primary antibodies depp1  (Cusabio)

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF reverses a cross-tissue stress–autophagy signature and highlights DEPP1 as a shared node. (A) Venn diagram of TFDF-reversal DEGs in bone and the hippocampus (reversal defined as Model vs. Sham significant change, directionally opposed in TFDF-H vs. Model). (B) Pathway enrichment of reversal genes highlighting FOXO signaling (database and statistics in Materials and Methods). (C) Cross-filtering for Model↑ and TFDF-H↓ genes in both tissues identifies DEPP1 as a shared, treatment-reversed transcript (expression changes shown for bone and hippocampus). (D) Complementary enrichment of the reversal sets prioritizes FOXO signaling in both tissues. (E) GSEA plots demonstrating negative enrichment of FOXO programs in the Model vs. Sham groups and a positive shift in TFDF-H vs. Model (bone and hippocampus) groups.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Expressing

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF reduces DEPP1 expression and normalizes the expression of autophagy–mitochondrial markers in the hippocampus and bone of OVX–CUMS mice. (A) Representative hippocampal immunofluorescence images of NeuN (neurons, red) and DEPP1 (green) in the Sham, Model, TFDF-L, and TFDF-H groups; nuclei are stained with DAPI (blue). (B) Quantification of the DEPP1/NeuN double-positive area in the hippocampus. (C) Representative DEPP1 immunohistochemistry in trabecular bone. (D) Quantification of the DEPP1-positive area in bone. (E and F) Representative Western blots for SIRT1, FOXO3, and DEPP1 in the hippocampus (E) and bone (F). (G and H) Densitometric analysis of SIRT1, FOXO3, and DEPP1 expression in the hippocampus (G) and bone (H) normalized to that of β-actin. (I) Representative hippocampal immunofluorescence for NeuN (red) and LC3B (green). (J) Quantification of the hippocampal LC3B/NeuN double-positive area. (K) Representative LC3 immunohistochemistry in trabecular bone. (L) Quantification of the LC3-positive area in bone. (M and N) Western blots for p62, LC3B, and TOM20 in the hippocampus (M) and bone (N). (O and P) Densitometric analysis of p62, LC3B, and TOM20 expression in the hippocampus (O) and bone (P), normalized to that of β-actin.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Expressing, Immunofluorescence, Staining, Immunohistochemistry, Western Blot

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF normalizes the SIRT1–FOXO3–DEPP1 axis, rebalances excessive autophagy, and rescues mitochondrial and osteogenic functions in MC3T3-E1 cells. MC3T3-E1 cells were assigned to Control, Model (H 2 O 2 injury), TFDF, or NAC (positive antioxidant control) groups. Immunoblotting revealed that SIRT1 down-regulation, FOXO3 hyperacetylation, and DEPP1 up-regulation after H 2 O 2 were reversed by TFDF (A), as determined by densitometry (B). TMRE microscopy and quantification revealed ΔΨm loss in the Model group and restoration by TFDF (C and D). Autophagy/mitochondrial markers indicated over-autophagy under injury that were recalibrated toward baseline by TFDF (E and F). TEM revealed autophagosome accumulation and swollen mitochondria in the Model group, which were mitigated by TFDF (G). Multicolor IF (LC3B/TOM20/DEPP1) revealed increased LC3B puncta, TOM20 fragmentation, and DEPP1 elevation in the Model group, all of which improved with TFDF (H); colocalization analyses confirmed normalization of LC3B–TOM20 (mitophagy coupling) and a reduction in TOM20–DEPP1 coupling by TFDF (I and J). Osteogenic function assays demonstrated TFDF-mediated recovery of ALP activity and ARS mineral deposition (K), accompanied by increased RUNX2 expression and OCN/ALP expression, as determined by Western blotting (L and M). Data are presented as the mean ± SEM, with n indicated on the plots; the statistical tests and multiple-comparison procedures are described in Materials and Methods. Abbreviations: TFDF, total flavonoids of Drynaria fortunei ; NAC, N-acetyl-L-cysteine; TMRE, tetramethylrhodamine ethyl ester; LC3, microtubule-associated protein 1 light chain 3; OCN, osteocalcin.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Control, Western Blot, Microscopy, Activity Assay, Expressing, Comparison

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF mitigates oxidative injury in HT22 cells by restoring SIRT1–FOXO3–DEPP1 signaling and autophagy–mitochondrial homeostasis, thereby improving neuroplasticity. Cells were assigned to Control, Model (H 2 O 2 ), TFDF, or NAC (antioxidant control) groups. Western blots showing SIRT1–FOXO3–DEPP1↑ after injury and reversal by TFDF (A and B). TMRE imaging revealed ΔΨm loss in the Model group and rescue by TFDF (C and D). Autophagy/mitochondrial markers were recalibrated toward baseline by TFDF (E and F), which is consistent with TEM showing fewer autophagosomes and preserved cristae (G). IF (DAPI/LC3B/TOM20/DEPP1) demonstrated reduced LC3B puncta, increased TOM20 integrity, and decreased DEPP1 with TFDF (H), which was supported by colocalization readouts (DEPP1–TOM20 and LC3B–TOM20) (I and J). Quantification of DEPP1–TOM20 colocalization, and the mitophagy index is shown in (K). TFDF further increased the expression of BDNF and p-CREB/CREB and restored the expression of synapsin I and PSD-95 (L and M). Data are presented as the mean ± SEM; statistics and replicate numbers are provided in Materials and Methods. Abbreviations: TFDF, total flavonoids of Drynaria fortunei ; NAC, N-acetyl-L-cysteine; TMRE, tetramethylrhodamine ethyl ester.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Control, Western Blot, Imaging, Expressing

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: DEPP1 bidirectionally alters autophagy–mitochondrial coupling under oxidative injury in HT22 and MC3T3-E1 cells. DEPP1 expression was reduced by siRNA (KD) or increased by plasmid (OE); cells were exposed to H 2 O 2 to model injury. Protein and mRNA assays confirmed effective KD/OE in both lines (A and B). TMRE imaging revealed ΔΨm loss in the Model group, partial recovery in the KD+Model group, and a further decrease in the OE+Model group (C and D). TEM revealed swollen mitochondria and autophagosomes in the Model group, fewer autophagosomes after KD, and abundant autophagosomes after OE (E). Western blots demonstrated model-associated LC3-II accumulation, p62 depletion, and TOM20 reduction; KD shifted these toward control, whereas OE intensified them (F) with densitometry in (G). LC3B/TOM20 immunofluorescence revealed parallel changes in puncta burden and mitochondrial network integrity, as shown by the statistical data in (J) (H to J). Group labels: Control, Model, KD+Model, and OE+Model. Data are presented as the mean ± SEM; replicate numbers and statistics are provided in Materials and Methods.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Expressing, Plasmid Preparation, Imaging, Western Blot, Control, Immunofluorescence

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: DEPP1 knockdown with TFDF maintains and often augments pathway activity and functional rescue in injured osteoblasts and neurons. (A) Schematic of the gene–drug design and hypothesized placement of DEPP1 downstream of TFDF-responsive signaling. (B) Autophagy/mitochondrial Western blots (LC3-I/II, p62, and TOM20) in MC3T3-E1 and HT22 cells under Control, Model, siDEPP1+Model, TFDF+Model, and siDEPP1+TFDF+Model conditions. (C) Densitometry for panel (B). (D) Representative ROS fluorescence micrographs. (E) Quantification of intracellular ROS levels. (F) Osteogenic function of MC3T3-E1 cells: ALP staining (day 7) and ARS mineralization (days 14 to 21). (G) Osteogenic markers (RUNX2, OCN, and ALP) were measured by Western blotting. (H) Corresponding quantification. (I) Neuronal plasticity markers in HT22 cells (BDNF, p-CREB/CREB, Synapsin I, and PSD-95) were measured by Western blotting. (J) Quantification. In both cell types, siDEPP1 and TFDF each improved the injury phenotype, and siDEPP1+TFDF achieved comparable or greater improvement without occluding the effects of TFDF. Statistical tests and n values are provided in Materials and Methods and on the plots.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Knockdown, Activity Assay, Functional Assay, Western Blot, Control, Fluorescence, Staining

