Journal: BMC Cancer

Article Title: Effects of small interfering RNA targeting thymidylate synthase on survival of ACC3 cells from salivary adenoid cystic carcinoma

doi: 10.1186/1471-2407-8-348

Figure Lengend Snippet: Evaluation of TS mRNA transcript expression in the ACC3 cell line . Expression was compared with that in keratinocytes and bronchial epithelial cells as normal cell controls using real-time RT-PCR.

Article Snippet: RNA samples from human keratinocytes and human adult bronchial epithelial cells (Cell Applications, San Diego, CA, USA) were applied to real-time RT-PCR as control samples from non-cancer cells.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Effects of Glycyrrhizic Acid on Dry Eye Disease: Targeting Pyroptosis, Oxidative Stress, and Epithelial Barrier Dysfunction

doi: 10.3390/ijms27094153

Figure Lengend Snippet: The therapeutic efficacy of GA eye drops in DED mice. ( A ) The diagram of DED modeling, treatment, and ocular assessment timeline. ( B ) Photographs of corneal fluorescein staining (CFS) of mice across all designated groups at days 0, 7, and 14. ( C ) Quantitative analysis of CFS scores (n = 5). ( D ) Measurement of tear volume (n = 5). ( E – G ) RT-qPCR was employed to determine the mRNA levels of key pro-inflammatory cytokines including TNF-α , IL-1β , and IL-6 , within the corneal tissues (n = 3). ( H ) H&E staining of corneas in different groups (n = 4, Scale bar = 50 μm). ( I ) Measurement of corneal epithelial layer thickness (n = 4). ( J , K ) Morphological evaluation of the conjunctiva via PAS staining in each group of mice (Scale bar = 100 μm) and quantification of conjunctival goblet cell density (n = 4). The data were presented as mean ± SD of at least three independent experiments (ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Human corneal epithelial cells (HCECs; CRL-11135, ATCC, Manassas, VA, USA) were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 37 °C, 5% CO 2 incubator.

Techniques: Drug discovery, Eye Drops, Staining, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Effects of Glycyrrhizic Acid on Dry Eye Disease: Targeting Pyroptosis, Oxidative Stress, and Epithelial Barrier Dysfunction

doi: 10.3390/ijms27094153

Figure Lengend Snippet: GA protects the epithelial barrier via suppressing TNF-α and MMP9. ( A ) Immunofluorescence staining showing ZO-1 in HCECs (n = 3, scale bar = 50 μm). ( B ) Quantification of ZO-1-positive staining area in vitro (n = 3). ( C ) Wound-healing assays were employed to assess cell migration ability (n = 3, scale bar = 100 μm). ( D ) Measurement of HCECs migration (n = 3). ( E ) HCECs were pretreated with GA for 24 h followed by hyperosmotic stress for 24 h. Western blot analysis of TNF- α and MMP9 levels. ( F , G ) Quantification of TNF-α and MMP9 expression in vitro (n = 3). ( H ) Western blot analysis of TNF-α and MMP9 in different groups with each sample comprising five corneas. ( I – K ) Quantification of TNF-α, MMP9 and ZO-1 expressions in vivo (n = 3). The data were presented as mean ± SD of at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Human corneal epithelial cells (HCECs; CRL-11135, ATCC, Manassas, VA, USA) were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 37 °C, 5% CO 2 incubator.

Techniques: Immunofluorescence, Staining, In Vitro, Migration, Western Blot, Expressing, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Effects of Glycyrrhizic Acid on Dry Eye Disease: Targeting Pyroptosis, Oxidative Stress, and Epithelial Barrier Dysfunction

doi: 10.3390/ijms27094153

Figure Lengend Snippet: Schematic illustration of the multifaceted protective mechanisms of GA in DED. Hyperosmotic stress activates HMGB1, which induces lysosomal membrane permeabilization to promote pyroptosis via cathepsin B. This stress also impairs mitochondrial function, resulting in robust ROS production, and elevates TNF-α and MMP9 levels to disrupt ZO-1 tight junctions. GA effectively counteracts these pathological processes by: (1) inhibiting the HMGB1/LMP/CTSB pathway to suppress pyroptosis; (2) activating the Nrf2/HO-1/NQO1 antioxidant pathway to restore mitochondrial homeostasis and neutralize ROS; and (3) downregulating the TNF- α /MMP9 pathway to preserve the ocular surface epithelial barrier. Collectively, GA protects the ocular surface from oxidative damage, chronic inflammation, and barrier dysfunction.

