anti inos nos2  (Novus Biologicals)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safety, efficacy, and distal nerve Schwann cell biodistribution in mice and NHPs to support translation of AAV9 RNAi therapy for CMT1A

doi: 10.1016/j.omtn.2026.102881

Figure Lengend Snippet: Biodistribution, transduction efficiency, and molecular effects of AAV9.U6.miR871 dose response (A) Experimental design for in vivo testing of four different AAV9.U6.miR871 doses. (B–D) Vector genome copy number (VGCN) measurements showed dose-dependent transduction of PNS tissues ( n = 5/group). (E) ddPCR detected mature miR871 sequence in the PNS and peripheral tissues. (F–H) Human (hu) PMP22 , murine (mu) Pmp22 , and mu Mpz gene expression detected in spinal nerve roots, sciatic nerve, and femoral nerve ( n = 4/group). Fold changes in relative mRNA expression of CMT1A animals treated with different AAV9.U6.miR871 doses were calculated in comparison with expression levels of PBS-injected CMT1A mice. All samples were normalized to endogenous mouse Gapdh . (I–K) Western blot analysis showing protein optical densities (ODs) of human PMP22, murine Pmp22, and murine Mpz from CMT1A animals treated with different AAV9.U6.miR871 doses normalized to tubulin and compared to expression levels in age-matched PBS-injected CMT1A mice. Western blot images and unnormalized optical density calculations are shown in . Values are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p ≤ 0.0001 by one-way ANOVA with Tukey’s multiple-comparison test. R, lumbar roots; S, sciatic nerves; F, femoral nerves.

Article Snippet: Immunoblots were incubated with rabbit antisera hu PMP22 (1:500; Abcam, ab90782), mu/hu PMP22 (1:500; Novus, NBP2-67068), against EGFP (1:1,000; Abcam, ab6556), and mouse β-tubulin (1:4,000; Developmental Studies Hybridoma Bank, ε-7) at 4°C overnight.

Techniques: Transduction, In Vivo, Plasmid Preparation, Sequencing, Gene Expression, Expressing, Comparison, Injection, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safety, efficacy, and distal nerve Schwann cell biodistribution in mice and NHPs to support translation of AAV9 RNAi therapy for CMT1A

doi: 10.1016/j.omtn.2026.102881

Figure Lengend Snippet: RNAscope Plus analysis for the co-detection of miR871, hu PMP22 , and mu Pmp22 in the sciatic nerves of CMT1A mice treated with AAV9.U6.miR871 (A–C) Representative images of miR871, hu PMP22 , and mu Pmp22 staining in the sciatic nerves of CMT1A mice treated with 5E11 or 1E12 vg/animal of AAV9.U6.miR871 ( n = 6 animals/group). (D and E) Quantitative image analysis was performed using HALO software and the average number of miR871-, hu PMP22 -, and mu Pmp22 -positive cells ( n ≥ 147,000 cells/group), as well the average number of corresponding miR871, hu PMP22 , and mu Pmp22 dots/cell, reflecting expression at the single cell level, in CMT1A animals treated with AAV9.U6.miR871 (5E11 or 1E12 vg/animal) or PBS. Results in (E) demonstrate that PMP22 reductions do not lead to oversilencing on a cell-by-cell basis. Values are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p ≤ 0.0001 by one-way ANOVA with Tukey’s multiple-comparison test. Scale bars: 50 μm and 15 μm (enlarged inserts).

Article Snippet: Immunoblots were incubated with rabbit antisera hu PMP22 (1:500; Abcam, ab90782), mu/hu PMP22 (1:500; Novus, NBP2-67068), against EGFP (1:1,000; Abcam, ab6556), and mouse β-tubulin (1:4,000; Developmental Studies Hybridoma Bank, ε-7) at 4°C overnight.

Techniques: RNAscope, Staining, Software, Expressing, Single Cell, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safety, efficacy, and distal nerve Schwann cell biodistribution in mice and NHPs to support translation of AAV9 RNAi therapy for CMT1A

doi: 10.1016/j.omtn.2026.102881

Figure Lengend Snippet: Widespread AAV biodistribution in NHP nerves following lumbar intrathecal delivery leads to PMP22 silencing in NHPs (A) AAV vector genomes were detected in both the proximal and distal portions of all NHP nerves at both time points tested. Vector genomes were assessed by dPCR using a probe to the AAV2 ITR. Data were analyzed as vector copies normalized to host diploid genomes. While all AAV-treated samples showed detectable AAV genomes above PBS-treated controls, there were no significant differences between low- and high-dose groups except in the distal femoral nerve (FN-D). Together, these results indicate a lack of dose-response on AAV biodistribution to peripheral nerves in NHPs. (B) RT-dPCR detection of mature miR871 transcript at 6 weeks (left) and 12 weeks (right) indicated biodistribution and miR871 RNA accumulation in all nerve tissues at low (6E13) and high (1.2E14) doses. Absolute copies of mature miR871 were normalized to the housekeeping gene, beta-2-mocroglobulin ( B2M ). (C) The PMP22 transcript was reduced at 6 (left) and 12 weeks (right) in most AAV9.U6.miR871-treated nerve segments, with significant reductions indicated by asterisks. Monkey PMP22 mRNA was quantified by RT-dPCR, normalized to B2M, and then values plotted as relative to saline controls for each nerve segment, which we considered to represent normal PMP22 levels (100%). Data are displayed as means with error bars representing SEM. n = 2 control or 4 treated animals per dosing group. Each point represents a single animal’s averaged samples per tissue designation including two segments from the right nerve and two from the left nerve. Two-way ANOVA shows a significant treatment effect ( p < 0.0001) at both time points. Tukey’s multiple comparison tests represented as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. SN, sciatic nerve; FN, femoral nerve; UN, ulnar nerve; MN, median nerve; P, proximal; D, distal.

Article Snippet: Immunoblots were incubated with rabbit antisera hu PMP22 (1:500; Abcam, ab90782), mu/hu PMP22 (1:500; Novus, NBP2-67068), against EGFP (1:1,000; Abcam, ab6556), and mouse β-tubulin (1:4,000; Developmental Studies Hybridoma Bank, ε-7) at 4°C overnight.

Techniques: Plasmid Preparation, Saline, Control, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Safety, efficacy, and distal nerve Schwann cell biodistribution in mice and NHPs to support translation of AAV9 RNAi therapy for CMT1A

doi: 10.1016/j.omtn.2026.102881

Figure Lengend Snippet: Reduction of PMP22 protein demonstrates target engagement of miR871 in NHPs (A) Representative western blots using extracts from one side of the 12-week sciatic distal nerves showed PMP22 protein reduction in treated NHP nerves while MPZ was unaffected. Beta actin served as the loading control. (B) Quantification of all sciatic tissues demonstrated an average reduction of 16%–53% from normal PMP22 levels. (C) Similar PMP22 reduction is evident across all time points in both the upper and lower extremity nerves. Data are displayed as means with error bars representing SEM. n = 4–8 nerve segments per tissue designation from either two control or four treated animals per dosing group. One segment from the right nerve and one from the left nerve were analyzed per animal. SN = sciatic nerve, FN = femoral nerve, UN = ulnar nerve, MN = median nerve, P = proximal, D = distal. ∗ No sig except 12wk UN-D ∗.Two-way ANOVA with Tukey’s multiple comparison tests represented as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Immunoblots were incubated with rabbit antisera hu PMP22 (1:500; Abcam, ab90782), mu/hu PMP22 (1:500; Novus, NBP2-67068), against EGFP (1:1,000; Abcam, ab6556), and mouse β-tubulin (1:4,000; Developmental Studies Hybridoma Bank, ε-7) at 4°C overnight.

Techniques: Drug discovery, Western Blot, Control, Comparison

Journal: Biomaterials

Article Title: A comparative study assessing neural recording quality and inflammatory tissue response between stiff and flexible microelectrode arrays

doi: 10.1016/j.biomaterials.2025.123929

Figure Lengend Snippet: Heightened piezo1 expression around the stiff implant. A) Representative images showing Piezo1 expression around the flexible (left) and stiff (right) implant site (*). B) Quantification of normalized intensity as a function of distance from the implant (μm). C) Normalized intensity averaged across the first 50 μm from the implant site. 19 images for the flexible and 22 images for the stiff group were analyzed. D) Representative images showing Piezo1 (cyan), Iba-1 (green), GFAP (red), and merged expression around a stiff implant, with the zoomed inset with DAPI (blue) overlay. E) Correlation of total piezo1 expression with total Iba-1 expression per image for flexible (left, blue) and stiff (right, red) groups. The slope is significant for the stiff group, indicating a strong correlation between Piezo1 expression and Iba-1 intensity. F) Correlation of total piezo1 expression with total GFAP expression per image for flexible (left, blue) and stiff (right, red). 4 animals were implanted in each group. C) Two-way ANOVA with Sidak’s multiple comparison test was used, E-F) Data was normalized per image and the mean set to 0. A simple linear regression was performed, dashed lines indicate 95 % confidence intervals, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The scale bar is 100 μm in A) and D) and 50 μm in the zoomed inset in D). C) Data are mean ± SEM. E-F) Data is sum.

