human airway epithelial cell line calu 3  (ATCC)

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Concentration‐response analysis of cAMP elevating agents on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to cAMP elevating agent (a and b) forskolin and (c and d) isoproterenol in the basolateral (bottom) compartment for 3 h at various concentrations. (a and c) A dose–response curve for measured apical fluid pH is depicted and (b and d) comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4–5, FSK; n = 5, ISO). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Concentration Assay

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Control experiment in Ringer's solution to validate drug‐induced pH changes in the absence of cells. Various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) were administered to the basolateral (bottom) compartment for 3 h to evaluate the change in pH in the absence of human airway epithelial (Calu‐3) cells. Data presented as means ± SD ( n = 3). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Control

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of various pharmacological interventions on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 7). A two‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001; #### p ≤ 0.0001.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques:

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: CFTR inhibitors administered in the apical or basolateral compartment do not impact apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to CFTR inhibitors (a and c) CFTRinh‐172 and (b and d) GlyH‐101 in the apical (a and b, top) or basolateral (c and d, bottom) compartment for 3 h at various concentrations. cAMP elevating agent forskolin (0.1 μM) was used as a positive control and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 5). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Positive Control

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of cAMP elevating agents FSK and ISO post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Inhibition

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of CFTR potentiator VX‐770 post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with CFTR potentiator VX‐770 (1 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Inhibition

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) Schematic representation of the time-of-drug-addition assay. U2OS cells were treated with (B) 2 µM JG98 or (C) 1 µM JG345 at the indicated time points (conditions 1-8) or with the DMSO control. Virus inoculum (CHIKV-LR at MOI 1) was present for 1.5 h, after which the inoculum was removed, and fresh medium was added with or without the Hsp70 inhibitors or DMSO control. At 9 hpi, the infectious virus particle production was assessed using the plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the non-treated control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) U2OS cells were treated with increasing concentrations of the Hsp70 inhibitors or the equivalent volume of the DMSO control. The metabolic activity of the cells was assessed after 16 hours (h) of treatment using the MTS assay and was normalized to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (B-F) U2OS cells were infected with CHIKV-LR OPY1 at multiplicity of infection (MOI) 1 in the presence of increasing concentrations of the (B) DMSO control (0.002% matches 0.1 µM JG-compound, 0.01% DMSO matches 0.5 µM JG-compound, etc.) or the Hsp70 inhibitors concentrations of (C) JG18, (D) JG40, (E) JG98, (F) JG345. Supernatants were collected at 9 hpi, and the number of infectious CHIKV particles was quantified using plaque assay on Vero-WHO cells. (B-F) Percentage inhibition was determined relative to the DMSO control, and IC50 (which corresponds to a 50% reduction in viral titer) per Hsp70 inhibitor was determined via non-linear regression. Data are presented as mean±SEM from three independent experiments. (B) Statistical differences were determined using One-way ANOVA and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Activity Assay, MTS Assay, Metabolic Labelling, Infection, Plaque Assay, Inhibition

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A) African S27 CHIKV strain or the (B) Caribbean 99659 strain at MOI 1 and simultaneously treated with 10 µM, 10 µM, 2 µM, or 1 µM of JG18, JG40, JG98, or JG345, respectively, or the DMSO control. Production of infectious virus particles was assessed at 9 hpi. (C) U2OS cells were infected with DENV A2 (16681) at MOI 0.5 and treated with 10 µM JG18 or JG40, or the vehicle control. 24 hpi, supernatants were collected and DENV progeny production was analyzed via plaque assay on BHK-21 cells. Data are presented as mean±SEM from three independent experiments, and statistical differences were determined via one-way ANOVA and presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were treated with 2 µM JG98, 1 µM JG345, or the DMSO control during CHIKV infection at MOI 10. Virus production was measured at 9 hpi using a plaque assay. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Control, Infection, Virus, Plaque Assay

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A) The total intracellular vRNA and (B) genomic vRNA copy number were assessed at 4, 6, and 8 hpi in U2OS cells infected with CHIKV at MOI 10 and treated with vehicle control DMSO or 2μM JG98. Intracellular vRNA copies were quantified by RT-qPCR using specific primers against (A) E1 and (B) nsP1. (C) The number of subgenomic RNA copies was determined by subtracting the genomic vRNA copies from the total vRNA copies. Data is presented as mean ± SEM from at least three independent experiments. Student T-test was used to evaluate statistical differences from the DMSO control per timepoint and were presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Gating strategy to determine the percentage of cells positive for CHIKV E2 and capsid expression in U2OS cells treated with JG98, JG345, or the DMSO control. (A) Gating for cells and exclusion of doublets. (B and C) Gating to determine the percentage of cells positive for (B) capsid and (C) E2 based on mock-infected cells.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Expressing, Control, Infection