Article Snippet: Human corneal epithelial cells (HCECs; CRL-11135, ATCC, Manassas, VA, USA) were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 37 °C, 5% CO 2 incubator.

Techniques: Membrane

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 4 ch5E6 treatment leads to a substantial decrease in the growth of organoids derived from PC patients or pancreatic and lung cancer genetically engineered mouse models. a PC patient and b KPC mouse organoid staining with ch5E6 indicating MUC16 expression (green) and specific binding of mAb compared to no binding with isotype control mAb huIgG1. The binding of ch5E6 to the ductal cells (yellow) was illustrated by colocalization with CK-19 (red) staining in human and mouse pancreatic tumor organoids. Scale bar 10 µm.The representative images for ch5E6 treated. c Human PC patient. d KPC and (e) KPA mouse-derived organoids obtained by real-time kinetics using Incucyte live cell imaging system. The data were quantitated for organoid counts using essence incucyte software, plotted as a graph of change in organoid counts or area over time (3–5 days) for both ch5E6 and huIgG1 treatments, and is shown in parallel. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Derivative Assay, Staining, Expressing, Binding Assay, Control, Live Cell Imaging, Software

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 6 Inhibition of EMT by ch5E6 is validated in PC and NSCLC cell line-derived xenografts. a Immunofluorescence analysis showing a decrease in pFAK(Y397) and N-cadherin expression in xenograft tumors of SW1990 cells treated with ch5E6 compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: three animals). The data was plotted for changes in fluorescence intensity using GraphPad Prism 9 and is shown in parallel. Nuclei were stained with DAPI. b Immunofluorescence analysis of ch5E6 treated SW1573 cell line-derived xenografts showing a reduction in pFAK(Y397) and N-cadherin levels compared to isotype control mAb huIgG1 group (n = 6–8 fields/tissue: 3 animals). Scale bar, 10 µm; magnified images, 2 µm. No significant changes in the intensity of MUC16 were seen in the ch5E6 treated versus isotype control tumors derived from both cancers. c, d Immunoblot analysis of ch5E6 treated PDAC and NSCLC tumor lysates showing a substantial decrease in phosphorylated levels of FAK(Y397), p70S6K(T389) and N-cadherin as compared to huIgG1 treatment. Error bars indicate SEM. Scale bar, 400 μm; magnified images, 100 μm; *P < 0.05; **P < 0.01.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Inhibition, Derivative Assay, Expressing, Control, Staining, Western Blot

Journal: NPJ precision oncology

Article Title: Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

doi: 10.1038/s41698-023-00423-7

Figure Lengend Snippet: Fig. 7 MUC16 and N-cadherin are clinically correlated in patient tumors. a, b Representative images and quantitative illustration of immunohistochemical analyses demonstrate a strong positive correlation between MUC16 and N-cadherin (R = 0.84) in both primary PC tumors (n = 10) and liver metastasis (R = 0.99) samples (n = 8). Scale bar, 400 µm; magnified images, 100 µm. *P < 0.05; **P < 0.01. c Representative images of immunofluorescence analysis showing coexpression of MUC16 (green) and N-cadherin (red) in primary PDAC tumors compared to no MUC16 and N-cadherin in normal pancreatic sections. Scale bar, 20 µm; magnified images, 5 µm. d Schematic diagram representing ch5E6 induced downregulation of MUC16 mediated EMT resulting in its anti-tumor potential in PC and NSCLC. Overall, anti- MUC16 chimeric mAb5E6 (ch5E6) binds to the cell surface-tethered domain of MUC16, interferes with oncogenic pFAK/p70S6K/N-cadherin signaling associated with MUC16-mediated EMT, and reduces tumor burden in both PC and NSCLC models.

