lc3  (Cell Signaling Technology Inc)

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

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: Cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, IPVH00010), and probed with primary antibodies against: RUNX-2 (Abcam, ab236639), ALP (Affinity, DF6225), P53 (Affinity, AF0879), P21 (Affinity, DF6423), P16 (Abcam, ab51243), PINK1 (HUABIO, ER1706-27), PARKIN (HUABIO, ET1702-60), P62 (Abcam, ab109012), LC3 (NOVUS, NB100-2220), FTMT (Abmart, PC20086S), Phospho-PINK1[Ser228] (Cell Signaling, 89010T), Phospho-PINK1[Ser402] (Absin, abs148820), and GAPDH (Affinity, AF7021).

Techniques: Western Blot, Expressing, Marker

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B induces PINK1/Parkin pathway-dependent mitophagy initiation in PDAC cells. (A) Western blot analysis of PINK1, Parkin, and LC3 in the mitochondria of PANC-1 and AsPC-1 cells. (B) Western blot analysis of Parkin in the mitochondria and cytoplasm of PANC-1 and AsPC-1 cells. (C) Western blot analysis of LC3 in the presence or absence of Agrimol B in the presence or absence of Mdivi-1 for 24 h. (D, E) Western blot analysis of LC3 in PDAC cells transfected with siScramble, siPINK1, or siParkin following treatment with or without Agrimol B. (F) Western blot analysis of LC3 in PANC-1 and AsPC-1 cells with or without Agrimol B in the presence or absence of wortmannin. (G-I) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of wortmannin. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Transfection, Immunofluorescence

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B blocks autophagic flux in PDAC cells. (A, B) Western blot analysis of P62 and CTSD in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (C, E, F) Immunofluorescence analysis of RFP-GFP-LC3 after PANC-1 and AsPC-1 cells were transfected with RFP-GFP-LC3 for 48 h, followed by treatment with or without Agrimol B for another 24 h. Scale bars, 10 μm. (D, G-L) Immunofluorescence analysis of the colocalization of endogenous LC3 with LAMP2 after treatment with Agrimol B or rapamycin for 24 h in PANC-1 and AsPC-1 cells. Scale bars, 10 μm. (M-O) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of HCQ. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Immunofluorescence, Transfection

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B regulates mitophagy by downregulating MTERF3 expression. (A) Venn diagram showing the overlap of differentially expressed proteins (fold-change ≥ 1.3 or ≤ 0.76) between PANC-1 and AsPC-1 cells. (B, C) Volcano plots of DEGs identified via label-free quantitative proteomics in PANC-1 and AsPC-1 cells. (D) Western blot analysis of MTERF3 in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (E) Differences in MTERF3 expression between normal tissues and cancer tissues in the UCSC Xena database. (F) Kaplan-Meier analysis of MTERF3 expression and overall survival in 64 patients with PDAC. (G, H) CCK-8 assay in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (I) Western blot analysis of LC3 in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (J) Western blot analysis of PINK1 and Parkin in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (K) Immunohistochemical analyses of PINK1 and MTERF3 expression in PDAC tissues. Scale bars, 100 μm. (L) Correlation of the immunostaining intensities of PINK1 and MTERF3. (M) Western blot analysis of TIM23, SOD2, and HADHA in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (N) Molecular docking suggests that Agrimol B can bind to MTERF3 with a binding energy of -6.085 kcal/mol. (O) Western blot analysis of MTERF3 in cells treated with or without Agrimol B in the presence or absence of MG132.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Expressing, Quantitative Proteomics, Western Blot, CCK-8 Assay, Transfection, Plasmid Preparation, Immunohistochemical staining, Immunostaining, Binding Assay

Journal: Molecular Metabolism

Article Title: Picalm coordinates clathrin-mediated endocytosis and actin remodeling during myogenesis

doi: 10.1016/j.molmet.2026.102351

Figure Lengend Snippet: Picalm is required for lysosomal degradation of autophagic vesicles. (A) Western blot analysis of LC3-I and LC3-II protein levels in si Picalm and siNT C2C12 cells during differentiation. (B) Quantification of LC3-II/I ratio relative to levels of siNT at day 0, 2 and 4 (n = 3 independent experiments, performed in duplicates or triplicates). (C) Treatment with autophagic modulators (Rapamycin: RAPA; 3-Methyladenine: 3-MA; Chloroquine: CQ; Bafilomycin-A1: BAFA1) was performed on day 0 for 6h. ∗3-MA induces autophagy under nutrient-rich conditions but inhibits autophagy under nutrient-deprived conditions (n = 3 independent experiments, performed in duplicates or triplicates). (D) Quantification of LC3-II/I ratio normalized to the respective untreated siNT condition for each individual experiment. (E–G) Western blot analysis of LC3-II and LC3-I, and Gapdh as loading control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student's t-test with Welch correction.

Article Snippet: LC3 , Polyclonal , Rabbit , Cell signaling , 2775.

Techniques: Western Blot, Control