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: Docking and functional perturbation support SIRT1 as a key TFDF-responsive effector. (A) Docked pose of naringenin in the SIRT1 activator pocket with key hydrogen-bond and hydrophobic contacts indicated. (B) Radius of gyration, (C) number of protein–ligand hydrogen bonds, (D) backbone RMSF, (E) solvent-accessible surface area, and (F) ligand RMSD, all indicating rapid stabilization after ~10 ns and maintenance of a compact, well-behaved complex. (G) Free-energy landscape of the SIRT1–naringenin trajectory plotted along PC1 and PC2 (Δ G = − k B T ln P ), showing a dominant low-energy basin corresponding to the bound state. (H) SPR analysis of SIRT1–naringenin binding, showing concentration-dependent sensorgrams and a 1:1 Langmuir fit consistent with specific interaction. (I) Western blots of FOXO3 and Depp1 (HT22 and MC3T3-E1) after Sirt1 knockdown (KD) or overexpression (OE). (J) TMRE staining (ΔΨm) and (K) corresponding quantification: ΔΨm decreases with KD and increases with OE. (L) Triplex IF (LC3, TOM20, and DEPP1) showing the autophagy burden, mitochondrial network integrity, and DEPP1 levels across KD/OE conditions. (M) Quantifications of puncta burden, TOM20 continuity, and DEPP1 intensity. Docking to DEPP1 with neoeriocitrin, naringin, and naringenin produced low-affinity, nonconvergent poses (not shown); docking and molecular dynamics results for naringin, naringenin, and the positive control are provided in the Supplementary Materials. Abbreviations: R g , radius of gyration; RMSF, root mean square fluctuation; SASA, solvent-accessible surface area; RMSD, root mean square deviation.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Functional Assay, Solvent, Binding Assay, Concentration Assay, Western Blot, Knockdown, Over Expression, Staining, Produced, Positive Control

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: SIRT1 determines cellular responsiveness to TFDF in neurons and osteoblasts. Under H 2 O 2 injury, cells were treated with TFDF alone or in combination with Sirt1 knockdown (KD) or overexpression (OE). Western blots showed that TFDF decreased acetyl-FOXO3 and DEPP1 levels and that these effects were abrogated by KD and strengthened by OE in HT22 and MC3T3-E1 cells (A, densitometry in B). ROS imaging/quantification demonstrated that injury-induced oxidative stress was reduced by TFDF, partially reversed by KD, and further reduced by OE (C and D). Immunofluorescence staining for LC3B/TOM20/DEPP1 revealed a TFDF-driven improvement in autophagy–mitochondrial morphology that was attenuated by KD and potentiated by OE in both cell types (E and F). Autophagy immunoblots (LC3-II and p62) confirmed the same interaction pattern (G, densitometry in H).

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Knockdown, Over Expression, Western Blot, Imaging, Immunofluorescence, Staining

Journal: Research

Article Title: Targeting a Shared Mitophagy Regulator: The SIRT1–FOXO3–DEPP1 Axis Underpins the Dual Bone and Brain Benefits of Total Flavonoids from Drynaria fortunei

doi: 10.34133/research.1125

Figure Lengend Snippet: TFDF alleviates OVX–CUMS-associated bone–brain comorbidity by engaging SIRT1-centered stress–autophagy signaling. Left: Experimental framework: 7-week-old female mice underwent ovariectomy combined with chronic unpredictable mild stress (OVX+CUMS, 12 weeks) and received total flavonoids of Drynaria fortunei (TFDF), resulting in improved bone density and depression-like behaviors. Right: Working model: OVX+CUMS-related cellular stress elevates reactive oxygen species (ROS) and disrupts autophagy–mitochondrial homeostasis. TFDF activates SIRT1, reduces FOXO3 acetylation, suppresses stress-responsive DEPP1, and restores antioxidant capacity (e.g., catalase [CAT]), thereby lowering ROS (with N-acetyl-L-cysteine [NAC], shown as an antioxidant control) and rebalancing autophagy to support organelle quality control. These coordinated effects ultimately improve cellular function and the observed functional phenotype in bone- and brain-relevant cells.

Article Snippet: IF was performed on mouse hippocampal sections and on MC3T3-E1 and HT22 cells using primary antibodies DEPP1 (CUSABIO, CSB-PA865135LA01HU), LC3 (Immunoway, PT0235R), and TOM20 (Immunoway, PT0287R).

Techniques: Control, Cell Function Assay, Functional Assay