Article Snippet: Antibodies to visualize astrocytes (GFAP, 1:500, Z033401 Dako), macrophage/microglia IBA-1(microglia, 1:500, NC9288364 Fisher), Piezo1 mechanosensitive ion channels (Piezo1,1:100, NBP275617 Novus Biologicals), neuronal nuclei (NeuN, 1:750, CAD69 Abcam), and neural filament (NF200, 1:250 MAB5256 Millipore) were used.

Techniques: Expressing, Comparison

Journal: Biomaterials

Article Title: A comparative study assessing neural recording quality and inflammatory tissue response between stiff and flexible microelectrode arrays

doi: 10.1016/j.biomaterials.2025.123929

Figure Lengend Snippet: Cell-specific intensity analysis. A) Microglia mask of a sample image using Iba-1. Black portions represent the extracted cell structures. This mask was used to identify the sample microglia shown in B). B) Piezo1 (blue) stained image showing activated microglia expressing Piezo1. C) Average within-microglia Piezo1 intensity with distance from the probe center. D) Average within-microglia Iba-1 intensity with distance from the probe center. E) Astrocyte mask of the above image using GFAP. Black portions represent the extracted cell structures. This mask was used to identify the sample astrocyte processes shown in F). F) Piezo1 (blue) stained image showing astrocyte processes expressing Piezo1. G) Average within-astrocyte Piezo1 intensity with distance from the probe center. H) Average within-astrocyte GFAP intensity with distance from the probe center. For all microglia and astrocyte analysis, the increase in intensity with proximity to the injury is highly significant for both probe substrates, and the cells surrounding stiff probes have higher intensity expression than the flexible. I) Piezo1 (red) stained image overlayed with NeuN ROIs produced by Cellpose. J) Piezo1 (red) stained image merged with NeuN (green). K) Average within-neuron Piezo1 intensity with distance from the probe center. Sample DAPI (white) stained image for a L) stiff and M) flexible probe implant. N) Average DAPI circularity index for all images analyzed above. Bar plot is the same data, but binned into two large bins rather than 10 evenly spaced bins. For A) and E), DAPI is overlayed on the mask. DAPI that passes through masked cell structures is seen as red, while DAPI covered by the mask is seen as yellow. Scale bar is 100 μm and 50 μm in zoomed insets in M&N. * Indicates implantation site. For comparisons between stiff and flexible, a two-way ANOVA with Sidak’s multiple comparison test was used. For correlation of intensity with distance from the probe, a Spearman’s r correlation test was used due to the non-normality of many of the datasets, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data is mean ± SEM.

Article Snippet: Antibodies to visualize astrocytes (GFAP, 1:500, Z033401 Dako), macrophage/microglia IBA-1(microglia, 1:500, NC9288364 Fisher), Piezo1 mechanosensitive ion channels (Piezo1,1:100, NBP275617 Novus Biologicals), neuronal nuclei (NeuN, 1:750, CAD69 Abcam), and neural filament (NF200, 1:250 MAB5256 Millipore) were used.

Techniques: Staining, Expressing, Produced, Comparison

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: International Dental Journal

Article Title: A Novel Nonsense Variant in TBX15 Transcription Factor Suggests an Expanded Genetic Spectrum Of Submucous Cleft Palate

doi: 10.1016/j.identj.2026.109520

Figure Lengend Snippet: Pedigrees and facial photographs of the proband. (A) Clinical features of submucous cleft palate. The black upward arrow indicates the triad of a bifid uvula, zona pellucida, and a notch in the posterior surface of the hard palate. (B) Pedigree of the family with inherited TBX15 pathogenic variants. Solid symbols indicate affected family members, and the proband is marked with a black upward arrow. Co-segregation analysis for the family members are shown in the pedigree. Solid symbols indicate affected family members, and the proband is marked with a black upward arrow. (C) Sanger-sequencing chromatograms for the family members. The red arrow indicates the mutation site. (D) Schematic representation of the variant in TBX15 is shown. Domains/motifs in TBX15 are indicated with different colored squares. Cross-species alignment of amino acid sequences of TBX15 shows a high level of conservation in this variant. The mutated residue within the TBX15 proteins is highlighted.

Article Snippet: The tissues were blocked with blocking reagent for 30 min and incubated with the primary antibodies: TBX15 (NBP2-49036, Novus; diluted 1:200), OSR2(NBP3-12328, Novus; diluted 1:200), COL1A1 (ab270993, Abcam; diluted 1:200), MYOD(BM5759, Boster, diluted 1:50), OCN(PB1008, Boster, diluted 1:50).

Techniques: Sequencing, Mutagenesis, Variant Assay, Residue

Journal: International Dental Journal

Article Title: A Novel Nonsense Variant in TBX15 Transcription Factor Suggests an Expanded Genetic Spectrum Of Submucous Cleft Palate

doi: 10.1016/j.identj.2026.109520

Figure Lengend Snippet: Genotypic features and relative transcription activity of TBX15 variant. (A) Schematic representation of the TBX15 gene-disruptive variant. Three-dimensional structure modeling of wild-type TBX15 and the substitution variant (c.C1231T, p.Gln411*) was performed using the AlphaFold Protein Structure Database. (B) Intracellular distribution of mutant TBX15 proteins. The cellular nucleus was visualized by DAPI (blue). The wild‐type TBX15 protein (GFP‐tagged) localized exclusively within the cell nucleus (green signal fused with blue signal), while the mutated TBX15 (GFP‐tagged; c.C1231T, p.Gln411*) was localized at both cell nucleus and extra‐nuclear regions. Co-transfection of a control vector and a GFP‐tagged mutant TBX15 vector was performed in a 293T cell line to mimic the heterozygous state. Quantification of fluorescence intensity revealed a significant reduction in the nuclear/cytoplasmic ratio for mutant TBX15 compared to wild-type ( p < .001). (C) The variant identified in the SMCP patients disrupt the promoter activities of TBX15. The 293T cells were transfected with 0.2 μg TBX15 expression plasmids ( pCMV-TBX15-wt, pCMV-TBX15-C1231T ) respectively as indicated. The Tbx15 promoter activity was significantly decreased when was co-transfected with pCMV-TBX15-C1231T and pGL3-TBX-promoter c ompared to co-transfected with pCMV-TBX15-wt . The pGL3-basic vector served as a negative control, and the pRL-TK plasmid was employed as an internal control for transfection efficiency. The relative luciferase activities of the TBX15 -luciferase plasmids were determined by luciferase assay. Luciferase activity was measured 48 h post transfection. All transfection experiments were performed in triplicate. The relative luciferase activity was 1.7 ± 0.12 for wild-type TBX15, compared to 0.63 ± 0.05 for the mutant ( p < .01)

Article Snippet: The tissues were blocked with blocking reagent for 30 min and incubated with the primary antibodies: TBX15 (NBP2-49036, Novus; diluted 1:200), OSR2(NBP3-12328, Novus; diluted 1:200), COL1A1 (ab270993, Abcam; diluted 1:200), MYOD(BM5759, Boster, diluted 1:50), OCN(PB1008, Boster, diluted 1:50).

Techniques: Activity Assay, Variant Assay, Mutagenesis, Cotransfection, Control, Plasmid Preparation, Fluorescence, Transfection, Expressing, Negative Control, Luciferase

Journal: International Dental Journal

Article Title: A Novel Nonsense Variant in TBX15 Transcription Factor Suggests an Expanded Genetic Spectrum Of Submucous Cleft Palate

doi: 10.1016/j.identj.2026.109520

Figure Lengend Snippet: Single-nucleus RNA-seq experiments revealed spatiotemporal expression variations of TBX15 during palatogenesis. (A) Uniform Manifold Approximation and Projection (UMAP) labeled by initial cluster assignment per cell, from Seurat analysis of ∼8,755 mouse palate cells from E13.5, E14.5 and E15.5. (B) Bubble plot showing the expression of marker genes of each cell cluster. The size of bubbles indicates the proportion of cells within a cell population that express a given gene. The color indicates the average expression of each gene. (C) Feature plot showing Tbx15 expression during palate development. (D) UMAP shows the distribution of mouse palate cells from E13.5, E14.5, and E15.5. red for E13.5, green for E14.5, and blue for E15.5. (E) UMAP plot showing Tbx15 expression from E13.5, E14.5 and E15.5 during palate development. Statistical significance was assessed by Wilcoxon rank-sum test with Benjamini-Hochberg correction. (F) Double immunofluorescence staining of TBX15 (green) and MyoD (red) at E15.5 revealed TBX15 localization in the fused palatal shelves during palate development. p, palatal shelf; t, tongue. Scale bar, 50 μm.

