Journal: The Kaohsiung journal of medical sciences

Article Title: Paeoniflorin inhibits colorectal cancer cell stemness through the miR-3194-5p/catenin beta-interacting protein 1 axis.

doi: 10.1002/kjm2.12736

Figure Lengend Snippet: FIGURE 1 PF suppressed CRC cell viability, migration, invasion, stemness, and EMT by deactivating Wnt/β-catenin signaling. HCT116 and SW480 cells were treated with PF (0, 5, 10, 30, and 60 μM). (A) Cell viability was measured using CCK-8 assay. (B–C) Cell migration and invasion were measured using transwell assays. (D–E) β-Catenin, p-β-catenin, Axin2, C-myc, OCT4, Nanog, and Sox2 levels were analyzed using western blot assay. (F) Sphere-formation capacity was measured using the sphere-formation assay. (G) Protein levels of MMP2, MMP9, E-cadherin, and vimentin in CRC cells were determined using western blot assay. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Axin2, axis inhibition protein 2; CCK-8, cell counting kit-8; CRC, colorectal cancer; EMT, epithelial–mesenchymal transition; MMP2, matrix metalloproteinase-2; MMP9, metalloproteinase-9; PF, paeoniflorin; OCT4, octamer binding factor 4; Sox2, SRY-box transcription factor 2.

Article Snippet: Normal human colon epithelial cells and human CRC cells (HCT116 and SW480) were obtained from ATCC (VA, USA) and cultured in DMEM (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco) at 37 C with 5% CO2.

Techniques: Migration, CCK-8 Assay, Western Blot, Tube Formation Assay, Inhibition, Cell Counting, Binding Assay

Journal: The Kaohsiung journal of medical sciences

Article Title: Paeoniflorin inhibits colorectal cancer cell stemness through the miR-3194-5p/catenin beta-interacting protein 1 axis.

doi: 10.1002/kjm2.12736

Figure Lengend Snippet: FIGURE 2 MiR-3194-5p enhanced CRC cell viability, migration, invasion, EMT, and stemness. (A) qRT-PCR was employed to detect miR- 3194-5p expression in CRC cells. We induced miR-3194-5p knockdown/overexpression in CRC cells by transfecting miR-3194-5p inhibitor/ mimics into the cells. (B) miR-3194-5p expression in cells was examined using qRT-PCR. (C) CCK-8 assay was conducted to examine cell viability. (D–E) Cell migration and invasion were measured using transwell assays. (F) Protein levels of MMP2, MMP9, E-cadherin, and vimentin in CRC cells were measured using western blot assay. (G) OCT4, Nanog, and Sox2 protein levels in CRC cells were measured using western blot assay. (H) Sphere-formation capacity was determined using the sphere-formation assay. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001. CCK-8, cell counting kit-8; CRC, colorectal cancer; EMT, epithelial–mesenchymal transition; MMP2, matrix metalloproteinase-2; MMP9, metalloproteinase-9; OCT4, octamer binding factor 4; qRT- PCR, quantitative real-time polymerase chain reaction; Sox2, SRY-box transcription factor 2.

Article Snippet: Normal human colon epithelial cells and human CRC cells (HCT116 and SW480) were obtained from ATCC (VA, USA) and cultured in DMEM (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco) at 37 C with 5% CO2.

Techniques: Migration, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, CCK-8 Assay, Western Blot, Tube Formation Assay, Cell Counting, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 3. NHE1 stimulation leads to ERM activation. A, HRPT cell NHE1 was stimulated by an established NH4Cl (50 mM for 25 min at 37 °C) pulse protocol. NH4Cl causes cytosol acidification and, after placement in serum-free, NH4Cl-free medium (time 0), NHE activa- tion. Whole cell lysates were harvested at the indicated times, resolved by 4–20% SDS-PAGE to allow separation between individual ERM protein bands, and immunoblotted with anti-phospho-ERM antibodies (upper panel). The blots were stripped and reprobed with anti-ezrin IgG (lower panel). B, because NH4Cl may activate NHEs other than NHE1, HRPT cells were osmotically stimulated with isotonic medium with 100 mM sucrose, which activates NHE1 but inhibits NHE3. Whole cell lysates were then resolved by 8% SDS-PAGE and probed for the ERM- activated state by immunoblotting with anti-phospho-ERM antibodies (upper panel, doublets appear as a single large band) and for ezrin expression with anti-ezrin IgG (lower panel). C, PS120 cells, which do not express NHEs (upper panel) or PS120 transiently transfected to express wild-type NHE1 (lower panel), were preincubated with EIPA (5 M, 1 h). NHE1 was then stimulated by NH4Cl protocol. Total cell lysates were harvested at indicated times and immunoblotted for phos- pho-ERM expression. Both blots contain equal protein content/lane and were developed from the same film.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activation Assay, SDS Page, Western Blot, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 4. NHE1 and ERM directly interact in the cytoskeleton fraction. To determine whether NHE1 and ERM physically interact, HRPT NHE1 was stimulated by NH4Cl and then permitted to recover for indicated time periods (A) or by sucrose for the indicated times (B). The cells were lysed in Nonidet P-40 buffer and centrifuged, and cy- toskeleton-rich pellets were lysed in 1% SDS buffer. The lysates were immunoprecipitated with affinity-purified anti-NHE1 IgG, resolved by 8% SDS-PAGE, and immunoblotted with anti-phospho-ERM IgG.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Immunoprecipitation, Affinity Purification, SDS Page

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 5. NHE1-ezrin interaction protects against apoptosis. A, to determine whether NHE1 regulates ERM proteins and PI3K, NHE1 was stimulated with sucrose (6 h at 37 °C) addition to medium in LLC-PK1 cell lines stably transfected with wild-type ezrin, constitutively active (T567D) ezrin, dominant negative ezrin (T567A), amino-terminal ezrin (N-term) ezrin, or Y353F ezrin that prevents PI3K interaction. B, wild-type, T567D, and T567A cells were incubated with or without STS (5 M for 6 h). *, p 0.05 compared with wild-type, STS-treated cells by ANOVA. C, HRPT cells were preincubated with PI3K inhibitor wortmannin (300 nM for 30 min at 37 °C) or LY294002 (20 M for 30 min at 37 °C), and then treated with STS (5 M for 6 h at 37 °C). Apoptosis was assayed by annexin V labeling in all experiments. *, p 0.05 compared with STS-treated cells by ANOVA.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Stable Transfection, Transfection, Dominant Negative Mutation, Incubation, Labeling

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 6. Apoptosis is regulated by Akt activity. A, HRPT cells were pretreated with Akt inhibitor (Akt I; 30 M at 37 °C) for 1 h and then incubated with STS (5 M for 6 h at 37 °C). The cells were stained with annexin V for apoptosis analysis. Similar results were observed in hypertonic sucrose-treated LLC-PK1 and HRPT cells (not shown). B, LLC-PK1 cells were transfected with 50 nM Akt siRNA or 50 nM 8 integrin siRNA (control siRNA) for 48 h. The cells were then incubated with or without STS (5 M for 6 h at 37 °C), and lysates were probed for Akt expression by immunoblot analysis. The blots were stripped and reprobed for -tubulin as a control for protein loading and nonspecific RNA interference effects. C, LLC-PK1 cells were treated as described in B, but intact cells were instead assayed for apoptosis by annexin V labeling. Similar results were observed when apoptosis was determined by DAPI staining of chromatin (not shown).

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activity Assay, Incubation, Staining, Transfection, Control, Expressing, Western Blot, Labeling

Journal: Journal of Biological Chemistry

Article Title: The NHE1 Na+/H+ Exchanger Recruits Ezrin/Radixin/Moesin Proteins to Regulate Akt-dependent Cell Survival

doi: 10.1074/jbc.m400814200

Figure Lengend Snippet: FIG. 7. NHE1 stimulation regulates Akt activity. A, HRPT cell NHE1 was activated by NH4Cl pulse protocol. Whole cell lysates were harvested at indicated times and immunoblotted with anti-phospho- Akt antibodies (upper panel). The blots were stripped and reprobed with anti-Akt1 IgG (lower panel). B, HRPT cells were preincubated with EIPA (5 M, 1 h, 37 °C), and then NHE1 was activated by sucrose (100 mM for 1 h at 37 °C) addition to medium. Whole cell lysates were probed for Akt activity by immunoblotting with anti-phospho-Akt antibodies (upper panel); the blots were stripped and reprobed with anti-Akt1 antibodies (lower panel) as a loading control. The histogram depicts densitometry data from three phospho-Akt blots, expressed as the means S.E. *, p 0.05 compared with other groups by ANOVA.

