteogenic marker proteins (RUNX2, ALP). (n) RT-qPCR analysis of osteogenic genes ( Runx2, Alpl, Bglap, Sp7 ). Data are presented as mean ± SEM; One-way ANOVA (Tukey's multiple-comparison test); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Over Expression, Western Blot, Transduction, Control, Membrane, Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: FTMT upregulation during iron accumulation impairs mitophagy by inhibiting PINK1 phosphorylation. (a) Immunofluorescence detection of intracellular and mitochondrial iron levels in BMSCs. Scale bar: 10 μm. (b) Western blot analysis of FTMT expression in BMSCs with or without FAC treatment. (c) Co-immunoprecipitation analysis of PINK1-FTMT interaction in BMSCs treated with FAC. (d) Schematic diagram of full-length and domain-deletion mutants of PINK1 (MTS: mitochondrial targeting sequence; TM: transmembrane domain; KD: kinase domain). (e) Co-immunoprecipitation using anti-Flag antibody in BMSCs transfected with WT-PINK1 or PINK1 deletion mutants and treated with FAC, followed by FTMT detection. (f) Western blot analysis of PINK1 phosphorylation at Ser228 and Ser402 in BMSCs. (g) Western blot analysis of mitophagy/autophagy-related proteins (PINK1, p-PINK1(Ser228), PARKIN, P62, LC3) in control and FTMT-knockdown BMSCs under iron accumulation. (h) Cellular ATP content measurement. (i) Mitochondrial membrane potential assessment by MT-1 immunofluorescence staining. Scale bar: 20 μm. (j, k) Flow cytometric analysis of (j) intracellular ROS and (k) mitochondrial superoxide levels. (l) Western blot analysis of mitophagy/autophagy-related proteins (p-PINK1(Ser228), PARKIN, P62, LC3) in BMSCs expressing PINK1 with S228A point mutation. Data are presented as mean ± SEM; Unpaired 2-tailed Student's t -test (a), One-way ANOVA (Tukey's multiple-comparison test) (h, j and k); ** P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Phospho-proteomics, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Sequencing, Transfection, Control, Knockdown, Membrane, Staining, Mutagenesis, Comparison

Journal: Redox Biology

Article Title: FTMT-mediated suppression of mitophagy links iron accumulation to osteoporosis

doi: 10.1016/j.redox.2026.104157

Figure Lengend Snippet: Impaired mitophagy in BMSCs from osteoporosis patients with iron accumulation. (a) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs from normal controls, postmenopausal osteoporosis patients and osteoporosis patients with iron accumulation. (b) Western blot analysis of mitochondrial ferritin (FTMT) expression levels in BMSCs. (c) Western blot analysis of mitophagy/autophagy-related proteins PINK1, p-PINK1(Ser228), PARKIN, P62, and LC3 in BMSCs. (d) Western blot analysis of mitophagy/autophagy-related proteins PINK1, PARKIN, P62, and LC3 in BMSCs of PMOP and IOP group with or without CCCP intervention. (e) Western blot analysis of senescence-related proteins (P53, P21, P16) in BMSCs of PMOP and IOP group with or without CCCP intervention. (f) Western blot analysis of osteogenic marker proteins (RUNX2, ALP) in BMSCs of PMOP and IOP group with or without CCCP intervention.

Article Snippet: Then, 100 nmol/L Mitophagy Dye (containing 100 nmol/L MitoTracker Green Probe [Dojindo, MT10]) was added to each group, and the cells were incubated for 30 min. After experimentation, the cells were washed twice with Hank's solution.

Techniques: Western Blot, Expressing, Marker

Journal: Biotechnology & Biotechnological Equipment

Article Title: Restoring mitophagy in prostate cancer cells: the role of miR-141 rescue in counteracting MAPK1/ERK2-dependent autophagy suppression

doi: 10.1080/13102818.2023.2293055

Figure Lengend Snippet: Figure 4. Impact of miR-141 mimic and siMAPK1 on mitophagy flux: assessments under normal, starvation, and bafilomycin A treat ment conditions using MtPhagy and Lyso dyes (Dojindo) for mitophagy and lysosomes, respectively. (A) Flow cytometry histogram analysis of MtPhagy and Lyso dyes intensity in LNCaP and PC3 cells in non-treated (nt), starvation (nt_starvation), starvation after bafilomycin A treatment (nt_starv_Baf), miR-141 mimic transfected in high glucose (miR-141), and after bafilomycin A treatment (miR-141_Baf), and miR-141 mimic transfected in low glucose (miR-141_starv) conditions, and after bafilomycin A treatment (miR-141_starv_Baf). (B) Median with error bars plot of the MFI data obtained from non-treated (nt), starvation (nt_starvation), star vation after bafilomycin A treatment (nt_starvation_Baf), miR-141 mimic transfected in low glucose (miR-141_starvation) conditions, and after bafilomycin A treatment (miR-141_starvation_Baf), and miR-141 mimic transfected in high glucose (miR-141), and after bafilomycin A treatment (miR-141_Baf). statistically significant differences (p < 0.05) are shown using asterisks. (C) Flow cytometry histogram analysis of MtPhagy and Lyso dyes intensity in LNCaP and PC3 cells in intact (nt), starvation (nt_starvation), starvation after bafilomycin A treatment (nt_starv_Baf), siMAPK1 transfected in high glucose (siMAPK1), and after bafilomycin A treatment (siMAPK1_ baf), and siMAPK1 transfected in low glucose (siMAPK1_starv) conditions, and after bafilomycin A treatment (siMAPK1_starv_baf). (D) Median with error bars plot of the MFI data obtained from intact (nt), starvation (nt_starvation), starvation after bafilomycin A treatment (nt_starvation_Baf), siMAPK1 transfected in low glucose (siMAPK1_starvation) conditions, and after bafilomycin A treat ment (siMAPK1_starvation_Baf), and siMAPK1 transfected in high glucose (siMAPK1), and after bafilomycin A treatment (siMAPK1_ Baf). statistically significant differences (p < 0.05) are shown using asterisks. (E) and (F) Non-treated (nt), starved (starv), miR-141 mimic transfected in high glucose (miR-141) and miR-141 mimic transfected in low glucose (miR-141 starv) conditions PC3 cells, showing cell morphology, Lyso dye fluorescence (green) and Mtphagy dye fluorescence (red) on Etaluma LS620 inverted microscope. Cells with co-localisation are shown with white full triangle, cells with lacking colocalization are shown with transparent triangle. All microscopy images have a scale bar of 25 µm. Expression in (B) and (F) is assessed using the MFI parameter from FCS histograms, with values normalized to a control value of 1. Asterisks above bars denote statistically significant differences as determined by ANOVA (*p < 0.05).

Article Snippet: 500–563 nm), mitophagy was tracked with 100 nmol/L MtPhagy dye (Dojindo Molecular Technologies, MD01.5; Ex.

Techniques: Flow Cytometry, Transfection, Fluorescence, Inverted Microscopy, Microscopy, Expressing, Control