pri mary antibody edar  (Proteintech)

Journal: Advanced Science

Article Title: Red Blood Cell‐Derived Exosomal miR‐93‐5p Promotes Lung Cancer Progression through PTEN Suppression

doi: 10.1002/advs.202511940

Figure Lengend Snippet: RBC‐Derived miRNA Transfer to Lung Cancer Cells via Exosomes. A) Schematic of a dual‐chamber Transwell system with a 0.4 µm membrane. RBCs and RBC‐conditioned medium (RBC‐medium) are maintained in the up chamber, while only the RBC‐medium passes to the low chamber, excluding intact RBCs. Lung cancer cells in the up chamber are exposed to both RBCs and RBC‐medium, whereas cells in the low chamber are exposed only to RBC‐medium. B) Expression levels of miR‐93‐5p in NSCLC cells after exposure to RBCs from cancer patients (Can. Pat.) and healthy controls (Healthy Con.), analyzed from the up chamber, low chamber, and regular medium as a negative control, indicating that RBC‐derived miRNAs are primarily transferred via the RBC‐ medium without direct contact. C) Relative expression levels of miR‐451 under the same conditions, supporting the same conclusion. (One‐way ANOVA) (Normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was verified using Levene's test prior to ANOVA). D) Transmission electron microscopy (TEM) images of exosomes isolated from RBC‐conditioned medium of a healthy control (upper panel) and a lung cancer patient (lower panel). Both images were captured at ×100 000 magnification, and the scale bar represents 200 nm. Exosomes appear as round, membrane‐bound vesicles with a typical diameter of 30–150 nm. These images confirm the successful isolation and morphology of exosomes from both groups, with increased vesicle abundance observed in the cancer patient sample. Annotations have been added to clearly identify the exosome source and distinguish the upper and lower panels. E) Western blot analysis confirming the presence of Ago2 protein in exosome fractions from RBC‐medium of ten lung cancer patines (Cs) and ten normal healthy controls (Ns) with GAPDH used as a loading control. The A549 NSCLC cell line was included as a control. F) Relative Ago protein expression was quantified and presented as a bar graph, with levels normalized to GAPDH (set as 100%). There is no difference in Ago expression between tumor and normal lung tissues (p = 0.356). G) miR‐93‐5p levels were significantly higher in both exosomes and Ago2‐immunoprecipitated complexes from cancer patients compared to healthy controls. H) miR‐451 levels were significantly higher in exosomes from cancer patients, but showed no significant difference in Ago2 complexes between the two groups. All tests assumed normal distribution and equal variances, confirmed prior to analysis using the Shapiro–Wilk and Levene's tests, respectively. All assays were conducted using a minimum of three independent biological replicates per group. Please note: * p < 0.05; * * p < 0.01; ** * p < 0.001; ns, not significant.

Article Snippet: Assay IDs for pri‐miR‐93‐1 (Hs03303305_pri), pri‐miR‐93‐2 (Hs03303627_pri), DROSHA (Hs00203008_m1), and DICER1 (Hs00229023_m1) were used to quantify the precursors and biogenesis transcripts, respectively.

Techniques: Derivative Assay, Membrane, Expressing, Negative Control, Transmission Assay, Electron Microscopy, Isolation, Control, Western Blot, Immunoprecipitation

Journal: Advanced Science

Article Title: Red Blood Cell‐Derived Exosomal miR‐93‐5p Promotes Lung Cancer Progression through PTEN Suppression

doi: 10.1002/advs.202511940

Figure Lengend Snippet: RBC‐Derived Exosomal Transfer of miR‐93‐5p and miR‐451 to Lung Cancer Cells. A) Fluorescent microscopy images displaying uptake of CD63‐positive (red) RBC‐derived exosomes from lung cancer patients by NSCLC cells, with nuclei stained blue (DAPI). Scale bar: 20 µm. B) Similar uptake of CD63‐positive (red) RBC‐derived exosomes from healthy controls by NSCLC cells. C,D) Quantitative analysis demonstrating a significant increase in the uptake of CD63‐positive RBC‐derived exosomes from cancer patients compared to healthy controls, with no uptake observed from basal medium (control medium). n = 3 independent experiments; data = mean ± SD, * p < 0.01, * * p < 0.001. E–H) A549 and H460 cancer cells showed increased levels of both miR‐93‐5p and miR‐451 after incubation with exosomes from cancer patients, but only miR‐451 was elevated following exposure to exosomes from cancer‐free controls (n = 3). Data = mean ± SD; * * p < 0.001. I) Fluorescent images show reduced CD63 staining in A549 cells treated with the exosome release inhibitor GW4869 compared to untreated cells, indicating decreased exosome secretion. Scale bar: 20 µm. J) Impact of GW4869 treatment on miR‐93‐5p and miR‐451 levels in A549 and H460 cells, displaying significantly reduced miRNA levels in treated cells compared to controls (n = 3; Student's t‐test). ns = not significant; * * p < 0.001. K) Impact of the exosome uptake inhibitor Dynasore on miR‐93‐5p and miR‐451 levels in A549 and H460 cells, displaying significantly reduced miRNA levels in treated cells compared to controls. ns = not significant; * * p < 0.001. Statistical analyses were performed using two‐tailed Student's t‐tests. Normality was confirmed using the Shapiro–Wilk test, and equality of variance was assessed using Levene's test. Data are presented as mean ± SD. Three human NSCLC cell lines were used in these experiments, consistently demonstrating similar results; data for A549 and H460 cell lines are presented. All assays were conducted using a minimum of three independent biological replicates per group (n ≥ 3).

Article Snippet: Assay IDs for pri‐miR‐93‐1 (Hs03303305_pri), pri‐miR‐93‐2 (Hs03303627_pri), DROSHA (Hs00203008_m1), and DICER1 (Hs00229023_m1) were used to quantify the precursors and biogenesis transcripts, respectively.

Techniques: Derivative Assay, Microscopy, Staining, Control, Incubation, Two Tailed Test