Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: NAD metabolism is disrupted in tendinopathic tendons in vivo . a . A schematic diagram of the harvested region of human Achilles tendons. (The mid-portion of Achilles tendon) and the gross image of healthy and AT tendons. b. NAD + /NADH ratio and quantification of NAD metabolism (including, NAD + , NADH, total NAD pool) in human Achilles tendons. c. Quantification of ATP production. d. GSEA revealing top NAD metabolism-related GO terms enriched in human Achilles tendons. e. GSEA revealing top degeneration-related GO terms enriched in human Achilles tendon. f. Images of H&E staining, AB (Alcian blue) staining and Masson staining of human normal tendons and tendinopathic tendons. Bonar scores. g. Immunohistochemical staining of COL1 and COL3. h. Immunohistochemical staining of PARP and NOX4. i. Quantitative analysis of COL1, COL3, PARP, NOX4, and ratio of COL3/COL1. j. Immunohistochemical staining and quantification of TUNEL. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: In Vivo, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB corrects NAD dysmetabolism and matrix degeneration in the rat tendinopathy model. a . A schematic illustration of the rat tendinopathy model. b. Gross image of the Rat AT model. c. Gait analysis results for the different groups. The blue dotted line represents stride length; the red dotted line represents step length. Blue print: forepaw; red print: hind paw. d. Quantification of stride length, step length, and the length of the front/rear paw prints. e. Hot plate test. f. Paw contraction thresholds were assessed utilizing von Frey fibers to gauge mechanical sensitivity (n = 6). g. Quantification of ATP in the rat AT model. h. NAD + /NADH ratio i. Quantification of NAD metabolism (including, NAD + , NADH, total NAD pool). j. H&E staining, AB staining and Masson staining. Bonar scores of rats AT model. k. Immunohistochemical staining of Col1 and Col3. l. Immunohistochemical staining of Parp and Nox4. m. Quantitative analysis of Col1, Col3, Parp, Nox4. n. TUNEL staining and quantitative analysis of apoptotic cells. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 5; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Hot Plate Test, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB reduces TBHP-induced NAD metabolism disorder in vitro and in vivo. a . The cell viability. b. Quantitation of NAD metabolism (including NAD + /NADH ratio, NAD + , NADH, total NAD pool) in rat tenocytes. c. Quantitation of ATP in rat tenocytes. d. Representative Western blot results for Col3, Col1, MMP3, and MMP13 in rat tenocytes. e. Ratio of Col3/Col1 by d. f. Representative Western blot results for Parp, c-Parp, Nox4, Bcl2, and Bax. g, h. Immunofluorescence and quantification analysis of Col1, Col3, Parp, and Nox4 expression in rat tenocytes. The ratio of Col1/Col3. DAPI, 4′,6-diamidino-2-phenylindole. i. Fluorescence images of the DCFH-DA probe for hydrogen peroxide in rat tenocytes. j. JC-1 staining in rat tenocytes. k. Quantitative analysis of ROS. l. Quantitative analysis of JC-1. m. Annexin V and activated caspase-3 staining and quantitative analysis of apoptotic cells in rat tenocytes. The results are presented as medians with 95% CIs. n = 3, 5, ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: In Vitro, In Vivo, Quantitation Assay, Western Blot, Immunofluorescence, Expressing, Fluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB alleviates TBHP-induced tendinopathy in human Achilles tendon explant. a . A schematic illustration of the human extra explant. b. Quantification of NAD metabolism in human extra explants under TBHP treatment combined with or without MB. (n = 3) c. H&E staining, AB staining, and Masson staining of human tendon explants. The Bonar scores. d. Quantification of ATP in human tendon explants. e. NFR. f. Immunohistochemical staining of COL1 and COL3. g. Immunohistochemical staining of PARP and NOX4. h. Immunohistochemical staining of AIF and NDUFB8. i. Quantification of the immunohistochemical staining data. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 5; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Staining, Immunohistochemical staining

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB alleviates oxidative stress in a MC1-dependent manner in rat tenocytes. a. Quantification of ATP in rat tenocytes after TBHP treatment combined with/without MB or rotenone (Rot, a complex I inhibitor). b. NFR. c. Quantification of NAD metabolism. d. Representative Western blot results for Parp, c-Parp, Nox4, Aif, Col3, Col1, and Ndufb8. e. Parp, Nox4, Aif expression by immunofluorescence microscopy (n = 3). f. Col3, Col1, Ndufb8 expression by immunofluorescence microscopy. g. Quantification of the immunofluorescence data from e and f . h. JC-1 staining. i. Quantitative analysis of JC-1. j, k. Fluorescence images and quantitative analysis of ROS. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB increases the expression of Aif under Aif knockdown in tenocytes. a. The NAD metabolism after Aif was knocked down in rat tenocytes. b. Quantification of ATP after Aif knockdown. c. NFR. d. Representative Western blot results for Parp, c-Parp, Nox4, Aif, Col3, Col1, and Ndufb8. e. Quantification of the Western blot data from d. f. Quantification of the immunofluorescence staining data . g. Col3, Col1, Parp, Nox4, Ndufb8, and Aif expression by immunofluorescence microscopy; h. Fluorescence images of ROS. i. JC-1 staining. j. Quantification of DCFH-DA staining data. k. Quantification of JC-1 staining data. l. Schematic illustration of the mechanism of MB in tendinopathy. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Expressing, Knockdown, Western Blot, Immunofluorescence, Staining, Microscopy, Fluorescence

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Probiotics Ameliorate Atrial Fibrillation-Associated Biomarkers and Inflammation in Rats via Modulation of the Gut Microbiota and NLRP3 Inflammasome Pathway

doi: 10.5812/ijpr-168612

Figure Lengend Snippet: Probiotics improved atrial fibrillation (AF) by regulating intestinal flora imbalance and the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway in rats. The expression of AF biomarkers pentraxin 3 (PTX3), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), chemerin, and Gal-3 was detected by Western blot (A) and real-time quantitative polymerase chain reaction (RT-qPCR) (B). n = 5; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1 h and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-NLRP3 antibody (68102-1-lg, Proteintech), Anti-caspase-1 antibody (HA722222, Huane Bio), Anti-IL-1β antibody (26048-1-ap, Proteintech), Anti-pentraxin 3 (PTX3) antibody (DF8762, Affinity), Anti-IL-6 antibody (ab9324, abcam), Anti-tumor necrosis factor-alpha (TNF-α) antibody (bs-0549R, Bioss), Anti-chemerin antibody (833550-4-RR, Proteintech), Anti-Galectin-3 antibody (60207-1-Ig, Proteintech), and Anti-GAPDH antibody (AF7021, Affinity).

Techniques: Probiotics, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: KRAS-dependent glycolytic reprogramming of endothelial cells in sporadic arteriovenous malformations

doi: 10.1038/s44321-026-00383-y

Figure Lengend Snippet: Experiments were conducted with IM-HUVEC KRAS WT and KRAS G12V cells (−/+ doxycycline [Dox] induction). ( A ) Representative immunofluorescence (IF) staining and quantification of VE-cadherin (Green). Mean ± standard deviations (SD). One-way ANOVA with Tukey’s post hoc tests. n (independent experiments) = 3 (3–4 fields of view per replicate). Scale bars = 100 μm. ( B ) Fluorescence measurements of 70 kDa FITC transwell leak assay. Two-tailed paired t test. n = 6. ( C ) Representative western blots and quantification of VE-cadherin. Mean ± SD. One-way ANOVA. n = 3. ( D ) Representative spheroid sprouting assay (24 h). Bar graph: mean ± SD. Box and whiskers: min to max. Kruskal–Wallis test with Dunn’s multiple comparisons. n = 5 (5–12 spheroids per condition per replicate). Scale bars = 200 μm. ( E ) Representative Angiogenesis Dot Blot profiling of cell culture media. Significantly changed proteins are highlighted using yellow dotted boxes. n = 3. ( F ) Representative image of wound migration assay (6 h). Yellow dotted lines highlight cell boundaries after migration. Mean ± SD. One-way ANOVA with Tukey’s post hoc tests. n = 4. Scale bars = 200 μm. ( G ) Representative IF staining and intensity quantification of hyaluronic acid binding protein (HABP). Mean ± SD. Two-tailed unpaired t tests (comparing Dox vs No Dox). n = 4. Scale bars = 100 μm. ( H ) Representative IF staining and intensity quantification of fibronectin (FN1). Mean ± SD. Two-tailed unpaired t tests (comparing Dox vs No Dox). n = 4. Scale bars = 50 μm. ( I ) Glycolysis stress test (left) and mitochondrial (mito) stress test (right). SL327 treatment = 2 μM. Mean ± standard error of means (SEM). Selected statistics shown represent significant changes between KRAS G12V +Dox condition compared to all other conditions. One-way ANOVA with Tukey’s post hoc tests. n = 6 (glycolysis test), n = 5 (mito test) and n = 4 for SL327 treatments. In this figure, * P value < 0.02 with exact P values shown in Appendix Table . .