Article Snippet: Cell Lines—The HRPT human renal proximal tubule epithelial cell line (a gift from Dr. L. Racusen, Johns Hopkins University) and LLCPK1 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (Invitrogen) plus 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin-fungizone solution (Sigma).

Techniques: Activity Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Development of high-affinity, single-domain protein binders for neutralizing household allergens

doi: 10.1101/2025.08.03.668213

Figure Lengend Snippet: (a) Thermostability profile of AVA-1-1-C3 as a representative protein, as determined by differential scanning fluorimetry. (b-g) Cytotoxicity of primary human cells following 48-hour exposure to AVA-1-1-C3 or an equivalent concentration of bovine serum albumin (BSA), evaluated using the CellTiter-Glo® (CTG) luminescent cell viability assay. Cell types included human epithelial keratinocytes (HEK, b), human dermal fibroblasts (HDF, c), human follicle dermal papilla cells (HFDPC, d), human renal cortical epithelial cells (HRCEPC, e), human skeletal muscle cells (HSKMC, f), and human dermal microvascular endothelial cells (HDMEC, g). No significant differences in cell viability were observed. Data in (b-g) represents mean ± s.d. and are representative of two experimental replicates.

Article Snippet: For in vitro toxicity studies, primary cells including human dermal fibroblasts (HDF, Cell Applications, Cat# 106K-05a, lot 1632), human epidermal keratinocytes (HEK, Cell Applications, Cat# 102-05a, lot 2146), human dermal microvascular endothelial cells (HDMEC, PromoCell, Cat# C-12210, lot 483Z001.3), human skeletal muscle cells (HSKMC, Cell Applications, Cat# 150K-05a, lot 3507), and human renal cortical epithelial cells (HRCEpC, Promocell, Cat# C-12660, lot 501Z019.23).

Techniques: Concentration Assay, Cell Viability Assay

Journal: Scientific Reports

Article Title: Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model

doi: 10.1038/s41598-019-48092-6

Figure Lengend Snippet: ( A ) Human corneal epithelial cells (HCEC) were subjected to in vitro scratch assay. Representative images from scratch wound healing assay of HCEC showing time course of corneal re-epithelialization (abrasion 1 mm) in vitro model at different time intervals (0, 6, 12, 18 & 24 hours) after post wounding. Scratch wounds were made in confluent cultures of corneal epithelial cells. The red lines define the area lacking cells where as arrows indicating the movement of cells towards closing the wound. The images were analyzed by Image J software (National Institutes of Health [NIH], Bethesda, MD, USA) with Scale bar = 100 µm. Images were captured at 4X magnification using camera-equipped inverted microscope (Olympus Onvented, DSR-012). ( B ) In vitro wound healing of migrating corneal epithelia in confluent monolayer of HCEC showing linear phase of wound healing at time intervals 6, 12, 18 and 24 hours while wound was closed at 24 hours of post wounding. Cellular migration was calculated using one way ANOVA by GraphPad (7.0) with significance of p < 0.001. Each value is representing three individual experiments, error bars indicates SDM.

Article Snippet: The Human corneal epithelial cell (HCEC), were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: In Vitro, Wound Healing Assay, Software, Inverted Microscopy, Migration

Journal: Scientific Reports

Article Title: Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model

doi: 10.1038/s41598-019-48092-6

Figure Lengend Snippet: Western Blot analysis detected expression of cdk10 and ETS2 in HCEC: ( A) The expression level of cdk10 and ETS2 at active hours of migration (18 hours) related to non-migrating sample with beta- Actin as loading control. lane 1 NM, non-migrating; lane 2, migrating at 18 hrs. (B) Quantification and intensity measurement of relative protein expression were analyzed by Quantity One software (Bio-Rad, USA). Histograms are generated using GraphPad Prism software (7.04).Values are expressed as ± SD, significance (P < 0.005) was calculated using one way ANOVA test statistically.

