human foetal human lung fibroblasts  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA induces a concentration-dependent increase in [Ca 2+ ] i in WI-38 human lung fibroblasts. ( A ) Representative [Ca 2+ ] i traces from Fura-2/AM-loaded WI-38 cells exposed to increasing AA concentrations: 1 µM (grey trace), 3 µM (pink trace) and 30 µM (blue trace). In this and the following figures, the horizontal lines indicate the duration of agonist and drug application. For clarity, the baseline of each Ca 2+ tracing has been normalised to zero. ( B ) The non-cumulative AA concentration–response relationship. The data points (squares) represent the mean ± standard error of the mean (SEM) of the [Ca 2+ ] i amplitudes, expressed in A.U., plotted against the logarithm of the AA concentration. The sigmoidal curve (blue line) was obtained by fitting the data to Equation (1) (see ), yielding an EC 50 value of 3.42 µM. The R 2 value for the curve fit was 0.95. AA concentrations are labelled above each data point, and n denotes the number of cells analysed, with the number of independent experimental replicates given in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA-induced Ca 2+ signalling in WI-38 human lung fibroblasts involves both intracellular Ca 2+ release and Ca 2+ extracellular entry. ( A ) Representative Ca 2+ traces from WI-38 cells stimulated with 30 µM AA in PSS (blue) or 0Ca 2+ (black). In 0Ca 2+ experiments, extracellular Ca 2+ was removed 200 s before the application of AA to prevent Ca 2+ entry (dotted line). For clarity, the baseline of the traces was adjusted to zero. ( B ) Mean ± SEM of peak [Ca 2+ ] i amplitudes obtained in 0Ca 2+ compared to PSS, expressed in A.U. ( C ) Representative traces showing the response to different AA concentrations (3 µM, pink trace; 30 µM, blue trace) in 0Ca 2+ , followed by the restoration of extracellular Ca 2+ in the continued presence of AA. For clarity, the baseline of the traces was adjusted to zero. The initial transient peak represents Ca 2+ release from intracellular stores, whereas the subsequent sustained response corresponds to Ca 2+ entry from the extracellular medium. ( D ) Mean ± SEM amplitudes of intracellular Ca 2+ release and extracellular Ca 2+ entry at the indicated AA concentrations, expressed in A.U. Statistical comparisons for panels ( B , D ) were performed using the Mann–Whitney U test (****, p < 0.0001). n denotes the number of cells analysed, and the number of independent experimental replicates used is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA evokes intracellular Ca 2+ release in WI-38 human lung fibroblasts via the GPR40–PLCβ–IP 3 Rs pathway. ( A ) Representative traces showing the Ca 2+ response to AA (30 µM) in PSS (blue), and after pretreatment with the GPR40 antagonist GW1100 (10 µM, 10 min, PSS + GW, pink trace). The traces also show the responses to AA in a 0Ca 2+ (black) and with GW1100 pretreatment in 0Ca 2+ (0Ca 2+ + GW, green trace). For clarity, the baseline of the traces was adjusted to zero. ( B ) Summary data (mean ± SEM) of peak amplitudes for the conditions shown in ( A ), expressed in A.U. Statistical analysis was performed using the Mann–Whitney U test (****, p < 0.0001). ( C ) Representative traces showing the response to AA (30 µM; blue) and after pretreatment with pertussis toxin (PTX; 100 ng/mL, 30 min, pink trace), a Gi/o protein inhibitor. For clarity, the baseline of the traces was adjusted to zero. ( D ) Summary data (mean ± SEM) for the conditions shown in ( C ), expressed in A.U. The Mann–Whitney U test was used (ns, p > 0.05). ( E ) Representative traces showing the response to AA (30 µM, blue) and following treatment with the PLC inhibitor U73122 (10 µM, 30 min, pink trace), or its inactive analogue U73343 (10 µM, 30 min, grey trace). For clarity, the baseline of the traces was adjusted to zero. ( F ) Summary data (mean ± SEM) for the conditions shown in ( E ), expressed in A.U. Statistical analysis was performed using the Kruskal–Wallis test (****, p < 0.0001; ns, p > 0.05). ( G ) Representative traces of the AA response in PSS (blue) and 0Ca 2+ (black), and after IP 3 Rs inhibition with 2-APB (50 µM, 20 min) in PSS (PSS + 2-APB, pink trace) and 0Ca 2+ (0Ca 2+ + 2-APB, green trace). For clarity, the baseline of the traces was adjusted to zero. ( H ) Summary data (mean ± SEM) for conditions in ( G ), expressed in A.U. Statistical analysis was performed using the Kruskal–Wallis test (ns, p > 0.05; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001). The n value represents the number of cells analysed, and the number of independent experimental replicates is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: MANN-WHITNEY, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA-induced intracellular Ca 2+ release in WI-38 human lung fibroblasts depends on IP 3 Rs in the ER and TPCs in lysosomes, but not on mitochondrial Ca 2+ uptake via the mitochondrial Ca 2+ uniporter. ( A ) A representative trace showing the AA-induced Ca 2+ signal following depletion of the ER stores with CPA (10 µM, orange trace) under 0Ca 2+ conditions. Complete ER depletion was verified by the absence of any detectable Ca 2+ response to 300 µM ATP. ( B ) Representative traces under 0Ca 2+ conditions following treatment with GPN (100 µM; 20 min, purple trace), a lysosomal disruptor. ( C ) Representative traces under 0Ca 2+ conditions following pre-incubation with the TPCs inhibitor Trans-Ned 19 (Trans-Ned; 100 µM, 45 min, grey trace). ( D ) A summary graph showing the mean ± SEM of the peak Ca 2+ amplitudes in the cells under 0Ca 2+ conditions: untreated control cells (black); CPA-treated cells (orange); GPN-treated cells (purple); and Trans-Ned–treated cells (grey), expressed in A.U. Statistical analysis using Kruskal–Wallis test revealed a significant reduction in Ca 2+ release for all treatment groups compared to the control group (****, p < 0.0001). ( E ) Representative traces comparing control cells (blue) and cells treated with ruthenium red (RR; 10 µM, pink), an inhibitor of mitochondrial Ca 2+ uptake. For clarity, the baseline of the traces was adjusted to zero. ( F ) Mean ± SEM peak Ca 2+ amplitudes in control and RR-treated cells, expressed in A.U. Statistical comparison using the Mann–Whitney U test indicated no significant difference (ns, p > 0.05). The n value represents the number of cells analysed, and the number of independent experimental replicates is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Incubation, Control, Comparison, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: Functional expression of TRPV4 channels and their contribution to AA-induced Ca 2+ signalling in WI-38 human lung fibroblasts. ( A ) Representative traces showing increases in [Ca 2+ ] i , evoked by the selective TRPV4 agonist GSK1016790A in PSS (GSK; 20 nM, purple trace), under 0Ca 2+ conditions (0Ca 2+ + GSK; 20 µM, black trace), and in the presence of the TRPV4 antagonist RN-1734 (RN; 20 µM, 60 min, green trace). The arrow indicates the time of GSK application. For clarity, the baseline of the traces has been adjusted to zero. ( B ) Summary data showing the mean ± SEM of peak [Ca 2+ ] i amplitudes in the cells under GSK conditions (purple bar), in 0Ca 2+ conditions plus GSK (black bar), and in the presence of the TRPV4 antagonist RN-1734 (green bar), expressed in A.U. ( C ) Representative [Ca 2+ ] i traces showing the Ca 2+ response to AA (blue trace) in the absence or presence of RN-1734 (RN; 20 µM, 60 min, pink trace). The arrow indicates the time of AA application. For clarity, the baseline of the traces has been adjusted to zero. ( D ) Summary data showing the mean ± SEM of peak [Ca 2+ ] i amplitudes in the absence (blue bar) or presence (pink bar)of RN-1734 ( C ), expressed in A.U. A statistical comparison was performed using the Mann–Whitney test (****, p < 0.0001) for panels ( B , D ). Numbers inside the bars indicate the number of cells analysed ( n ), as indicated in parentheses for three independent experiments.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Functional Assay, Expressing, Comparison, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA-activates Ca 2+ entry through TRPV4 and SOCE in WI-38 human lung fibroblasts. Representative traces showing [Ca 2+ ] i responses recorded in WI-38 cells evoked by AA 30 µM ( A , B ) or CPA ( C ) under 0Ca 2+ . Complete ER Ca 2+ depletion was confirmed by the absence of an intracellular Ca 2+ response to ATP (300 µM). After ATP washout, extracellular Ca 2+ was restored by re-addition of PSS, either in the continued presence of AA (PSS + AA, 30 µM, ( A )), alone (PSS, ( B )), or in the presence of CPA (PSS + CPA, 10 µM, ( C )). ( D ) Mean ± SEM of peak Ca 2+ amplitudes corresponding to Ca 2+ release under 0Ca 2+ conditions ( left panel) and Ca 2+ entry following Ca 2+ re-addition ( right panel) for the conditions showed in ( A – C ), expressed in A.U. Statistical analysis was performed using the Kruskal–Wallis test. Significance levels are indicated (ns, not significant; ** p < 0.001; **** p < 0.0001). ( E ) Representative traces illustrating AA-evoked extracellular Ca 2+ entry following restoration of extracellular Ca 2+ . The blue trace corresponds to PSS re-addition in the continued presence of AA and represents the control condition. The remaining traces show AA-induced Ca 2+ entry recorded in the presence of the SOCE inhibitor BTP-2 (20 µM, green trace), the selective TRPV4 inhibitor RN-1734 (RN, 20 µM, grey trace), or the combined treatment (RN + BTP-2; pink trace). For clarity, the baseline of the traces has been adjusted to zero. ( F ) Mean ± SEM of peak Ca 2+ entry amplitudes under the indicated conditions in ( E ), expressed in A.U. Statistical analysis was performed using the Kruskal–Wallis test (* p < 0.05; **** p < 0.0001). The n value represents the number of cells analysed, and the number of independent experimental replicates is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA stimulates NO production in WI-38 human lung fibroblasts via a Ca 2+ -dependent pathway. ( A ) Representative traces showing NO production, as measured by DAF-FM fluorescence, in response to AA (30 µM, blue). For comparison, traces