Article Snippet: For each mL of collagen matrix mixture, the mixture contained: 550 μL of methylcellulose solution with 40% FBS + 150 μL of 15.6 mg/mL NaHCO 3 in water + 10 μL of 1 M NaOH + 300 μL of Cultrex 3-D Culture Matrix Rat Collagen I (R&D Systems, Cat #: 3447-020-01).

Techniques: Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Western Blot, Dot Blot, Cell Culture, Migration, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: KRAS-dependent glycolytic reprogramming of endothelial cells in sporadic arteriovenous malformations

doi: 10.1038/s44321-026-00383-y

Figure Lengend Snippet: ( A ) Schematic of co-culturing IM-HUVEC KRAS and IM-HUVEC F-Tractin cells. Example images showing the difference between the F-Tractin and mScarlet-KRAS signals. ( B – G ) Representative IF staining of ( B ) pERK ( n = 3), ( C ) VE-cadherin ( n = 3), ( D ) Fibronectin (FN1) ( n = 3), ( E ) BrdU ( n = 3), ( F ) Ki67 ( n = 3), ( G ) TUNEL ( n = 3) within the co-culture system (−/+ Dox). Time of KRAS induction varies depending on the experiment: (i) proliferation assays (i.e., BrdU, Ki67) = 24 h; (ii) FN1 and TUNEL assays = 3 days; (iii) pERK and VE-cadherin assays = 4 days. White asterisks in ( C ) indicate examples of barrier gaps. Arrows in ( D ) indicate regions under WT ECs that have reduced FN1 levels. ( H ) Representative IF staining of pERK within the co-culture system showing changes to the areas occupied by IM-HUVEC F-Tractin cells after 4 days of co-culture with IM-HUVEC KRAS cells ( n = 3). In all images, “FT” marks regions of IM-HUVEC F-Tractin cells, while “K” marks regions of IM-HUVEC KRAS WT or G12V cells. White solid lines show the boundary between IM-HUVEC KRAS and F-Tractin cells. All scale bars = 100 μm. ( I , J ) Quantifications of cell area ( I ) and circularity ( J ) by tracing VE-cadherin staining images from the same experiments as ( C ). Violin plots with dotted lines show interquartile range. One-way ANOVA with Tukey’s post hoc tests. n = 3 (540 cells were quantified per condition). ( K , L ) Quantifications of proportions of ( K ) BrdU and ( L ) Ki67 positive IM-HUVEC KRAS cells from the same experiments as ( E , F ). Mean ± SD. One-way ANOVA tests. n = 3 (5 fields of view per condition per replicate). ( M ) Quantification of area occupied by IM-HUVEC F-Tractin cells after co-culture with IM-HUVEC KRAS cells (−/+ Dox). Two groups of experiments were conducted: (1) KRAS expression was induced right after seeding (prior to 100% confluency) (left); (2) KRAS expression was induced after the formation of confluent monolayers (right). Mean ± SD. Unpaired, two-tailed t tests. n = 3 (6 fields of view per condition per replicate).

Article Snippet: For each mL of collagen matrix mixture, the mixture contained: 550 μL of methylcellulose solution with 40% FBS + 150 μL of 15.6 mg/mL NaHCO 3 in water + 10 μL of 1 M NaOH + 300 μL of Cultrex 3-D Culture Matrix Rat Collagen I (R&D Systems, Cat #: 3447-020-01).

Techniques: Staining, TUNEL Assay, Co-Culture Assay, Expressing, Two Tailed Test