immunofluorescence staining with orm1  (Proteintech)

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 levels are elevated in both BALF and lung tissues of LPS‐induced ARDS Rats. (A, D, E) ELISA analysis of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) from control and LPS‐treated rats at 24 h post‐induction. (B, E, H) qRT‐PCR analysis of ORM1, TF, and PAI‐1 mRNA expression levels in lung tissues of control and LPS‐treated rats at 24 h post‐induction. (J) Western blot analysis of ORM1, TF, and PAI‐1 protein expression in lung tissues of LPS‐induced ARDS rats at 24 h post‐induction, with quantitative densitometry shown in (G, C, I). (K) Immunofluorescence co‐staining for ORM1 (red) and alveolar type II cell marker surfactant protein C (SP‐C, green). Yellow signal (white arrow) indicates ORM1/SP‐C co‐localization. Scale bar: 300 μm. Data represent mean ± SD ( n = 6 per group). Significance in (A–J) was calculated using an independent t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Downregulation of ORM1 Significantly Mitigates LPS‐Induced Lung Injury in ARDS Rats. (A) Hematoxylin and eosin (H&E) staining revealssubstantial histopathological damage in lung tissues at 24 h post‐LPS induction and significant attenuation of LPS‐induced pulmonary injury following ORM1 knockdown. Scale bars: 200 μm. (B) Histopathological injury scores corresponding to H&E staining for lung injury assessment. (C) Pulmonary edema assessed by lung wet/dry weight ratio. Data represent mean ± SD ( n = 6). Significance in (B) and (C) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Staining, Knockdown, Control

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Suppression Ameliorates Alveolar Hypercoagulation and Fibrinolysis Inhibition In Vivo. (A–C) ELISA quantification of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) showing: Significant elevation of all three biomarkers at 24 h post‐LPS airway nebulization inhalation compared to controls, and marked reduction in their levels following ORM1 knockdown. (D–F) qRT‐PCR analysis reveals: Significant elevation of ORM1, TF, and PAI‐1 mRNA levels in LPS‐induced ARDS lung tissues compared to control, Marked downregulation of these transcripts following ORM1 knockdown. (J) Representative Western blots showing upregulated ORM1, TF, and PAI‐1 protein expression in lung tissues at 24 h post‐LPS induction, with significant reduction following ORM1 knockdown, Quantitative densitometric analysis of (G) ORM1, (H) TF, and (I) PAI‐1 protein levels normalized to GAPDH. (K) Collagen III deposition in lung parenchyma visualized by immunohistochemistry (brown staining indicates positive expression). Scale bars: 300 μm. Data represent mean ± SD ( n = 6), Significance in (A–I) were calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Inhibition, In Vivo, Enzyme-linked Immunosorbent Assay, Knockdown, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances NF‐κB Signaling Pathway Activation In Vivo. (A) The Western blot method was used to detect the protein expression levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 in the lung tissues of rats in each group, with quantitative densitometry showing the expression levels of total IKKβ (B) and total p65 (D) in the lung tissues of each group of rats were not statistically significant, and the expression levels of phosphorylated IKKβ (C) and phosphorylated p65 (E) in the lung tissues of rats induced by LPS increased, while those in the lung tissues of rats induced by LPS decreased after knockdown of ORM1. Data represent mean ± SD ( n = 6). Significance in (B–E) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: Not significant ( p > 0.05).

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Promotes TF and PAI‐1 Expression in LPS‐Stimulated AEC II Cells. (A–C) qRT‐PCR analysis showed that, compared with the control group, the mRNA levels of ORM1, TF, and PAI‐1 in AEC II cells induced by LPS for 24 h were significantly increased, and the levels of these indicators were down‐regulated after the knockdown of ORM1. (D) Representative Western blot results showed that the protein expressions of ORM1, TF, and PAI‐1 in AEC II cells were upregulated after 24 h of LPS induction and significantly decreased after the knockdown of ORM1. Quantitative density analysis normalized the protein levels of (E) ORM1, (F) TF, and (G) PAI‐1 to GAPDH. Data represent mean ± SD ( n = 6). Significance in (A–C, E–G) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Western Blot

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances TF and PAI‐1 expression maybe through the NF‐κB pathway. (A) Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 were assessed using Western Blot analysis, followed by semiquantitative analysis (B–E) after ORM1 knockdown. Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), total p65, TF, and PAI‐1 were assessed using Western Blot analysis (F), followed by semiquantitative analysis (G–N) after NF‐κB inhibitor benzoxathiole treatment. Data represent mean ± SD ( n = 3). Significance in (B–E, G–N) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns: Not significant ( p > 0.05).

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Expressing, Western Blot, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Directly Activates the NF‐κB Pathway in LPS‐Exposed AEC II Cells. (A) Cell immunofluorescence co‐staining of ORM1 (red) and phosphorylated IKKβ (p‐IKKβ, green). The yellow signal (white arrow) indicates the co‐localization of ORM1/p‐IKKβ. After 24 h of LPS induction, the expression and co‐expression of ORM1 and p‐IKKβ in AEC II cells increased. After knockout of ORM1, In LPS‐induced AECII cells, the expressions of ORM1, p‐IKKβ and their co‐expressions were all decreased. (B) Co‐expression of ORM1 and p‐IKKβ in LPS‐Induced AEC II cells following NF‐κB pathway inhibitor (benzoxathiole) treatment were decreased. The yellow areas, indicated by white arrows, represent regions of co‐localization between ORM1 and p‐IKKβ (scale bar: 30 μm). (C and D) The immunoprecipitation results show that in LPS‐induced AEC II cells, ORM1 interacts with p‐IKKB.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Immunofluorescence, Staining, Expressing, Knock-Out, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Concentrations of ORM1 in BALF Are Significantly Elevated in Patients with ARDS. (A) ORM1, (B) TF, and (C) PAI‐1 concentrations in BALF from patients with ARDS ( n = 28) and non‐ARDS ( n = 26) were measured using specific human ORM1, TF, and PAI‐1 enzyme‐linked immunosorbent assay (ELISA) kits. (D) Oxygenation index, expressed as the ratio of arterial oxygen partial pressure to oxygen concentration (P/F), in ARDS patients ( n = 28) and non‐ARDS patients ( n = 26). Correlations between BALF concentrations of ORM1 and those of (E) TF, (F) PAI‐1, and (G) P/F ratio in patients with ARDS are shown. Data represent mean ± SD. Significance in (A–D) was calculated using an independent t test; significance in (A–D) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test, and (E–G) were evaluated with Pearson's correlation coefficients. **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay