Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal microvascular endothelial cells (HRMECs) were left untreated or stimulated with tumor necrosis factor-α (TNF-α) (5 ng/mL) for 24 h with or without apigenin (10 µg/mL). Protein expression of intercellular adhesion molecule-1 (ICAM-1) (A) and vascular cell adhesion molecule-1 (VCAM-1) (B) was determined by Western blot analysis. Adhesion of fluorescently labeled THP-1 monocytic cells to HRMECs monolayer was quantified (C) . Results are expressed as mean ± standard deviation or standard error of mean from three different experiments each performed in triplicate. One-way ANOVA and independent t-test were used for comparisons between three groups and two groups, respectively. *p < 0.05 compared with values obtained from untreated cells. #p < 0.05 compared with values obtained from cells treated with TNF-α (RFU = relative fluorescence unit).

Article Snippet: After reaching a confluent monolayer, overnight starved endothelial cells were 1h pretreated with apigenin (Cat No 1227/10, R&D Systems) followed by 5 ng/ml TNF-α treatment for 16 h. Next, 5 x 10 fluorescent-LeukoTracker labelled monocytic THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were added on the top of treated HRMECs monolayer for 45 min. After washing 3 times, the remaining adherent THP-1 cells were lysed and fluorescence was measured using a spectraMax Gemini-XPS (Molecular Devices, CA, USA) with excitation and emission wavelengths of 485 nm and 538 nm, respectively.

Techniques: Expressing, Western Blot, Labeling, Standard Deviation, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

doi: 10.1002/jev2.12044

Figure Lengend Snippet: Purity of EV samples assessed by particle/protein ratio and Western blotting. (a)‐(c) The protein concentrations of EV preparations separated by different methods from CCM, urine and plasma. The protein concentrations have been corrected for initial sample input volume. (d)‐(f) The particle/protein ratios of EV preparations separated by different methods from CCM, urine and plasma. The particle numbers were measured by nFCM. The error bars represented the standard deviation of three repetitive experiments. * P < 0.05, ** P < 0.01, one‐way ANOVA analysis with Tukey multiple comparison test as well as a variance‐covariance model. (g) Western blots of flotillin‐1, CD63, CD81, and calnexin for EV preparations separated from CCM by different methods. 9 μg of protein from each EV preparation was loaded. (h) Western blots of flotillin‐1, CD63, CD81, THP and calnexin for EV preparations separated from urine by different methods. 3.3 μg of protein from each EV preparation was loaded. (i) Western blots of flotillin‐1, CD81, APOA1 and calnexin for EV preparations separated from plasma by different methods. 25 μg of protein from each EV preparation was loaded. MCF7 membrane and cytosolic protein fractions served as positive and negative controls for flotillin‐1, CD63, CD81 and calnexin. Whole urine sample was used as positive control and MCF7 cellular fractions as negative control for THP. Recombinant human Apolipoprotein A1 served as positive control for APOA1.GAPDH staining was to show proper loading and running of the cytosolic MCF7 fraction

Article Snippet: Overnight incubation at 4°C of the membranes was performed with the following primary antibodies: THP (1:1,000, mouse monoclonal B‐2, #sc‐271022 Santa Cruz Biotechnology); and APOA1 (1:5,000, goat polyclonal, #ab7613, Abcam).

Techniques: Western Blot, Clinical Proteomics, Standard Deviation, Comparison, Membrane, Positive Control, Negative Control, Recombinant, Staining

Journal: Heliyon

Article Title: Association between TOP2A, RRM1, HER2, ERCC1 expression and response to chemotherapy in patients with non-muscle invasive bladder cancer.

doi: 10.1016/j.heliyon.2022.e09643

Figure Lengend Snippet: Figure 1. Expression levels of TOP2A and RRM1 in human NMIBC tissues and their significance in cancer recurrence. A and B, Immunohistochemistry show pos- itive and negative expression of TOP2A and RRM1 in NMIBC tissues. C, The mean survival time of the TOP2A-positive group was significantly shorter than that of the TOP2A-negative expression group (p ¼ 0.040). D, No significant difference was observed in the overall survival rates between the RRM1-positive and RRM1- negative expression groups (p > 0.05). E, In the pirarubicin-treatment group, 32 and 13 patients had positively and negatively expressed TOP2A, respec- tively. Tumours with high TOP2A expression showed a lower recurrence rate than those with low TOP2A expres- sion after one year of pirarubicin treat- ment (p ¼ 0.037). F, In the gemcitabine- treatment group, 18 and 22 patients had positive and negative RRM1 expression, respectively. Tumours with low expres- sion of RRM1 had lower recurrence rate than tumours with high expression of RRM1 after treatment with gemcitabine within one year (p ¼ 0.024).

Article Snippet: Gemcitabine (catalogue no. HYB0003, MedChemExpress, USA) and pirarubicin (catalogue no. HY13725A, MedChemExpress, USA) were used.