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: (A-D) U2OS cells were infected with CHIKV at MOI 10 and treated with JG98 (2μM), JG345 (1μM), or the DMSO control. At 9hpi, E2 and capsid expression were assessed using flow cytometry to determine the (A and C) percentage of positive cells and (B and D) mean fluorescent intensity (MFI; geometric mean). (E and F) Representative western blot of capsid, E2, or vinculin expression from protein lysates of U2OS cells infected with CHIKV at MOI 10 and treated for 9 h with Hsp70 inhibitor JG-98 (2μM), JG-345 (1μM), or the DMSO control. (G and H) Quantification of Western blots from three independent experiments. Protein levels are normalized to vinculin and are expressed as relative protein level to vehicle control DMSO. Data are presented as mean±SEM from at least three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Expressing, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: U2OS cells were infected with the (A-C, G-I) CHIKV-LR or the (D-F) CHIKV S27 strain for 9 h in the presence of (A-F) 2 µM JG98, 1 µM JG345, (G-I) 20 µM VER-155008, or the DMSO control. Supernatants were collected and the number of (A, D, and G) infectious particles and (B, E, and H) secreted genome equivalent copies (GECs) were measured using plaque assay and RT-qPCR, respectively. (C, F, and I) Specific infectivity is depicted as the ratio between produced infectious particles and GECs. Data are presented as mean± SEM from three independent experiments. One-way ANOVA was used to evaluate statistical differences from the DMSO control and was given when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Infection, Control, Plaque Assay, Quantitative RT-PCR, Produced

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of the U2OS cells was assessed after 16 hours of treatment with increasing concentrations of the VER155008 or the equivalent volume of the DMSO control. The percentage of metabolically active cells relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). Data are presented as mean±SEM from three independent experiments.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling

Journal: bioRxiv

Article Title: The Heat shock protein 70 machinery is crucial in the production of infectious chikungunya virus progeny

doi: 10.64898/2026.01.26.701664

Figure Lengend Snippet: Metabolic activity of (A) U2OS and (B) HFF-1 cells was assessed after 16 h of treatment with increasing concentrations of JG231 or the equivalent volume of the DMSO control. The percentage of metabolically active cells is presented relative to the DMSO control. A decrease of 15% in metabolically active cells was considered non-toxic (dotted line represents 85%). (C) U2OS cells and (D) HFF-1 cells were infected with CHIKV-LR for 9 h in the presence of 2 µM JG231 or the DMSO control. Supernatants were collected, and the number of infectious particles in the supernatant was determined using the plaque assay. (E and F) Mouse skin explants were infected with 10 6 PFU/mL of the CHIKV 899 strain during treatment with 2 µM JG231 or an equal volume of DMSO. (E) Infectious virus production (tissue culture infectious dose 50% (TCID 50 )) and (F) total virus production (GEC) at 2 dpi is displayed per mg tissue. The dotted line indicates the limit of quantification (LOQ). To evaluate statistical differences from the DMSO control, we used a Student T-test, and differences are presented when p≤0.05 with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Article Snippet: The human bone osteosarcoma epithelial cell line U2OS (ATCC HTB-96) and human foreskin fibroblast cell line HFF-1 (ATCC SCRC-1041) were maintained in Dulbecco’s minimal essential medium (DMEM), high glucose, GlutaMAX tm, and sodium pyruvate (Gibco), supplemented with penicillin-streptomycin (P/S; 100U/mL, Gibco) and 10% or 15% fetal bovine serum (FBS, Lonza), respectively.

Techniques: Activity Assay, Control, Metabolic Labelling, Infection, Plaque Assay, Virus

Journal: Frontiers in Immunology

Article Title: Anti-colorectal cancer effects of IRX4 and sensitivity studies to oxaliplatin

doi: 10.3389/fimmu.2025.1581244

Figure Lengend Snippet: IRX4 Expression is Significantly Downregulated in CRC. (A, B) Immunofluorescence staining of IRX4 (red) and the cell nucleus (blue), showing that IRX4 is primarily localized in the nucleus, with significantly reduced average fluorescence intensity in CRC cells compared to normal intestinal epithelial cells. Scale bar: 50 μm. (C, D) Protein expression of IRX4 in CRC tissues and adjacent normal tissues with statistical analysis. (E, F) Proin expression of IRX4 in CRC cells and normal intestinal epithelial cells. (G) RT-qPCR analysis of IRX4 expression in CRC tissues and corresponding adjacent normal tissues. (H) mRNA expression of IRX4 in four CRC cell lines compared to FHC cells. Data are presented as the mean ± SEM (n = 3). P < 0.05 is considered the difference to be statistically significant.

Article Snippet: The normal human intestinal epithelial cell line FHC and CRC cell lines HCT116, SW480, HCT8, and Caco2 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR