non tumorigenic wi 38 cell lines  (ATCC)


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    Structured Review

    ATCC non tumorigenic wi 38 cell lines
    Anti -SARS-CoV-2 activity and cytotoxicity against <t>normal</t> <t>Vero-E6</t> <t>cells</t> for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).
    Non Tumorigenic Wi 38 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Quercitrin loaded cyclodextrin based nanosponge as a promising approach for management of lung cancer and COVID-19"

    Article Title: Quercitrin loaded cyclodextrin based nanosponge as a promising approach for management of lung cancer and COVID-19

    Journal: Journal of Drug Delivery Science and Technology

    doi: 10.1016/j.jddst.2022.103921

    Anti -SARS-CoV-2 activity and cytotoxicity against normal Vero-E6 cells for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).
    Figure Legend Snippet: Anti -SARS-CoV-2 activity and cytotoxicity against normal Vero-E6 cells for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).

    Techniques Used: Activity Assay

    2) Product Images from "A BDNF-TrkB autocrine loop enhances senescent cell viability"

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33709-8

    ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p
    Figure Legend Snippet: ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p

    Techniques Used: Activation Assay, Western Blot, Transfection, Cell Counting, RNA Sequencing Assay, Quantitative RT-PCR, Blocking Assay

    BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p
    Figure Legend Snippet: BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p

    Techniques Used: Marker, Quantitative RT-PCR, Transfection, Western Blot, Expressing, RNA Sequencing Assay, Immunofluorescence, Staining

    Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .
    Figure Legend Snippet: Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .

    Techniques Used: Concentration Assay

    Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Inhibition, Activity Assay

    BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Transfection, Blocking Assay, Microscopy, Activity Assay, Western Blot

    Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Western Blot, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence

    3) Product Images from "A BDNF-TrkB autocrine loop enhances senescent cell viability"

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33709-8

    ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p
    Figure Legend Snippet: ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p

    Techniques Used: Activation Assay, Western Blot, Transfection, Cell Counting, RNA Sequencing Assay, Quantitative RT-PCR, Blocking Assay

    BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p
    Figure Legend Snippet: BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p

    Techniques Used: Marker, Quantitative RT-PCR, Transfection, Western Blot, Expressing, RNA Sequencing Assay, Immunofluorescence, Staining

    Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .
    Figure Legend Snippet: Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .

    Techniques Used: Concentration Assay

    Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Inhibition, Activity Assay

    BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Transfection, Blocking Assay, Microscopy, Activity Assay, Western Blot

    Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p
    Figure Legend Snippet: Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p

    Techniques Used: Western Blot, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence

    4) Product Images from "Clathrin-associated AP-1 controls termination of STING signalling"