Techniques: Expressing, Immunohistochemistry

Journal: Heliyon

Article Title: Association between TOP2A, RRM1, HER2, ERCC1 expression and response to chemotherapy in patients with non-muscle invasive bladder cancer.

doi: 10.1016/j.heliyon.2022.e09643

Figure Lengend Snippet: Figure 2. mRNA and protein levels of TOP2A and RRM1 in bladder cancer cell line cells. A, RT-PCR and Western blot analysis showed that the mRNA and protein levels of TOP2Ain the BIU- 87 cell line were higher than those in the CCC-HB-2 and KK47 cell line (p < 0.01), whereas the mRNA and protein levels of RRM1were lower in the KK47 cells (p < 0.05) (The original blots images were provided in supplement figure1A-TOP2A, supplement figure1A-RRM1 and supplement figure1A-GAPDH). B, The mRNA and protein levels of TOP2A in BIU-87 cells (The original blots images were pro- vided in supplement figure1B-TOP2A, supplement figure1B-GAPDH-TOP2A, supplement figure1B-RRM1 and sup- plement figure1B-GAPDH-RRM1) were significantly decreased by the transfection with TOP2A shRNA, and RRM1 was increased in KK47 cells by the transfection with the PCDNA3 RRM1 plasmid. C, Survival curves of BIU-87 and KK47 cells treated with different concentrations of pirarubicin (0, 0.5, 1, 2, 4 and 8 μg/ml) for 48 h. The IC50 values of pirarubicin in BIU- 87 and KK47 cells were 0.84 0.22 μg/ml and 1.82 0.31 μg/ml, respectively. The IC50 values of pirarubicin in the shTOP2A group were significantly higher than those in the vector control group of BIU-87 cells (p < 0.05). D, Survival curves of BIU-87 and KK47 cells treated with different concentrations of gemcita- bine (0, 1, 3, 5, 7 and 10 μg/ml) for 48 h the IC50 values of gemcitabine in BIU-87 and KK47 cells were 4.94 0.31 μg/ml and 3.45 0.32 μg/ml, respectively. The IC50 values of gem- citabine in the PCDNA RRM1 group were significantly higher than those in the vector control group of KK47 cells (p < 0.05). Each experiment was repeated three times.

Article Snippet: Gemcitabine (catalogue no. HYB0003, MedChemExpress, USA) and pirarubicin (catalogue no. HY13725A, MedChemExpress, USA) were used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Plasmid Preparation, Control

Journal: Heliyon

Article Title: Association between TOP2A, RRM1, HER2, ERCC1 expression and response to chemotherapy in patients with non-muscle invasive bladder cancer.

doi: 10.1016/j.heliyon.2022.e09643

Figure Lengend Snippet: Figure 3. Expression levels of HER2 and ERCC1 in human NMIBC tissues and their significance in cancer recurrence. A, Immunohistochemistry shows positive and negative expression of HER2 in NMIBC tissues. B, Immunohistochemistry shows positive and negative expression of ERCC1 in NMIBC tissues. C, No significant difference was observed in the overall survival rates between the HER2-positive and HER2-negative expression groups (p > 0.05). D, No significant difference was observed in the overall survival rates between the ERCC1-positive and ERCC1-negative expression groups (p > 0.05). E, In the pirarubicin-treatment group, HER2 was positively expressed in 20 of the 45 patients. The recurrence rate in the patients with positive TOP2A and HER2 co-expression was higher than that in those with TOP2A expression alone (p ¼ 0.002). F, In the gemcitabine-treatment group, ERCC1 was negatively expressed in 17 of the 40 cases. Patients with both the negative expression of both RRM1 and ERCC1 had lower recurrence rates than those with only a low expression of RRM1 (p ¼ 0.023).

Article Snippet: Gemcitabine (catalogue no. HYB0003, MedChemExpress, USA) and pirarubicin (catalogue no. HY13725A, MedChemExpress, USA) were used.

Techniques: Expressing, Immunohistochemistry

Journal: Heliyon

Article Title: Association between TOP2A, RRM1, HER2, ERCC1 expression and response to chemotherapy in patients with non-muscle invasive bladder cancer.

doi: 10.1016/j.heliyon.2022.e09643

Figure Lengend Snippet: Figure 4. The sensitivity of different subpopulations of cells to chemotherapeutic drugs. A, RT-PCR and Western blot analysis showed that the mRNA and protein levels of HER2 in the BIU-87 cell line were higher than those in the CCC-HB-2 and KK47 cell line, whereas the mRNA and protein levels of RRM1 were no significant different in these cells (The original blots images were provided in supplement figure2A-HER2, supplement figure2A-ERCC1 and supplement figure2A-GAPDH). B, TOP2AþHER2- cells, TOP2AþHER2þ cells, TOP2AHER2þ cells and TOP2AHER2- cells were sorted from BIU-87 cells by FACS. C, Separated TOP2Aþ HER2- cells were more sensitive to pirarubicin than TOP2AþHER2þ positive cells (p < 0.05). D, RRM1þERCC1- cells, RRM1þERCC1þ cells, RRM1ERCC1þcells and RRM1ERCC1- cells were also sorted from the BIU-87 cell line. E, RRM1ERCC1- cells were more sensitive to gemcitabine chemotherapy than the RRM1ERCC1þ

Article Snippet: Gemcitabine (catalogue no. HYB0003, MedChemExpress, USA) and pirarubicin (catalogue no. HY13725A, MedChemExpress, USA) were used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot