Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly reduces melanoma cell viability and proliferation. STAT1 mRNA expression was analyzed in (A) A375 and (B) RPMI-7951 melanoma cells post-STAT1 knockdown using siRNA. (C) Western blotting was used to assess STAT1 protein levels in melanoma cell lines following siRNA-mediated knockdown. Cell viability was evaluated after STAT1 knockdown in (D) A375 and (E) RPMI-7951 cells using Cell Counting Kit-8 assays. (F) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown. (G) Quantification of colony formation assay results. **P<0.01 vs. si-NC group. siRNA, small interfering RNA; STAT1, signal transducer and activator of transcription 1; si-STAT1, siRNA targeting STAT1; si-NC, negative control siRNA.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Knockdown, Expressing, Western Blot, Cell Counting, Colony Assay, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly promotes melanoma cell apoptosis and inhibits migration. (A and B) Apoptosis was assessed and quantified in A375 and RPMI-7951 cells following STAT1 knockdown using flow cytometry. (C) Transwell migration assays were conducted to evaluate cell migration following STAT1 knockdown. Magnification, ×200. (D) Quantification of migration capacity in A375 and RPMI-7951 cells following STAT1 knockdown. *P<0.05 vs. si-NC. STAT1, signal transducer and activator of transcription 1; si-STAT1, small interfering RNA targeting STAT1; si-NC, negative control small interfering RNA.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Knockdown, Migration, Flow Cytometry, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 regulates TUBB4A expression at the transcription level. (A) STAT1 mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (B) TUBB4A mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (C) Specific fragments of the TUBB4A promoter region were cloned into the luciferase reporter plasmids upstream of the firefly luciferase gene. (D) Transcriptional activity of various TUBB4A promoter fragments was analyzed by luciferase reporter assay in 293T cells, with the −1,783 and −1,771 fragments exhibiting the highest activity. (E) STAT1 siRNA-mediated knockdown significantly reduced STAT1 mRNA levels in A375 cells. (F) STAT1 knockdown significantly reduced the luciferase activity of the −1,783 fragment of the TUBB4A promoter, but not the −1,771 fragment. (G) Chromatin immunoprecipitation assays were performed in A375 and RPMI-7951 cells targeting the −1,783 binding site in the TUBB4A promoter region. Quantitative PCR provided evidence of STAT1 binding to this region. Genomic DNA input was set to 100%. **P<0.01 vs. si-NC; ## P<0.01 vs. PGL3; && P<0.01 vs. IgG. STAT1, signal transducer and activator of transcription 1; siRNA, small interfering RNA; si-NC, negative control siRNA; si-STAT1, siRNA targeting STAT1; si-STAT1-1, siRNA targeting STAT1 sequence 1; si-STAT1-2, siRNA targeting STAT1 sequence 2; TUBB4A, tubulin β4A; PGL3, promoter-gluc luciferase 3; LUC, firefly luciferase gene.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Expressing, Knockdown, Transfection, Clone Assay, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Sequencing

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression mitigates the effects of STAT1 knockdown on cell viability and proliferation. TUBB4A mRNA levels were measured in (A) A375 and (B) RPMI-7951 cells following TUBB4A overexpression mediated by a lentiviral vector. (C) TUBB4A protein expression was analyzed after its overexpression. Combined STAT1 knockdown and TUBB4A overexpression transfections were performed, followed by a western blot analysis of TUBB4A protein levels in (D) A375 and (E) RPMI-7951 cells. Cell viability was assessed via Cell Counting Kit-8 assays following combined STAT1 knockdown and TUBB4A overexpression in (F) A375 and (G) RPMI-7951 cells. (H) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown and TUBB4A overexpression. (I) Quantification of colony formation assay results. **P<0.01 vs. Ov-NC; ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; STAT1, signal transducer and activator of transcription 1; si-NC, negative control small interfering RNA; si-STAT1, small interfering RNA targeting STAT1; Ov-NC, negative control lentiviral overexpression vector; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Over Expression, Knockdown, Plasmid Preparation, Expressing, Transfection, Western Blot, Cell Counting, Colony Assay, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression reverses the effects of STAT1 knockdown on apoptosis, migration and tumor growth. (A-D) Apoptosis and migration were evaluated in A375 and RPMI-7951 cells following STAT1 knockdown and TUBB4A overexpression. Magnification, ×200. (E) Representative images of isolated xenograft tumors in mice. Tumor volumes were measured in nude mice injected subcutaneously with 2×10 6 A375 cells that had been subject to STAT1 knockdown and TUBB4A overexpression. (F) Quantification of mouse tumor volumes showed that STAT1 knockdown significantly suppressed tumor growth, whereas TUBB4A overexpression reversed this inhibitory effect. ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; si-NC, negative control small interfering RNA; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Melanoma cells (A375 and RPMI-7951) were seeded into 6-well plates (cat. no. 3516; Corning, Inc.) at a density of 500–1,000 cells per well and allowed to adhere for 24 h. Cells were then transfected with si-NC (cat. no. A09010) or si-STAT1 (cat. no. A09009; GenePharma), as well as transfected using a lentiviral vector for TUBB4A overexpression (Ov-TUBB4A; pReceiver-Lv105; GeneCopoeia, Inc.) or Ov-NC (pReceiver-Lv105 Empty Vector; GeneCopoeia, Inc.).