    Article Title: Clathrin-associated AP-1 controls termination of STING signalling

    Journal: Nature

    doi: 10.1038/s41586-022-05354-0

    AP-1 binding directs STING degradation to limit immune activation. a , Airyscan imaging of HeLa STING cells that were stimulated for 2.5 h with diABZI. The colocalization of pSTING with AP-1γ is quantified by Manders’ colocalization coefficients. One representative cell is shown, and the quantification is the mean ± s.e.m. of n = 7 cells from one out of four independent experiments. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). b , HeLa STING cells were stimulated with diABZI. After immunoprecipitation (IP) with anti-Flag antibody, cells were analysed by western blot. c , HeLa STING cells were stimulated with diABZI for 2 h, infected with HSV-1 (multiplicity of infection (MOI) = 10) for 6 h or transfected with 90mer dsDNA (1 µg) for 3 h or 1 µM 2’3’-cGAMP for 6 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. d , HeLa cells transfected with siRNAs were stimulated with diABZI and analysed by western blot. Vinculin was used as a loading control. e , Induction of IFNB1 , IFIT1 , IFIT2 and IFIT3 expression was assessed by quantitative PCR with reverse transcription (RT–qPCR) in HeLa cells transfected with siRNAs and treated with diABZI for 3 h. Ratios of IFNB1 , IFIT1 , IFIT2 and IFIT3 mRNA versus GAPDH mRNA normalized to the untreated groups of each condition. Data are mean ± s.d. of three technical replicates. P values were obtained by two-tailed Student’s t -test. f , WI-38 human fibroblasts transfected with siRNAs for three days were stimulated with diABZI and analysed by western blot. GAPDH was used as a loading control. One representative example of three ( a , d – f ) or two ( b , c ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition ( d , f ). Source Data
    Figure Legend Snippet: AP-1 binding directs STING degradation to limit immune activation. a , Airyscan imaging of HeLa STING cells that were stimulated for 2.5 h with diABZI. The colocalization of pSTING with AP-1γ is quantified by Manders’ colocalization coefficients. One representative cell is shown, and the quantification is the mean ± s.e.m. of n = 7 cells from one out of four independent experiments. White arrows point at occurrences of pSTING. Scale bars, 4 µm (left) or 1 µm (magnified panels). b , HeLa STING cells were stimulated with diABZI. After immunoprecipitation (IP) with anti-Flag antibody, cells were analysed by western blot. c , HeLa STING cells were stimulated with diABZI for 2 h, infected with HSV-1 (multiplicity of infection (MOI) = 10) for 6 h or transfected with 90mer dsDNA (1 µg) for 3 h or 1 µM 2’3’-cGAMP for 6 h. After immunoprecipitation with anti-Flag antibody, samples were analysed by western blot. d , HeLa cells transfected with siRNAs were stimulated with diABZI and analysed by western blot. Vinculin was used as a loading control. e , Induction of IFNB1 , IFIT1 , IFIT2 and IFIT3 expression was assessed by quantitative PCR with reverse transcription (RT–qPCR) in HeLa cells transfected with siRNAs and treated with diABZI for 3 h. Ratios of IFNB1 , IFIT1 , IFIT2 and IFIT3 mRNA versus GAPDH mRNA normalized to the untreated groups of each condition. Data are mean ± s.d. of three technical replicates. P values were obtained by two-tailed Student’s t -test. f , WI-38 human fibroblasts transfected with siRNAs for three days were stimulated with diABZI and analysed by western blot. GAPDH was used as a loading control. One representative example of three ( a , d – f ) or two ( b , c ) independent experiments is shown. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition ( d , f ). Source Data

    Techniques Used: Binding Assay, Activation Assay, Imaging, Immunoprecipitation, Western Blot, Infection, Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    5) Product Images from "Antimicrobial, Antiviral, and In-Vitro Cytotoxicity and Mosquitocidal Activities of Portulaca oleracea-Based Green Synthesis of Selenium Nanoparticles"

    Article Title: Antimicrobial, Antiviral, and In-Vitro Cytotoxicity and Mosquitocidal Activities of Portulaca oleracea-Based Green Synthesis of Selenium Nanoparticles

    Journal: Journal of Functional Biomaterials

    doi: 10.3390/jfb13030157

    Cell viability percentages of WI-38 and HepG2 cells after treatment with various concentrations of Se-NPs. The data are represented as mean ± SE ( n = 3). Different letters on the column at the same concentration indicate that mean values are significantly different ( p ≤ 0.05).
    Figure Legend Snippet: Cell viability percentages of WI-38 and HepG2 cells after treatment with various concentrations of Se-NPs. The data are represented as mean ± SE ( n = 3). Different letters on the column at the same concentration indicate that mean values are significantly different ( p ≤ 0.05).

    Techniques Used: Concentration Assay

    Morphological alterations of WI-38 and HepG2 cells in response to treatment with various concentrations of Se-NPs for 24 h under inverted microscopy. Magnification: 60×.
    Figure Legend Snippet: Morphological alterations of WI-38 and HepG2 cells in response to treatment with various concentrations of Se-NPs for 24 h under inverted microscopy. Magnification: 60×.

    Techniques Used: Inverted Microscopy

    6) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    7) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    8) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    9) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    10) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    11) Product Images from "TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line"

    Article Title: TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line

    Journal: Biomedicines

    doi: 10.3390/biomedicines10092240

    Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.
    Figure Legend Snippet: Immunoblot analysis of expression of NF-κB and AP-1 signaling pathways members: pERK, p-p38 and IKBα on ( A ) WI-38 cells infected with TLR5 WT and TLR5 N592S adenoviral constructs and ( B ) H1299 cells infected with TLR5 N592S and TLR5 N592S adenoviral constructs. Cells were infected with an adenoviral vector carrying either TLR5 WT or TLR5 N592S gene and stimulated with a flagellin (50 ng/mL) at five different time points (15, 30, 60, and 120 min). Vinculin was used as endogenous control and for normalization. Biological replicates were made and data shown are representative of two independent experiments.