Article Snippet: We performed Real time MT-glo assay to determine the effect of anti-MUC16 chimeric mAb5E6 (ch5E6) and isotype control mAb (huIgG1; Bio X Cell).

Techniques: Immunohistochemical staining

Journal: Journal of Medical Virology

Article Title: The Open Reading Frame 7b of the SARS‐CoV‐2 Disperse Trans‐Golgi and Activate the NLRP3 Inflammasome

doi: 10.1002/jmv.70894

Figure Lengend Snippet: IL‐1β release is potentiated in human primary small airway epithelial cells by SARS‐CoV‐2 ORF7b expression. (A) Expression of inflammasome genes in human primary small airway epithelial cells (HSAEC). (B) Expression of inflammasome genes in HSAEC cells transfected with plasmids encoding for NLRP3 and SARS‐CoV‐2 ORF7a or ORF7b as indicated. (C) ELISA of IL‐1β in HSAEC supernatants transfected for 24 h with NLRP3 and SARS‐CoV‐2 ORF7a or ORF7b as indicated and then treated or not with nigericin (10 μM) for 24 h. Each symbol represents an independent experiment performed in duplicate biological replicates, and data are represented as mean ± s.e.m; Paired t ‐test was used for B,C; ** p > 0.001 < 0.01, * p > 0.01 < 0.05, p > 0.05 not significant ( ns ).

Article Snippet: Human primary small airway epithelial cells (HSAEC, ATCC PCS‐301‐010) were cultured in Airway Epithelial Cell Basal Medium (ATCC) complemented with hyaluronic acid supplement (500 mg/mL), linoleic acid (0.6 mM), lecithin (0.6 mg/mL), L‐glutamine (6 mM), P extract (0.4%) and with the Bronchial Epithelial Growth Kit containing 1.0 mM epinephrine, 5 mg/mL transferrin, 10 nM T3, 5 mg/mL hydrocortisone, 5 ng/mL recombinant human epithelial growth factor (EGF), and 5 mg/mL recombinant human insulin (ATCC).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Pilot Proteomic Analysis of Urinary Extracellular Vesicles Supports the “Toxic Urine Hypothesis” as a Vicious Cycle in Refractory IC/BPS Pathogenesis

doi: 10.3390/ijms27010130

Figure Lengend Snippet: Urinary extracellular vesicles from IC/BPS patients enhance NF-κB activity in HEK293T reporter cells and human bladder epithelial cells. ( A ) Schematic representation of the dual-luciferase reporter assay workflow. HEK293T cells were seeded at 0 h, transfected with NF-κB reporter (Firefly luciferase) and internal control (Renilla luciferase) plasmids at 16 h, stimulated with uEVs (50 µg/mL) or TNFα (10 ng/mL) at 32 h, and assessed for luciferase activity at 56 h. pGL3-Basic vector lacking NF-κB binding sites served as a negative control. ( B ) NF-κB activity represented as normalized Firefly/Renilla luciferase ratios. Data are presented as the mean ± SEM. Statistical significance was determined between the indicated groups: *, p < 0.05. ( C ) Here, 50 µg/mL of uEVs from a healthy donor (H) or an IC patient (Pt) was used to treat human bladder epithelial cells (HBlEpC) for 48 h. The protein expressions of 14-3-3ζ, p-NF-κB p65 at serine536, and total NF-κB p65 were determined via Western blot. GAPDH was used as an internal protein loading control. The numbers below the blots indicate expression levels relative to H uEV–treated cells after normalization to GAPDH.

Article Snippet: For the detection of cellular proteins, human bladder epithelial cells (HBlEpC; Cell Applications, Inc., San Diego, CA, USA) were treated with uEVs for 48 h and lysed with RIPA buffer, followed by SDS-PAGE and immunoblot analysis using the indicated antibodies.

Techniques: Activity Assay, Luciferase, Reporter Assay, Transfection, Control, Plasmid Preparation, Binding Assay, Negative Control, Western Blot, Expressing