Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: Constitutive iNOS expression in ovarian cancer cells (A) mRNA expression levels of iNOS across an ovarian cancer cell lines from the CCLE database. (B) Assessment of the relative NOS2 mRNA expression in ovarian cancer cell lines from the NCI-60 database. (C) Protein expression levels of iNOS in ovarian cancer cells were determined by western blot. (D) RT qPCR analyses of NOS2 mRNA in ovarian cancer cell lines. (E) Constitutive protein expression of iNOS is maintained regardless of stimulation with LPS or a cytokine mix in OVCAR8 and A2780 cells. (F) Inducible expression of iNOS upon cytokine stimulation in A2780 wild-type cells lacks basal expression. Experiments were performed in biological duplicate or triplicate.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS increases sensitivity to cisplatin in ovarian cancer (A) The cell viability of OVCAR8 cells was assessed using RealTime-Glo MT Cell Viability Assay after culturing in increasing concentrations of cisplatin for 72 h. (B) OVCAR8 cells incubated with or without L-NMMA for 48 h, measured by RealTime-Glo MT Cell Viability Assay. (C) Cell viability after either no treatment (control), cisplatin, and a combination of cisplatin and L-NMMA by RealTime-Glo MT Cell Viability Assay. (D) Western blot analysis of vimentin protein expression in ovarian cancer cell lines. (E) Ovarian cancer cell lines were analyzed for VIM mRNA levels by qPCR. (F) OVCAR8 cells were treated with or without L-NMMA (4, 6, 8, and 10 mM) for 48 h, and western blot was used to analyze the effect of L-NMMA on the vimentin protein expression. B-actin was used as a loading control. Band densities were quantified using ImageJ analysis. Error bars, SEM. ∗ p < 0.05; ∗∗ p < 0.01 (compared with the control group, using two-way ANOVA). All experiments were independently repeated three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Viability Assay, Incubation, Control, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS knockout increased chemosensitivity and impaired cell motility and migration of ovarian cancer cells (A) Immunoblotting showing reduced iNOS (left) and vimentin (right) expression levels following NOS2 knockdown in the OVCAR8 cells. (B) NOS2 knockdown sensitized OVCAR8 cells to cisplatin, reducing its IC 50 value. Log-logistic model was used to analyze the data. The group comparison between control group and KO-1 had a p value = 8.98e-07. The comparison between control and KO-2 had a p value = 0.0132. The detected EC50 for control group was 0.8554, for KO-1 was 0.6037, for KO2 was 0.7688. (C and D) Analysis of reduced protein (left) and mRNA (right) expression of iNOS and vimentin following siRNA transfection in OVCAR8 and A2780cis cells for 72 h, assessed by western blot and RT-qPCR. (E) OVCAR8 cells were transfected with 2 different siRNAs or the scramble siRNA and were assessed for migration using the scratch wound assay. The area of the wound was measured at 0, 12, 24, and 36 h by the IncuCyte live-cell analysis system. Two-way repeated measures ANOVA was used to analyze the data. After 6 h, siRNA1 had p value = 1, siRNA2 had p value = 0.23. After 12 h, siRNA1 had p value = 0.71 and siRNA 2 had p value = 0.16. After 24 h, siRNA1 had a p value = 0.39 and siRNA2 had a p value = 0.05. After 36 h, siRNA1 had a p value = 0.86 and siRNA2 had a p value = 0.04. (F) NOS2 KO-1 and KO-2 OVCAR8 cells formed significantly fewer colonies compared to parental OVCAR8. The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. (G) Silencing of NOS2 by two different siRNAs significantly reduced the number of colonies formed by OVCAR8 cells. Clonogenic growth was measured after 10 days, quantified using ImageJ, and represented as a bar graph (mean ± SEM). The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. All experiments were independently repeated two to three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Knock-Out, Migration, Western Blot, Expressing, Knockdown, Comparison, Control, Transfection, Quantitative RT-PCR, Scratch Wound Assay Assay, Cell Analysis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: Knockdown of NOS2 impaired the expression of genes involved in epithelial-to-mesenchymal transition (A) Heatmap showing genes whose expression differed significantly between NOS2 KO-1 and parental OVCAR8 cells. Top 15 altered genes are presented; red indicates upregulated genes, while green indicates downregulated genes. (B) Volcano plot showing the genes that altered significantly between NOS2 knockdown OVCAR8 and parental cells based on RNA-seq analysis. (C) Bubble plot of KEGG enrichment terms based on RNA-seq results showing enrichment of pathways related to wound healing, extracellular matrix organization, and regulation of cell shape. All the experiments were performed in triplicates.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Knockdown, Expressing, RNA Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: L-NMMA promotes vimentin destabilization by enhancing its ubiquitination (A) CHX chase assay showing vimentin protein levels in OVCAR8 cells treated with 10 mM L-NMMA at different time points. (B) Vimentin ubiquitination was assessed in OVCAR8 cells treated with L-NMMA for 24 h, in the presence of the proteasome inhibitor MG-132 (10 μM) for the final 4 h, followed by immunoprecipitation and western blot using an anti-ubiquitin antibody. (C) Measurement of vimentin S-nitrosylation levels was performed by immunoprecipitation in OVCAR8 cells. (D) Western blot analysis of vimentin expression in OVCAR8 cells treated with 10 mM L-NMMA alone or in combination with MG132 at 1, 5, or 10 μM. (E) Tumor growth and proliferation were monitored in mice bearing parental and NOS2 KO OVCAR8 tumors, evaluated by ROI measurements every 4 days ( n = 6). (F) Kaplan-Meier survival curves of mice bearing parental and NOS2 KO OVCAR8 tumors following cisplatin treatment ( n = 6). Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA for growth curves and the Kaplan-Meier method for survival analysis ( p < 0.05, ∗ p < 0.01). Experiments were performed in duplicate or triplicate.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing

Journal: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: Bioactive Materials

Article Title: Skin-mimetic bilayer hydrogel normalizes diabetic wound healing by orchestrating inflammatory cell dynamics: An early intervention strategy

doi: 10.1016/j.bioactmat.2026.02.025

Figure Lengend Snippet: In vitro assay of inflammation cell modulation under stimulation of SP-loaded Gel/HA and IL-10-loaded Ker/Cu. a Schematic of neutrophil migration test using a transwell system after treatment with the leaching solution of SP@Gel/HA. Gel/HA and blank culture medium were set for comparison. b Wright-Giemsa staining of HL-60 cells before and after differentiation. c Photograph of dHL-60 cells migrating to the lower chamber. d Quantitative analysis of neutrophil migration after treatments with SP@Gel/HA and Gel/HA. Untreated group serves as a control. e Schematic of macrophage polarization after treatment with LPS, IL-10, or IL-10/LPS. f Representative fluorescence images of macrophages after different treatment. Red: iNOS (M1 marker); Green: CD163 (M2c marker); Blue: DAPI (nuclear staining). g Schematic of macrophage efferocytosis test toward apoptotic dHL-60 cells under different treatments. h Flow cytometry plots of dHL-60 cells before and after apoptosis induction. i Representative fluorescent images of macrophage efferocytosis toward apoptotic dHL-60 cells under different treatments. Macrophages and apoptotic cells were stained green and red, respectively. All data were generated from at least three independent experiments and presented as the means ± standard deviation. Statistical analysis was performed by one-way ANOVA. ns, not significant; ∗∗∗∗p < 0.0001.

Article Snippet: After another 48 h, macrophage cells were harvested and stained with antibodies against human iNOS (Servicebio, GB11119) and CD163 (Abcam, ab182422) for 30 min. Fluorescence images were acquired using fluorescence microscope.

Techniques: In Vitro, Migration, Comparison, Staining, Control, Fluorescence, Marker, Flow Cytometry, Generated, Standard Deviation

Journal: Bioactive Materials

Article Title: Skin-mimetic bilayer hydrogel normalizes diabetic wound healing by orchestrating inflammatory cell dynamics: An early intervention strategy

doi: 10.1016/j.bioactmat.2026.02.025

Figure Lengend Snippet: Bilayer hydrogel orchestrates inflammatory cell dynamics during the early inflammation phase of diabetic wound healing. a Experimental timeline for assay of early neutrophil recruitment. b Immunohistochemical staining for Ly-6G in wounds at 8 h, 1 d and 3 d after injury. Diabetic wounds were treated with SP/IL-10@Bilayer, SP@Bilayer, IL-10@Bilayer, and saline solution (Model), respectively. Healthy mice treated with saline solution were set as Normal. c Quantitative analysis of Ly-6G + cells in each group. d Relative expression of CXCL-1 on day 1. e Relative expression of MCP-1 on day 1. f Experimental timeline for assay of M1 macrophage infiltration. g Immunofluorescence staining for iNOS in wounds on days 1, 3 and 6 after injury. h Quantitative analysis of iNOS + cells in each group. i-k Relative expressions of macrophage-associated pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 on day 3. l Schematic illustrating the dynamic modulation of inflammatory cells during the early inflammation phase of diabetic wounds by SP/IL-10@Bilayer. All data were generated from at least three independent experiments and presented as the means ± standard deviation. Statistical analysis was performed by one-way ANOVA. # means significant difference compared to the normal group. #p < 0.05, ##p < 0.01 and ###p < 0.001; ∗ means significant difference compared to the model group. ∗p < 0.05; & means significant difference compared to SP/IL-10@Bilayer. & p < 0.05 and && p < 0.01.