    Techniques Used: Expressing, Infection, Construct, Plasmid Preparation

    Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.
    Figure Legend Snippet: Gen set enrichment analysis showing the positive or negative effect of TLR5 N592S variant on gene set enrichment in H1299 and WI-38 cell line. Size of the dots represents ratio of DEGs in comparison to total number of genes in a given set.

    Techniques Used: Variant Assay

    Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.
    Figure Legend Snippet: Results of the gene ontology analysis of the differentially expressed genes in H1299 and WI-38 cell lines overexpressing TLR5 N592S variant. Statistically enriched process, function and component GO terms are shown and ranked based on the false discovery q-value.

    Techniques Used: Variant Assay

    NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values
    Figure Legend Snippet: NF-κB and AP-1 transcription factors activity in WI-38 cell lines transfected with WT and N592S TLR5-variant that are stimulated with flagellin (flag) or unstimulated (NS). Positive control (PC) is a mixture of a constitutively expressing GFP construct, constitutively expressing Firefly luciferase construct, and constitutively expressing Renilla luciferase construct, while negative control (NC) is a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct. Data shown are representative of three independent experiments. Statistical analysis was performed by using GraphPad, t -test; p -values

    Techniques Used: Activity Assay, Transfection, Variant Assay, Positive Control, Expressing, Construct, Luciferase, Negative Control

    Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.
    Figure Legend Snippet: Comprehensive transcriptome analysis of the H1299 and WI-38 cell lines overexpressing TLR5 WT or TLR5 N592S gene variants. Volcano plot of differentially expressed transcripts in H122 ( A ) and WI-38 ( B ) cell line. Heat map describing expression of statistically significant DEGs with dendograms indicating hierarchical clustering between H1299 ( C ) and WI-38 ( D ) cell lines.

    Techniques Used: Expressing

    12) Product Images from "Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells"

    Article Title: Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.991659

    Effect of consecutive histamine applications on Ca 2+ signal in WI-38 human lung fibroblasts WI-38. (A) Representative recording of a triple application of histamine (300 μM) in one cell, each followed by a washout with physiological saline solution (PSS). (B) Mean ± SE of peak and plateau amplitudes of the Ca 2+ transient evoked by 3 consecutive histamine applications: first application (1st), second application (2nd), third application (3rd). (C) Mean ± SE of the number of oscillations measured over 400 s after each histamine application. The number in the figure represents the number of cells studied. Comparison between groups was performed using the Kruskal–Wallis test (* = p ≤ 0.05; ns = no statistically relevant differences between groups).
    Figure Legend Snippet: Effect of consecutive histamine applications on Ca 2+ signal in WI-38 human lung fibroblasts WI-38. (A) Representative recording of a triple application of histamine (300 μM) in one cell, each followed by a washout with physiological saline solution (PSS). (B) Mean ± SE of peak and plateau amplitudes of the Ca 2+ transient evoked by 3 consecutive histamine applications: first application (1st), second application (2nd), third application (3rd). (C) Mean ± SE of the number of oscillations measured over 400 s after each histamine application. The number in the figure represents the number of cells studied. Comparison between groups was performed using the Kruskal–Wallis test (* = p ≤ 0.05; ns = no statistically relevant differences between groups).

    Techniques Used:

    Blocking Ca 2+ entry through VOCs does not affect histamine-evoked intracellular Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals in the presence of nickel (10 μM). (B) Typical recording of histamine-evoked Ca 2+ signals in the presence of nifedipine (10 μM). Comparison between groups was performed using the Mann-Whitney test (ns = not statistics differences between groups). (C) Mean ± SE of the peak and plateau amplitudes of the Ca 2+ response to histamine (300 μM) in absence (dark green bar) or presence of the non-specific VOC inhibitor, nickel 10 μM (blue bar). (D) Mean ± SE of the peak and plateau amplitudes of the Ca 2+ response to histamine (300 μM) in absence (dark green bar) or presence of the specific VOC inhibitor, nifedipine 10 μM (blue bar). (E) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in presence (dark green bar) or presence of the unspecific VOC inhibitor, nickel 10 μM (blue bar). (F) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in presence (dark green bar) or absence of the specific VOC inhibitor, nifedipine 10 μM (blue bar).
    Figure Legend Snippet: Blocking Ca 2+ entry through VOCs does not affect histamine-evoked intracellular Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals in the presence of nickel (10 μM). (B) Typical recording of histamine-evoked Ca 2+ signals in the presence of nifedipine (10 μM). Comparison between groups was performed using the Mann-Whitney test (ns = not statistics differences between groups). (C) Mean ± SE of the peak and plateau amplitudes of the Ca 2+ response to histamine (300 μM) in absence (dark green bar) or presence of the non-specific VOC inhibitor, nickel 10 μM (blue bar). (D) Mean ± SE of the peak and plateau amplitudes of the Ca 2+ response to histamine (300 μM) in absence (dark green bar) or presence of the specific VOC inhibitor, nifedipine 10 μM (blue bar). (E) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in presence (dark green bar) or presence of the unspecific VOC inhibitor, nickel 10 μM (blue bar). (F) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in presence (dark green bar) or absence of the specific VOC inhibitor, nifedipine 10 μM (blue bar).

    Techniques Used: Blocking Assay, MANN-WHITNEY

    Effect of SOCE blockade on histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals and effect of the application of 10 µM La 3+ ( n = 79). (B) Typical recording of histamine-evoked Ca 2+ signals and effect of the application of 10 μM Gd 3+ ( n = 40).
    Figure Legend Snippet: Effect of SOCE blockade on histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals and effect of the application of 10 µM La 3+ ( n = 79). (B) Typical recording of histamine-evoked Ca 2+ signals and effect of the application of 10 μM Gd 3+ ( n = 40).

    Techniques Used:

    Concentration-dependent effect of histamine on Ca 2+ signalling in WI-38 human lung fibroblasts. (A) Typical intracellular Ca 2+ recordings in WI-38 cells loaded with FURA-2/AM exposed to different histamine concentrations ranging from 100 nM to 1 mM. The baseline of Ca 2+ tracings has been shifted to avoid their overlapping for representation proposes. (B) Non-cumulative concentration-response relationship. Data points are the mean ± SE of the initial Ca 2+ peak amplitude (black circles) or plateau amplitude (blue circles) plotted against the logarithm of histamine concentration. The continuous curves were obtained by fitting the data to Eq. 1 , as shown in Materials and methods, which yielded EC 50 values of 4.96 and 5.25 µM for peak amplitude (black line) and plateau amplitude (blue line), respectively. Data points were obtained of at least 19 cells. (C) Percentage of cells that presented each of the Ca 2+ response patterns indicated in function of histamine concentrations applicated to WI-38 fibroblasts. (D) Data points are the mean ± SE of the number of oscillations measured over the first 400 s after histamine application, plotted against the logarithm of histamine concentration. The continuous curve was obtained by fitting the data to Eq. 1 , as shown in Materials and methods, which yielded EC 50 values of 2.38 µM.
    Figure Legend Snippet: Concentration-dependent effect of histamine on Ca 2+ signalling in WI-38 human lung fibroblasts. (A) Typical intracellular Ca 2+ recordings in WI-38 cells loaded with FURA-2/AM exposed to different histamine concentrations ranging from 100 nM to 1 mM. The baseline of Ca 2+ tracings has been shifted to avoid their overlapping for representation proposes. (B) Non-cumulative concentration-response relationship. Data points are the mean ± SE of the initial Ca 2+ peak amplitude (black circles) or plateau amplitude (blue circles) plotted against the logarithm of histamine concentration. The continuous curves were obtained by fitting the data to Eq. 1 , as shown in Materials and methods, which yielded EC 50 values of 4.96 and 5.25 µM for peak amplitude (black line) and plateau amplitude (blue line), respectively. Data points were obtained of at least 19 cells. (C) Percentage of cells that presented each of the Ca 2+ response patterns indicated in function of histamine concentrations applicated to WI-38 fibroblasts. (D) Data points are the mean ± SE of the number of oscillations measured over the first 400 s after histamine application, plotted against the logarithm of histamine concentration. The continuous curve was obtained by fitting the data to Eq. 1 , as shown in Materials and methods, which yielded EC 50 values of 2.38 µM.