Article Snippet: The Human corneal epithelial cell (HCEC), were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Western Blot, Expressing, Migration, Control, Software, Generated

Journal: Thoracic Cancer

Article Title: S‐ and R‐Carvedilol Prevent Benzo(a)pyrene‐Induced Lung Carcinogenesis

doi: 10.1111/1759-7714.70109

Figure Lengend Snippet: Effects of S‐ and R‐carvedilol on BPDE‐induced lung epithelial cell transformation: Benzo(a)pyrene diol epoxide (BPDE), an active metabolite of B(a)P, was used to transform BEAS‐2B cells. (A) & (B) The cells were pre‐treated with 5 μM S‐ and R‐carvedilol, 5 μM atenolol, 5 μM propranolol (Prop), and 5 μM ICI‐118551 (ICI) for 2 h, and then treated with 0.2 μM of BPDE for 1 h. The cells were then cultured with 5 μM of the drugs for 7 days. Afterward, they were seeded in soft agar in a 96‐well plate (2000 cells/well) containing 5 μM of the drugs in the top layer of agar. The cell colonies were counted under a microscope after 10 days of incubation. (C) Representative images were taken using GelCount of the wells containing colonies that grew on agar after 10 days of incubation. The plotted data are represented as mean ± SD, n = 6 to 8. The one‐way ANOVA followed by Dunnett's multiple comparison test was used to assess statistical differences. Statistical significance was denoted as ****: P < 0.0001 and **: P < 0.01.

Article Snippet: The BEAS‐2B cell line, originating from normal human bronchial epithelial cells, was obtained from ATCC (catalog number CRL‐9609).

Techniques: Transformation Assay, Cell Culture, Microscopy, Incubation, Comparison

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Basic concept of human lung-on-a-chip that accurately mimics structural and multicellular complexities of lung tissue. The figure details the core concept of the multi-compartmentalized lung-on-a-chip platform, illustrating the interactive multicellular communication between the respiratory airways and the adjacent supporting stromal chambers. This platform integrates various cellular elements and 3D structural complexities composed of natural polymers. A thin porous barrier embedded with vascular endothelial cells links the respiratory airway chamber with the supporting stromal chambers, enabling bidirectional cellular communication through the exchange of growth factors and cytokines. Furthermore, to accurately mimic the intricate multicellular interactions within lung tissue microenvironments, the two chambers are encompassed by media channels lined with human vascular cells (A) . The 3D mold for the human lung-on-a-chip system, designed to accurately replicate the structural characteristics of the lung tissue. The mold was produced using PLA-based 3D printing technology. Polydimethylsiloxane (PDMS) was subsequently injected into the 3D printed mold, and the resulting chip platform was extracted following polymerization process (B) . To replicate the physiological characteristics and multicellular diversity of lung tissue, the PDMS-based lung-on-a-chip platform was populated with multiple human lung cell types, including small airway epithelial cells, stromal cells, vascular endothelial cells, and macrophages. These cells were combined with a natural polymer mixture (hyaluronic acid and collagen), along with blood coagulating factors (thrombin and fibrinogen). The spindle-shaped respiratory airway chamber, which spans the entire chip, is populated with human small airway epithelial cells. This airway chamber is encircled by adjacent stromal chambers that house human stromal cells and macrophages, facilitating a comprehensive representation of the lung tissue microenvironment (C) . The PDMS-based lung-on-a-chip platform was designed in a rectangular shape, measuring 100 mm in length, 70 mm in central diameter, and 7 mm in height. The chip platform was fabricated to replicate the microenvironment of the lung tissue and facilitate multicellular communication between the respiratory airways and adjacent supporting stromal chambers (D) .

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Produced, Injection, Polymer

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Examination of the sustained viability and metabolic functions of multiple cells embedded within each segment of the lung-on-a-chip model. The spatial arrangement of diverse cells integrated into each section of the chip was investigated by first cultivating the tissue structures in a cell-specific culture medium for 24 h, followed by staining with the DNA-targeting fluorochrome 4′,6-diamidino-2-phenylindole (DAPI). The patterns of cellular distribution across each tissue structure were assessed with by fluorescent microscopy. Different types of lung tissue cells are integrated within each compartment of the lung-on-a-chip, including small airway epithelial cells (A) , stromal cells (B) , vascular endothelial cells (C) , and macrophages (D) . Following this, the cells were cultured in specialized medium tailored for each cell type and incubated for 1, 7, 14, 21, or 28 days post-integration. Cell viability was assayed using the live & death analysis, using fluorescent dyes to distinctly mark live (green) and dead (red) cells. The prolonged viability of cells in each chamber was then assessed by fluorescent imaging. Seventy or greater percent of the cells embedded in each compartment of the chip remained viable for 28 days, as indicated by the aforementioned green and red fluorescence. Additionally, each compartment of the chip was incubated in a serum-free setting with CCK-8 solution for 48 h. The metabolic functions of the integrated cells were then evaluated by determining the optical density at 450 nm. All experiments were performed in triplicate. Significant differences are indicated as *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Staining, Microscopy, Cell Culture, Incubation, Imaging, Fluorescence, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Preservation of distinct cellular characteristics in the natural polymer-based tissue architecture. An assessment was performed to determine if different cells retained their molecular properties following integration into each compartment of the chip. This involved cultivating the cells in tailored medium for a week and subsequently examining them with established biomarkers specific to each cell type. Staining was performed on human small airway epithelial cells for cytokeratin 18 and 19 (A) , while lung stromal cells were examined for fibronectin and vimentin (B) . Additionally, vascular endothelial cells were labeled for PECAM1 and vWF (C) , and macrophages were analyzed using CD11b and CD68 (D) . Each experiment was conducted three times. The nuclei in each field were stained with DAPI.

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Preserving, Polymer, Staining, Labeling

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Functional assessments of the respiratory airway and adjacent stromal chambers within the chip: critical protein expression and secretion, and mucin production. To assess whether incorporated cells retained their distinctive properties within respiratory airway chamber in the chip, samples were cultured in a specific culture medium for 7 days before being evaluated with targeted biomarkers (A) . Within the natural polymer-based 3D microenvironment of the chip, human small airway epithelial cells exhibited the expression of critical proteins, including MUC5AC, MUC5B, and SPDEF, at both the protein (B) and mRNA (C) levels. Periodic acid-Schiff (PAS) staining to evaluate whether embedded human airway epithelial cells within a chip platform could properly produce glycogen, mucopolysaccharides, and other carbohydrate-rich macromolecules (D). To determine whether the integrated cells preserved their unique characteristics within the stromal compartment of the chip, the samples were maintained in a designated culture medium for a week and subsequently analyzed using specific biomarkers (E) . In the chip's 3D microenvironment constructed from natural polymers, human stromal cells showed expression levels of key proteins, such as COL1A1, fibronectin 1, and laminin α1, at the protein (F) and mRNA (G) levels. To evaluate if human stromal cells embedded in a chip platform were capable of adequately secreting essential proteins into the chamber, each cell-loaded and cell-free chamber was cultured in a specific medium tailored for the respective cell types for 7 days. Following this, the medium was switched to a serum-free variant. After 48 h of incubation, the medium was collected and tested for the presence of secreted fibronectin and laminin from the stromal chamber (H). All experiments were performed in triplicate. Significant differences are indicated as * p < 0.05, ** p < 0.005, and *** p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Functional Assay, Expressing, Cell Culture, Polymer, Staining, Construct, Variant Assay, Incubation