are shown for the NO donor sodium nitroprusside (SNP; 500 µM, pink), AA stimulation in the presence of the NO scavenger cPTIO (10 µM, green), the non-selective eNOS inhibitor L-NAME (100 µM, 60 min, grey), and the selective eNOS inhibitor L-NIO (50 µM, 60 min, red). The arrow indicates the time of AA (30 µM) stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( B ) Summary bar graph of the peak NO production amplitudes from the experiments shown in ( A ). Data are presented as mean ± SEM, expressed in A.U. Statistical significance was determined by a Kruskal–Wallis test (****, p < 0.0001). ( C ) Representative traces showing AA-induced NO production under control conditions (AA; blue) and following the inhibition of key Ca 2+ signalling components with: GW1100 (10 µM, 10 min, purple) and 2-APB (50 µM, 20 min, brown). The arrow indicates the time of AA (30 µM) stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( D ) Summary bar graph of the peak NO production amplitudes from experiments in ( C ), expressed in A.U. Data are presented as mean ± SEM. Statistical significance was determined by a Kruskal–Wallis test compared to the control (**, p < 0.01; ****, p < 0.0001). ( E ) Representative traces of AA-induced NO production in the presence of a 0Ca 2+ solution (black trace); of SOCE inhibition by BTP-2 (20 µM, 20 min, orange trace); and TRPV4 inhibition by RN-1734 (20 µM, 60 min, magenta trace); and under combined inhibition with RN-1734 plus BTP-2 at the same concentrations described above (yellow trace). The arrow indicates the time of AA (30 µM) stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( F ) Summary bar graph of the peak NO production amplitudes under the conditions shown in ( E ) expressed in A.U. Data are presented as mean ± SEM. Statistical significance was determined by a Kruskal–Wallis test compared to the control (**, p < 0.01; ****, p < 0.0001). The n value represents the number of cells analysed, and the number of independent experimental replicates is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Fluorescence, Comparison, Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: The Ca 2+ –NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

doi: 10.3390/ijms27094016

Figure Lengend Snippet: AA-induced ROS generation in WI-38 human lung fibroblasts requires intracellular Ca 2+ release, Ca 2+ influx, and NO production. ( A ) Representative traces showing ROS production induced by AA (30 µM, blue) and H 2 O 2 (100 µM, pink). Pre-treatment with the antioxidant NAC (1 mM, 60 min, cyan). The arrow indicates the time of stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( B ) Quantification of peak ROS fluorescence from ( A ), expressed in A.U. Mean ± SEM. Kruskal–Wallis test; ****, p < 0.0001. ( C ) Representative traces of AA-induced ROS production and its modulation by Ca 2+ signalling inhibitors. Cells were stimulated with AA (30 µM) in PSS (blue trace), following pretreatment with the GPR40 antagonist GW1100 (GW; 10 µM, 10 min, purple), the IP 3 Rs inhibitor 2-APB (50 µM; 20 min, brown). The arrow indicates the time of stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( D ) Quantification of peak ROS fluorescence intensities from ( C ), expressed in A.U. Data are the mean ± SEM of ROS production peak amplitudes. The statistical test used was the Kruskal–Wallis test: ****, p < 0.0001. ( E ) Representative traces of AA-induced ROS production under Ca 2+ -free solution (0Ca 2+ ; black) or in the presence of the SOCE inhibitor BTP-2 (20 µM, 20 min, orange) or the TRPV4 inhibitor RN-1734 (20 µM, 60 min, magenta), or the combined treatment (RN + BTP-2; yellow trace). The arrow indicates the time of AA (30 µM) stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( F ) Quantification of peak ROS fluorescence intensities from ( E ). Data are the mean ± SEM of the peak ROS production amplitudes, expressed in A.U. The statistical test used was the Kruskal–Wallis test: **, p < 0.01; ****, p < 0.0001. ( G ) A representative trace showing the effect of NO pathway inhibition on AA-induced ROS production. Signals were recorded in response to AA (30 µM) under control conditions (blue), after NO scavenging with cPTIO (10 µM, 60 min, green), followed by non-selective NOS inhibition with L-NAME (100 µM, 60 min, grey), and subsequently by selective eNOS inhibition with L-NIO (20 µM, 60 min, red). The arrow indicates the time of AA (30 µM) stimulation. For clarity, the baseline of the traces has been adjusted to zero. ( H ) Quantification of peak ROS fluorescence intensities from ( G ). The data are the mean ± SEM of the peak ROS production amplitudes, expressed in A.U. The statistical test used was the Kruskal–Wallis test: ***, p < 0.001; ****, p < 0.0001. For all panels, the n value represents the number of cells analysed, and the number of independent experimental replicates is indicated in parentheses.

Article Snippet: Human foetal human lung fibroblasts (WI-38; CCL-75TM) were obtained from the American Type Culture Collection (ATCC ® , Manassas, VA, USA).

Techniques: Fluorescence, Inhibition, Control