Techniques: Over Expression, Knockdown, Migration, Isolation, Injection, Negative Control, Small Interfering RNA, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: Single-cell transcriptomic landscape of the mid-secretory eutopic endometrium reveals receptivity defects in adenomyosis

doi: 10.1186/s12967-026-07866-z

Figure Lengend Snippet: Characterization of eutopic endometrial epithelial cells in adenomyosis (AM). ( A ) UMAP plot showing four epithelial cell subclusters (Epi1–Epi4) ( B ) Heatmap of the top marker genes for each epithelial subcluster. ( C–E ) Gene Ontology (GO) enrichment analyses of top marker genes in Epi1 ( C ), Epi2 ( D ), and Epi3 ( E ). ( F ) Box plots showing the relative proportions of AM and control cells within each epithelial subcluster. ( G ) LGR5 and SOX9 mRNA expressions in mid-secretory eutopic endometrium from AM and control patients examined by qPCR analysis ( n = 12 per group). ( H , I ) Immunofluorescence staining for the expression and localization of LGR5 and SOX9. Scale bars, 50 μm; n = 12 per group. ( J ) Immunohistochemical staining for the expression of proliferation marker Ki-67. Scale bars, 50 μm; n = 12 per group. ( K ) Cell proliferation following plasmid LGR5 and SOX9 (p(LGR5 + SOX9)) overexpression in human endometrial epithelial cells (HEECs) isolated from mid-secretory endometrial tissues of controls ( n = 3) measured by CCK-8 assay. ( L ) Proliferation of HEECs transfected with p(LGR5 + SOX9) determined via EdU assay. Actively proliferating cells are visualized by green fluorescence, with all nuclei counterstained in blue. Scale bar = 100 μm; n = 3. ( M ) RT-qPCR quantification of proliferation markers ( MKI67 , PCNA ) and differentiation markers ( LIF , HOXA10 ) levels after LGR5 and SOX9 overexpression in control HEECs ( n = 3). ( N ) Representative immunofluorescence images for Ki-67 (red) and corresponding quantification of positive cells in HEECs. Scale bar = 100 μm; n = 3. ( O ) Western blotting of LIF and PCNA protein levels in HEECs transfected with p(LGR5 + SOX9). Data are presented as the mean ± SD, * P < 0.05, ** P < 0.01

Article Snippet: Control, LGR5, SOX9, and DIO2 overexpression plasmids (GeneCopoeia, China) were introduced using X-tremeGENE 9 (Roche, USA).

Techniques: Marker, Control, Immunofluorescence, Staining, Expressing, Immunohistochemical staining, Plasmid Preparation, Over Expression, Isolation, CCK-8 Assay, Transfection, EdU Assay, Fluorescence, Quantitative RT-PCR, Western Blot

Journal: Journal of Translational Medicine

Article Title: Single-cell transcriptomic landscape of the mid-secretory eutopic endometrium reveals receptivity defects in adenomyosis

doi: 10.1186/s12967-026-07866-z

Figure Lengend Snippet: Identification, functional characterization, and trajectory analysis of stromal cell subclusters in adenomyosis (AM). ( A ) UMAP visualization of stromal subclusters (Str1–Str4). ( B ) Dot plots depicting the average expression of established markers indicated ecotype. ( C ) Pseudotime trajectory showing the progression of Str1, Str2, Str3 and Str4. ( D ) Gene Ontology (GO) terms significantly enriched across gene clusters with distinct pseudo-temporal patterns. ( E ) The distribution of DIO2 , PGR , and WNT5A in stromal cell subclusters by UMAP. ( F ) Violin plot of inflammatory scores across stromal subclusters. ( G ) Box plots of the relative proportions of AM and control cells in each stromal subcluster. ( H ) Separate pseudotime trajectories of stromal subclusters in control and AM groups

Article Snippet: Control, LGR5, SOX9, and DIO2 overexpression plasmids (GeneCopoeia, China) were introduced using X-tremeGENE 9 (Roche, USA).

Techniques: Functional Assay, Expressing, Control

Journal: Journal of Translational Medicine

Article Title: Single-cell transcriptomic landscape of the mid-secretory eutopic endometrium reveals receptivity defects in adenomyosis

doi: 10.1186/s12967-026-07866-z

Figure Lengend Snippet: DIO2 ⁺ Str4 stromal cells exhibit SASP-like activity and drive decidualization. ( A , B ) Representative immunohistochemistry (IHC) images of DIO2, CXCL12, IGFBP3, and MMP14 in mid-secretory eutopic endometrial tissues from controls and adenomyosis (AM) patients ( n = 12). Scale bars, 50 μm. ( C ) ELISA quantification of decidualization markers IGFBP1 and PRL in control and AM tissues ( n = 12). ( D ) Senescence-associated β-galactosidase (SA-β-gal) staining in control HESCs undergoing in vitro decidualization. ( E ) RT-qPCR analysis of SASP-related cytokines CXCL14, TIMP3, and IL15 in decidualized control HESCs ( n = 3). ( F ) ELISA measurement of secreted SASP cytokines in culture supernatant ( n = 3). ( G – K ) Effects of DIO2 knockdown on β-gal activity ( G ), SASP cytokine mRNA expression ( H ), and secreted protein levels ( I – K ) in control HESCs ( n = 3). ( L , M ) F-actin filaments staining in control HESCs following DIO2 knockdown during in vitro decidualization, visualized with Alexa Fluor 555–conjugated phalloidin. Scale bar = 50 μm, n = 3. Data are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Control, LGR5, SOX9, and DIO2 overexpression plasmids (GeneCopoeia, China) were introduced using X-tremeGENE 9 (Roche, USA).

Techniques: Activity Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Control, Staining, In Vitro, Quantitative RT-PCR, Knockdown, Expressing

Journal: bioRxiv

Article Title: A novel Gorilla-derived oncolytic Adenovirus with natural selective replication in cancer cells

doi: 10.64898/2026.02.26.708271

Figure Lengend Snippet: (A) Schematic representation of the genetic structure of the GRAd32Fk25 EV20 SC vector. (B) A549, NCI-H727 or NCI-H1975 were infected with GRAd32Fk25 EV20 SC expressing a single-chain antibody targeting HER3, based on the EV20 sequence. Infection was performed at MOI 1-10-50-100, and supernatant-containing antibodies were separated on SDS-PAGE and blotted with an anti-human antibody. (C) MRC5 cells were infected either with GRAd32Fk25 expressing the EV20 SC, or with GRAd25 expressing the same EV20 SC at an MOI of 10 or 100. The supernatant-containing antibody were separated on SDS-PAGE and blotted with an anti-human antibody. (D) Affinity for HER3 (ECD) of supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated through direct ELISA and absorbance values are expressed in OD (450nm) are reported. (E) Affinity for cell surface HER3 on A375m exposed to supernatants containing GRAd-produced EV20 SC derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC or mock control. Affinity was evaluated by FACS and levels are expressed in arbitrary units (A.U.) of mean fluorescence intensity (M.F.I.). (F) Western blotting images for the evaluation of p-HER3, total HER3, p-Akt, total Akt, and actin (loading control) protein levels in A375m exposed to supernatants containing GRAd-produced EV20 SC (1:2 or 1:10 diluted) derived from A549, NCI-H727, or NCI-H1975 infected with 100 MOI of GRAd32Fk25 EV20 SC and treated or not with neuregulin-1β (NRG-1β). Equal amounts of protein samples were loaded per lane. All adenoviruses contained in the supernatants were heat-inactivated for 30 minutes at 56°C; EV20 is the positive control, EV20 heated is the positive control treated for 30 minutes at 56°C.

Article Snippet: For the evaluation of CD46 and CAR cell surface levels, 2-3 x 10 5 cells (MRC5, HUVEC, A549, NCI-H1299, NCI-H1975, NCI-H727) were collected and incubated with anti-human CD46 (1:50; mouse monoclonal; #12239-MM05, Sino Biological) or with anti-human CAR (1:50; rabbit monoclonal; #10799-R271, Sino Biological) for 30 min at 4°C.

Techniques: Plasmid Preparation, Infection, Expressing, Sequencing, SDS Page, Produced, Derivative Assay, Control, Direct ELISA, Fluorescence, Western Blot, Positive Control