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Discovery and confirmation of a reliable indicator to anticipating lung toxicity in human small airway epithelial cells. Depiction of the primary stages for the RNA-Seq methodology, comprising the experimental layout, alignment of read, measurement, and graphical representation (A) . Comprehensive RNA-seq results displayed in a heatmap that illustrates variations in gene expression between the control groups and those exposed to low (5 ng/ml) and high (7 ng/ml) dioxin doses. The heatmap highlights genes with upregulated (red) or downregulated (green) expression relative to the mRNA levels in the control groups (B) . KEGG pathway analyses to identify the potential interconnected pathways and functionalities impacted by toxin exposure (C) . Within the array of genes showing differential expression, an observable positive correlation was detected between the marked elevation of SERPINB2 expression and toxic exposure in human small airway epithelial cells (D and E) . Using real-time PCR and western blot analysis confirmation of increased SERPINB2 levels following exposure to both low and high doses of the toxin (F) . β-Actin was used as the internal protein control, and PPIA was used as the housekeeping gene for real-time PCR. All experiments were performed in triplicate. The data are presented as the means ± SDs. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: RNA Sequencing Assay, Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Validating the consistency of the selected indicator (SERPINB2) for lung toxicity in human small airway epithelial cells. Diagrammatic representation outlining how SERPINB2 regulates toxicant-triggered detrimental impacts in small airway epithelial cells (A) . Human small airway epithelial cells were transfected with a specific SERPINB2 shRNA and were subsequently exposed or not exposed to dioxin (5 ng/ml) for 72 h. The detrimental impacts on cell growth were evaluated through an MTT assay (B) . Depleting SERPINB2 effectively neutralized the detrimental effects of dioxin on the migration of small airway epithelial cells. This outcome was confirmed using a Transwell migration/invasion assay (C) and western blot analysis with antibodies targeting MMP-2 and MMP-9 (D) . Human small airway epithelial cells underwent transfection with a targeted SERPINB2 shRNA and were either exposed or not exposed to 5 ng/ml of dioxin. Following this treatment, alterations in DNA fragmentation associated with apoptosis and caspase-3 mediated apoptotic activities were assessed through nuclear staining (E) and western blot analysis (F) , respectively. β-actin was used as an internal control. All experiments were performed in triplicate. Data are presented as means ± standard deviations. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Transfection, shRNA, MTT Assay, Migration, Invasion Assay, Western Blot, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Development of a fluorescent-based detection platform linked with a toxicity detection marker in the respiratory airway compartment of the lung-on-a-chip model. A fluorescent detection platform tagged with SERPINB2 was effectively incorporated into human small airway epithelial cells. Toxin-stimulated SERPINB2 activity was evident as fluorescence of into GFP. Consequently, the lung toxicity of specific drug candidates can be assessed in both qualitative and quantitative manner through the measurement of fluorescent signal strength (A) . Human small airway epithelial cells underwent stable transfection with a GFP-tagged SERPINB2 detection vector, which emits a green color. Following exposure to 5 ng/ml dioxin, immunostaining indicated a significant increase in SERPINB2 activity, which was manifest as green fluorescence in these cells (B) . This fluorescence reporting platform was implemented in human small airway epithelial cells accurately positioned within the respiratory airway compartment of the lung-on-a-chip. The spatial distribution patterns of the incorporated human small airway epithelial cells within respiratory airway chamber then examined through a fluorescent imaging system (C) . Exposure to 5 ng/ml dioxin markedly enhanced SERPINB2 activity, which was evident as green fluorescence within the respiratory airway compartment of the chip (D) . The data are presented as the means ± standard deviations. *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Marker, Activity Assay, Fluorescence, Stable Transfection, Plasmid Preparation, Immunostaining, Imaging

Journal: International Journal of Biological Sciences

Article Title: Advanced Lung-on-a-Chip Technology: Mimicking the Complex Human Lung Microenvironment

doi: 10.7150/ijbs.105702

Figure Lengend Snippet: Exposure to different types of toxins triggers the activation of SERPINB2, resulting in emission of green fluorescence within the respiratory airway chamber of the chip. A GFP-tagged SERPINB2 fluorescent detection platform was effectively incorporated into human small airway epithelial cells. Subsequently, these cells were positioned in the appropriate compartments of the chip (A) . To validate the consistency of the toxicity assessment marker (SERPINB2)-tagged fluorescent detection platform, toxins were added to the respiratory airway chamber of the chip. The toxins included aristolochic acid I (10 μM), benzidine (10 μM), benzo[a]pyrene (2 μM), semustine (0.5 mM), TPA (5 nM), 1,2-dichloropropane (100 mM), 1,3-butadiene (10 mM), and 4,4'-methylenebis (5 μM). The response of SERPINB2 to various types of toxic exposure was assessed both subjectively and quantitatively through the measurement of the resultant fluorescent signal strength (B) . Significant differences are indicated as follows: *, p < 0.05; **, p < 0.005; and ***, p < 0.001 (two-sample t-test).

Article Snippet: Human small airway epithelial cells were obtained from PromoCell (C-12642; Heidelberg, Germany) and expanded in Small Airway Epithelial Cell Growth Basal Medium (SAGM, Cat. No.: CC-4124; No.: CC-3119; Lonza, Basal, Switzerland) supplemented with SAGM (Cat. No.: CC-4124) and 10% FBS.

Techniques: Activation Assay, Fluorescence, Marker