Article Snippet: The tissues were blocked with blocking reagent for 30 min and incubated with the primary antibodies: TBX15 (NBP2-49036, Novus; diluted 1:200), OSR2(NBP3-12328, Novus; diluted 1:200), COL1A1 (ab270993, Abcam; diluted 1:200), MYOD(BM5759, Boster, diluted 1:50), OCN(PB1008, Boster, diluted 1:50).

Techniques: RNA Sequencing, Expressing, Labeling, Marker, Double Immunofluorescence Staining

Journal: International Dental Journal

Article Title: A Novel Nonsense Variant in TBX15 Transcription Factor Suggests an Expanded Genetic Spectrum Of Submucous Cleft Palate

doi: 10.1016/j.identj.2026.109520

Figure Lengend Snippet: A TBX15-enriched sub-cluster is transcriptionally distinct from other mesenchymal cell populations in the developing palate. (A) UMAP plots showing mesenchymal cell subclusters and sample conditions. The initial UMAP projection was generated by re-clustering isolated mesenchymal cells. (B) Dot plot displaying the expression of highly expressed genes across 12 cell subclusters. The size of each circle corresponds to the percentage of cells in the subtype expressing the gene, and the color indicates the average expression. (C) UMAP plot showing Tbx15 expression within mesenchymal cells subclusters. Tbx15 expression is enriched in Tbx15+ mesenchyme compared to all other cell types in the secondary palate snRNA-seq dataset. (D) UMAP plot showing the expression of Tbx15 in mouse palate from E13.5, E14.5 and E15.5. (E1-F8) Comparison of patterns of expression of TBX15 and OSR2 in the mice embryos at E13.5. Immunofluorescence images shows oral views of the palatal shelves. (F1-F8) Comparison of patterns of expression of TBX15 and COL1A1 in the mice embryos at E13.5. Immunofluorescence images shows oral views of the palatal shelves. The white boxes indicate regions shown at higher magnification in the insets. p, palatal shelf. Scale bar, 50 μm.

Article Snippet: The tissues were blocked with blocking reagent for 30 min and incubated with the primary antibodies: TBX15 (NBP2-49036, Novus; diluted 1:200), OSR2(NBP3-12328, Novus; diluted 1:200), COL1A1 (ab270993, Abcam; diluted 1:200), MYOD(BM5759, Boster, diluted 1:50), OCN(PB1008, Boster, diluted 1:50).

Techniques: Generated, Isolation, Expressing, Comparison, Immunofluorescence

Journal: International Dental Journal

Article Title: A Novel Nonsense Variant in TBX15 Transcription Factor Suggests an Expanded Genetic Spectrum Of Submucous Cleft Palate

doi: 10.1016/j.identj.2026.109520

Figure Lengend Snippet: TBX15 colocalizes with OCN in the developing mouse palate and activates BGLAP transcription. Double immunofluorescence staining of TBX15 (A1-F1) and OCN (A2-F2) at E15.5 reveals TBX15 is localization in the anterior and middle palate during palate development. Nuclei were visualized by DAPI staining in the palate shelves (A3-F3). White boxes indicate regions shown at higher magnification in the insets. p, palatal shelf; t, tongue. Scale bar, 50 μm. (G)TBX15 and TBX22 activate the BGLAP promoter in luciferase reporter assays. Schematic representation of the promoter reporter constructs used in this study. The regions 1 kb upstream of the transcription start sites of human COL1A1 and BGLAP were cloned into the pGL3-basic vector. 293T cells were co-transfected with the indicated reporter constructs and either TBX15 expression plasmid (pCMV3.1-TBX15), TBX22 expression plasmid (pCMV3.1-TBX22), or empty vector control. Firefly luciferase activity was normalized to Renilla luciferase activity and expressed relative to the empty vector control for each promoter. Data are presented as mean ± SD from 3 independent experiments, each performed in triplicate. ** p < .01 by one-way ANOVA with Tukey's post hoc test.

Article Snippet: The tissues were blocked with blocking reagent for 30 min and incubated with the primary antibodies: TBX15 (NBP2-49036, Novus; diluted 1:200), OSR2(NBP3-12328, Novus; diluted 1:200), COL1A1 (ab270993, Abcam; diluted 1:200), MYOD(BM5759, Boster, diluted 1:50), OCN(PB1008, Boster, diluted 1:50).

Techniques: Double Immunofluorescence Staining, Staining, Luciferase, Construct, Clone Assay, Plasmid Preparation, Transfection, Expressing, Control, Activity Assay

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: 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