Article Snippet: After another 48 h, macrophage cells were harvested and stained with antibodies against human iNOS (Servicebio, GB11119) and CD163 (Abcam, ab182422) for 30 min. Fluorescence images were acquired using fluorescence microscope.

Techniques: Immunohistochemical staining, Staining, Saline, Expressing, Immunofluorescence, Generated, Standard Deviation

Journal: Bioactive Materials

Article Title: Skin-mimetic bilayer hydrogel normalizes diabetic wound healing by orchestrating inflammatory cell dynamics: An early intervention strategy

doi: 10.1016/j.bioactmat.2026.02.025

Figure Lengend Snippet: In vitro assay of inflammation cell modulation under stimulation of SP-loaded Gel/HA and IL-10-loaded Ker/Cu. a Schematic of neutrophil migration test using a transwell system after treatment with the leaching solution of SP@Gel/HA. Gel/HA and blank culture medium were set for comparison. b Wright-Giemsa staining of HL-60 cells before and after differentiation. c Photograph of dHL-60 cells migrating to the lower chamber. d Quantitative analysis of neutrophil migration after treatments with SP@Gel/HA and Gel/HA. Untreated group serves as a control. e Schematic of macrophage polarization after treatment with LPS, IL-10, or IL-10/LPS. f Representative fluorescence images of macrophages after different treatment. Red: iNOS (M1 marker); Green: CD163 (M2c marker); Blue: DAPI (nuclear staining). g Schematic of macrophage efferocytosis test toward apoptotic dHL-60 cells under different treatments. h Flow cytometry plots of dHL-60 cells before and after apoptosis induction. i Representative fluorescent images of macrophage efferocytosis toward apoptotic dHL-60 cells under different treatments. Macrophages and apoptotic cells were stained green and red, respectively. All data were generated from at least three independent experiments and presented as the means ± standard deviation. Statistical analysis was performed by one-way ANOVA. ns, not significant; ∗∗∗∗p < 0.0001.

Article Snippet: The infiltration of pro-inflammatory (M1) macrophages and polarization of M2c macrophages were analyzed by immunofluorescence staining using antibodies against iNOS (Servicebio, GB11119) and CD163 (Servicebio, GB14027), respectively.

Techniques: In Vitro, Migration, Comparison, Staining, Control, Fluorescence, Marker, Flow Cytometry, Generated, Standard Deviation

Journal: Bioactive Materials

Article Title: Skin-mimetic bilayer hydrogel normalizes diabetic wound healing by orchestrating inflammatory cell dynamics: An early intervention strategy

doi: 10.1016/j.bioactmat.2026.02.025

Figure Lengend Snippet: Bilayer hydrogel orchestrates inflammatory cell dynamics during the early inflammation phase of diabetic wound healing. a Experimental timeline for assay of early neutrophil recruitment. b Immunohistochemical staining for Ly-6G in wounds at 8 h, 1 d and 3 d after injury. Diabetic wounds were treated with SP/IL-10@Bilayer, SP@Bilayer, IL-10@Bilayer, and saline solution (Model), respectively. Healthy mice treated with saline solution were set as Normal. c Quantitative analysis of Ly-6G + cells in each group. d Relative expression of CXCL-1 on day 1. e Relative expression of MCP-1 on day 1. f Experimental timeline for assay of M1 macrophage infiltration. g Immunofluorescence staining for iNOS in wounds on days 1, 3 and 6 after injury. h Quantitative analysis of iNOS + cells in each group. i-k Relative expressions of macrophage-associated pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 on day 3. l Schematic illustrating the dynamic modulation of inflammatory cells during the early inflammation phase of diabetic wounds by SP/IL-10@Bilayer. All data were generated from at least three independent experiments and presented as the means ± standard deviation. Statistical analysis was performed by one-way ANOVA. # means significant difference compared to the normal group. #p < 0.05, ##p < 0.01 and ###p < 0.001; ∗ means significant difference compared to the model group. ∗p < 0.05; & means significant difference compared to SP/IL-10@Bilayer. & p < 0.05 and && p < 0.01.

Article Snippet: The infiltration of pro-inflammatory (M1) macrophages and polarization of M2c macrophages were analyzed by immunofluorescence staining using antibodies against iNOS (Servicebio, GB11119) and CD163 (Servicebio, GB14027), respectively.

Techniques: Immunohistochemical staining, Staining, Saline, Expressing, Immunofluorescence, Generated, Standard Deviation