Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF promotes lung cancer cell malignancy and M2 macrophage polarization. A549 and H520 cells were transfected with control (Ctrl) or EHF-knockdown (KD-EHF) plasmids. (A-B) Western blot and quantification of EHF protein levels in A549 and H520 cells. (C-D) EdU staining and quantification of EdU + cells in A549 and H520 cells, assessing proliferation. (E-H) Glucose uptake and lactate production assays were conducted using commercial kits to evaluate glycolytic output. (I-J) Flow cytometric analysis of apoptosis. (K-L) Transwell assay was employed to examine cell migration. (M − P) THP-1 cells were differentiated into THP-1-M0 macrophages with 100 ng/mL PMA for 24 h. Culture supernatants from Ctrl- or KD-EHF–transfected A549 and H520 cells were collected and co-incubated with THP-1-M0 macrophages. (M − N) Flow cytometric analysis was performed to assess the proportion of CD206 + M2-polarized macrophages. (O–P) ELISA was conducted to quantify the secretion of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the supernatants of THP-1-M0 macrophages. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Transfection, Control, Knockdown, Western Blot, Staining, Transwell Assay, Migration, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomal EHF reprograms THP-1-M0 macrophages to promote lung cancer cell malignancy and angiogenesis via paracrine signaling. (A) Schematic of the paracrine co-culture system: Exosomes from control (Ctrl) or EHF-knockdown (KD-EHF) A549/H520 cells were co-incubated with THP-1-M0 macrophages. The medium from these macrophages was used to treat A549/H520 cells or HUVECs. (B-E) THP-1-M0 macrophages were treated with three conditions: blank control (no exosome), exosomes from control-transfected A549/H520 cells (Exo Ctrl ), or exosomes from EHF-knockdown A549/H520 cells (Exo KD−EHF ). (B–C) Flow cytometry analysis of CD206 + M2 macrophage proportions in THP-1-M0 cells. (D-E) ELISA measurement of M2-associated cytokines (TGF-β1, IL-10, and VEGFA). (F-M) The medium from exosome treated macrophages was then used to incubate A549/H520 cells or HUVECs. (F-G) EdU staining and quantification of EdU + proliferating A549 and H520 cells. (H–I) Flow cytometric analysis of apoptosis in A549 and H520 cells. (J-K) Transwell migration assays and quantification of migrated A549 and H520 cells. (L-M) Tube formation assays and quantification of tube numbers in HUVECs. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Co-Culture Assay, Control, Knockdown, Incubation, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: RNF41 mediates exosomal EHF-driven pro-tumorigenic effects in lung cancer. (A) Schematic of the rescue co-culture system: THP-1-M0 macrophages (control or RNF41-overexpressing) were incubated with exosomes from control (Exo Ctrl ) or EHF-knockdown (Exo KD−EHF ) A549/H520 cells. The medium from these co-cultures was used to treat A549/H520 cells or HUVECs. (B–C) Flow cytometry was used to analyze the proportion of CD206 + M2-polarized macrophages in THP-1-M0 cells. (D-E) ELISA was employed to quantify the levels of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the CM from macrophages. (F-G) EdU assays were carried out to measure the proliferation of A549 and H520 cells. (H–I) Annexin V/PI flow cytometry was used to assess the apoptosis of A549 and H520 cells treated with CM. (J-K) Transwell assays were performed to evaluate the migration of A549 and H520 cells. (L-M) Tube formation assays were conducted on HUVECs treated with CM. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Co-Culture Assay, Control, Incubation, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF knockdown inhibits tumor growth and downregulates M2-associated molecules in v ivo. Nude mice were subcutaneously implanted with Ctrl or KD-EHF A549 cells. (A) Tumor volume was measured every 5 days over 30 days. (B) Tumor weight. (C) Representative images of excised tumors. (D) Western blot analysis of RNF41, CD206, TGF-β1, and VEGFA expression levels in tumor tissues. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Membranes were blocked with 5% skim milk in TBST, incubated with primary antibodies against EHF (1:1000, Catalog #: 27195-1-AP, Proteintech, Wuhan, China), TSG101 (1:1000, Catalog #: 14497-1-AP, Proteintech), Calnexin (1:1000, Catalog #: 10427-2-AP, Proteintech), CD9 (1:2000, Catalog #: 60232-1-Ig, Proteintech), RNF41 (1:500, Catalog #: 17233-1-AP, Proteintech), CD206 (1:1000, Catalog #: 18704-1-AP, Proteintech), TGF-β1 (1:1000, Catalog #: 26155-1-AP, Proteintech), VEGFA (1:1000, Catalog #: ab46154, Abcam, Cambridge, UK), and β-actin (1:1000, Catalog #: 66009-1-Ig, Proteintech) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000, Catalog #: ab6721/ab205719, Abcam) for 1 h at room temperature.

Techniques: Knockdown, Western Blot, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Dual knockdown of Alox15 and TGF-β1 by lipid nanoparticle-delivered siRNA in bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2026.102534

Figure Lengend Snippet: RNA sequencing analysis of an idiopathic pulmonary fibrosis dataset revealed upregulation of ALOX15 and TGF-β1 mRNA compared to controls. RNA sequencing was performed to compare the expression of ALOX15 and TGF-β1 between idiopathic pulmonary fibrosis and normal samples from GSE134692 and GSE150910 datasets.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against Alox15 (ab244205, Abcam), 4-HNE (ab48506, Abcam), TGF-β1 (sc-130348, Santa Cruz Biotechnology, USA), E-cadherin (IR55-180, iReal Biotechnology), Vimentin (GTX100619, GeneTex, Taiwan), α-SMA (E-AB-34268, Elabscience, China), or GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA).

Techniques: RNA Sequencing, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Dual knockdown of Alox15 and TGF-β1 by lipid nanoparticle-delivered siRNA in bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2026.102534

Figure Lengend Snippet: Bleomycin-induced IPF is associated with elevated lung Alox15 and TGF-β1 expression. Mice were administered with bleomycin (BLM) or saline on day 0. The mice in the BLM group were sacrificed on days 2, 4, 7 or 14 after treatment, and lung specimens were harvested. n = 6 per group. (A) Lung function tests were performed on day 14 before sacrifice. Rrs, airway resistance; Ers, lung tissue elastance; Cst, Static lung compliance; IC, Inspiratory capacity. (B) Histological analysis with H&E staining and Picro Sirius Red staining. Scale bar = 200 μm. (C) Quantification of Picro Sirius Red staining. Scatter plot showing the percentage of the positive area of staining in lung tissue. (D) Quantification of hydroxyproline concentration. (E) Real-time PCR were performed to detect the expression of Alox15 and TGF-β1 in the pulmonary tissues from each group. (F) Western blot analysis and (G) IHC analysis and quantification to detect Alox15, 4-HNE, and TGF-β1 expression. Scale bar = 50 μm. Scatter plot showing the percentage of the positive area in lung tissue. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against Alox15 (ab244205, Abcam), 4-HNE (ab48506, Abcam), TGF-β1 (sc-130348, Santa Cruz Biotechnology, USA), E-cadherin (IR55-180, iReal Biotechnology), Vimentin (GTX100619, GeneTex, Taiwan), α-SMA (E-AB-34268, Elabscience, China), or GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Saline, Staining, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Dual knockdown of Alox15 and TGF-β1 by lipid nanoparticle-delivered siRNA in bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2026.102534

Figure Lengend Snippet: LNP-mediated delivery of siRNAs into lung epithelial and fibroblast cells effectively reduces mRNA levels both in vitro and in vivo . (A, B) MLE-12 cells and murine lung fibroblasts were treated with negative control (NC), Alox15, or TGF-β1 siRNA for 24 h, followed by treatment with bleomycin for an additional 24 h, then subjected to real-time PCR assay. Fluorescence images of (C) MLE-12 cells and (D) murine lung fibroblasts treated with 50 nM naked siRNA-Cy5 or siRNA-Cy5 encapsulated with LNPs (siRNA@LNP) for 6 h, followed by staining with Hoechst 33342. Scale bar = 20 μm. (E) Flow cytometry analysis showing the mean fluorescent intensity (MFI) of Cy5 positive cells. (F) Mice were injected with saline, naked siRNA, or siRNA@LNP and sacrificed at 24 h after treatment. Cy5 fluorescence signal in the heart, lung, liver, spleen and kidney was measured by The PhotonIMAGER Optima system immediately. Fluorescence intensity in the lung expressed as total radiant efficiency. n = 3 per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Scatter plots representing individual mice.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against Alox15 (ab244205, Abcam), 4-HNE (ab48506, Abcam), TGF-β1 (sc-130348, Santa Cruz Biotechnology, USA), E-cadherin (IR55-180, iReal Biotechnology), Vimentin (GTX100619, GeneTex, Taiwan), α-SMA (E-AB-34268, Elabscience, China), or GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, In Vivo, Negative Control, Real-time Polymerase Chain Reaction, Fluorescence, Staining, Flow Cytometry, Injection, Saline

Journal: Biochemistry and Biophysics Reports

Article Title: Dual knockdown of Alox15 and TGF-β1 by lipid nanoparticle-delivered siRNA in bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2026.102534

Figure Lengend Snippet: Administration of Alox15 and TGF-β1 siRNA encapsulated with LNPs prevented pulmonary fibrosis and improved lung function in bleomycin-induced IPF mice. Mice were administered bleomycin (BLM) or saline on day 0 and treated with siRNA encapsulated LNPs (si-NC@LNP, si-Alox15@LNP, si-TGF-β1@LNP, or si-Alox15/si-TGF-β1@LNP) on days 1, 4, and 7. The mice were sacrificed on day 14, and lung specimens were harvested. n = 6 per group. (A) Lung function tests were performed before sacrifice. Rrs, airway resistance; Ers, lung tissue elastance; Cst, Static lung compliance; IC, Inspiratory capacity. (B) Histological analysis with H&E staining and Picro Sirius Red staining. Scale bar = 200 μm. (C) Quantification of Picro Sirius Red staining. Scatter plot showing the percentage of the positive area of staining in lung tissue. (D) Quantification of hydroxyproline concentration. (E) Real-time PCR was performed to detect the expression of Alox15 and TGF-β1 in the pulmonary tissues from each group. (F) Western blot analysis and (G) IHC analysis and quantification to detect Alox15, 4-HNE, and TGF-β1 expression. Scale bar = 50 μm. Scatter plot showing the percentage of the positive area in lung tissue. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies against Alox15 (ab244205, Abcam), 4-HNE (ab48506, Abcam), TGF-β1 (sc-130348, Santa Cruz Biotechnology, USA), E-cadherin (IR55-180, iReal Biotechnology), Vimentin (GTX100619, GeneTex, Taiwan), α-SMA (E-AB-34268, Elabscience, China), or GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Saline, Staining, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: MMP9 regulates osteogenesis and MMP2 expression through the TGF-β1/SMAD2/3 signaling pathway in lipopolysaccharide-induced osteoblasts

doi: 10.1590/1414-431X2026e15172

Figure Lengend Snippet: MMP9 regulated MMP2, TGF-β1, SMAD2, and SMAD3 expression in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with or without MMP9 inhibitor for 1 h. After that, cells were transfected with pcDNA3.1 or pcDNA3.1-mMMP9 and cultured for 48 h. mRNA expression of ( A and B ) MMP9, ( C ) MMP2, ( D ) TGF-β1, ( E ) SMAD2, and ( F ) SMAD3 was measured by qRT-PCR. ( G ) Protein expression of MMP9, MMP2, TGF-β1, SMAD2, SMAD3, phospho-SMAD2, and phospho-SMAD3 was measured by western blot. Immunofluorescence analysis of ( H ) phospho-SMAD2 and ( I ) phospho-SMAD3 in MC3T3 cells; scale bar 50 μm. Data are representative of three independent experiments and are reported as means±SD. *P<0.05, **P<0.01; t -test or ANOVA.

Article Snippet: The anti-TGF-β1 antibody was purchased from Proteintech (USA).

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Brazilian Journal of Medical and Biological Research

Article Title: MMP9 regulates osteogenesis and MMP2 expression through the TGF-β1/SMAD2/3 signaling pathway in lipopolysaccharide-induced osteoblasts

doi: 10.1590/1414-431X2026e15172

Figure Lengend Snippet: MMP9 regulated RUNX2, OSX, ALP, COL I, and OCN expression in lipopolysaccharide (LPS)-induced MC3T3-E1 cells. After pretreatment with pcDNA3.1 or pcDNA3.1-mMMP9 for 48 h, MC3T3-E1 cells were stimulated or not with 20 μg/mL LPS and cultured for another 12 h. mRNA expression of ( A ) MMP2, ( B ) TGF-β1, ( C ) SMAD2, ( D ) SMAD3, ( E ) RUNX2, ( F ) OSX, ( G ) ALP, ( H ) COL I, and ( I ) OCN was measured by qRT-PCR. J , Protein expression of MMP2, TGF-β1, P-SMAD2, SMAD2, P-SMAD3, and SMAD3 was measured by western blot. K , Protein expression of RUNX2, OSX, ALP, COL I, and OCN was measured by western blot. Immunofluorescence analysis of ( L ) phospho-SMAD2 and ( M ) phospho-SMAD3 in MC3T3-E1 cells; scale bar 50 μm. N , Alkaline phosphatase (ALP) staining (a1-a3) and alizarin red S (ARS) staining (b1-b3). O , Quantification of ALP staining. P , Quantification of ARS staining. Data are representative of three independent experiments and are reported as means±SD. *P<0.05, **P<0.01; ANOVA.

Article Snippet: The anti-TGF-β1 antibody was purchased from Proteintech (USA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Brazilian Journal of Medical and Biological Research

Article Title: MMP9 regulates osteogenesis and MMP2 expression through the TGF-β1/SMAD2/3 signaling pathway in lipopolysaccharide-induced osteoblasts

doi: 10.1590/1414-431X2026e15172

Figure Lengend Snippet: MMP9-regulated MMP2 expression was inhibited by TGF-β1 or SMAD2/3 in MC3T3-E1 cells. MC3T3-E1 cells were pretreated in the presence or absence of TGF-β1 or SMAD2/3 inhibitor for 1 h. After that, cells were transfected with pcDNA3.1 or pcDNA3.1-mMMP9 and cultured for 48 h. mRNA expression of ( A ) MMP2, ( B ) TGF-β1, ( C ) SMAD2, and ( D ) SMAD3 was measured by qRT-PCR. E , Protein expression of MMP2, TGF-β1, SMAD2, SMAD3, phospho-SMAD2, and phospho-SMAD3 was measured by western blot. Immunofluorescence analysis of ( F ) phospho-SMAD2 and ( G ) phospho-SMAD3 in MC3T3-E1 cells; scale bar 50 μm. H , mRNA and ( I ) protein expression of MMP2 was measured by qRT-PCR and western blot. The data are representative of three independent experiments and are reported as means±SD. *P<0.05, **P<0.01; ANOVA.

Article Snippet: The anti-TGF-β1 antibody was purchased from Proteintech (USA).

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Immunofluorescence