    Techniques Used: Concentration Assay

    Histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts do not involve Gα i/o activation but require PLC and ER Ca 2+ release. (A) Typical recording of the Ca 2+ response to histamine (300 μM) in fibroblasts preincubated for 30 min with 100 ng/ml pertussis toxin (PT). (B) Mean ± SE of the peak amplitude of histamine-evoked Ca 2+ transients in the absence (green bar) and presence of pertussis toxin (gray bar). Comparison between groups was performed using the Student’s t-test (ns = no statistically relevant differences between groups). (C) Typical recording of the effect of histamine (300 μM) on Ca 2+ signal in cells pretreated for 15 min with U73122 (10 μM), a specific PLC inhibitor. (D) Typical recording of the Ca 2+ response to histamine (300 μM) in cells pretreated for 15 min with U73343 (10 μM), an inactive analog of U73122. (E) Representative recording of the Ca 2+ signal evoked by histamine (300 μM) in cells pretreated with CPA (10 μM) in the absence of extracellular Ca 2+ (0Ca 2+ ). (F) Mean ± SE of the peak Ca 2+ response to histamine (300 μM; green bar) in the presence of U73122 10 μM (black bar), U73343 10 μM (blue bar) and CPA 10 μM (orange bar not visible, marked with a red arrow). The numbers in the figure represent the number of cells studied. Statistical comparison between groups was performed using ANOVA test (* = p ≤ 0.05).
    Figure Legend Snippet: Histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts do not involve Gα i/o activation but require PLC and ER Ca 2+ release. (A) Typical recording of the Ca 2+ response to histamine (300 μM) in fibroblasts preincubated for 30 min with 100 ng/ml pertussis toxin (PT). (B) Mean ± SE of the peak amplitude of histamine-evoked Ca 2+ transients in the absence (green bar) and presence of pertussis toxin (gray bar). Comparison between groups was performed using the Student’s t-test (ns = no statistically relevant differences between groups). (C) Typical recording of the effect of histamine (300 μM) on Ca 2+ signal in cells pretreated for 15 min with U73122 (10 μM), a specific PLC inhibitor. (D) Typical recording of the Ca 2+ response to histamine (300 μM) in cells pretreated for 15 min with U73343 (10 μM), an inactive analog of U73122. (E) Representative recording of the Ca 2+ signal evoked by histamine (300 μM) in cells pretreated with CPA (10 μM) in the absence of extracellular Ca 2+ (0Ca 2+ ). (F) Mean ± SE of the peak Ca 2+ response to histamine (300 μM; green bar) in the presence of U73122 10 μM (black bar), U73343 10 μM (blue bar) and CPA 10 μM (orange bar not visible, marked with a red arrow). The numbers in the figure represent the number of cells studied. Statistical comparison between groups was performed using ANOVA test (* = p ≤ 0.05).

    Techniques Used: Activation Assay, Planar Chromatography

    Dissection of histaminergic receptors (HR) responsible for histamine-evoked Ca 2+ signal in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signal in the presence of the H1R antagonist, pyrilamine (100 μM). (B) Typical recording of the histamine-evoked Ca 2+ signal in the presence of the H2R antagonist, ranitidine (50 μM). (C) Typical recording of the histamine-evoked Ca 2+ signal in the presence of the H3R antagonist, clobenpropit (50 μM). (D) Typical recording of histamine-evoked Ca 2+ signal in the presence of H4R antagonist, JNJ-7777120 (10 μM). All histaminergic antagonists were preincubated for 30 min prior to histamine application (incubation time not shown). For experiments showed in (A,C) , arachidonic acid (AA 50 μM) was applied after histamine application to corroborate cell viability. (E) Mean ± SE of the peak amplitude of the Ca 2+ transient evoked by histamine (300 μM) in the absence (Ctrl) and presence of the HR antagonists: pyrilamine 100 μM (H1), ranitidine 50 μM (H2), clobenpropit 50 μM (H3) and JNJ-7777120 10 μM (H4). The numbers in the figure represents the number of cells studied. Comparison between groups was performed using the Kruskal–Wallis test (* = p ≤ 0.05) (ns = no statistically relevant differences between groups).
    Figure Legend Snippet: Dissection of histaminergic receptors (HR) responsible for histamine-evoked Ca 2+ signal in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signal in the presence of the H1R antagonist, pyrilamine (100 μM). (B) Typical recording of the histamine-evoked Ca 2+ signal in the presence of the H2R antagonist, ranitidine (50 μM). (C) Typical recording of the histamine-evoked Ca 2+ signal in the presence of the H3R antagonist, clobenpropit (50 μM). (D) Typical recording of histamine-evoked Ca 2+ signal in the presence of H4R antagonist, JNJ-7777120 (10 μM). All histaminergic antagonists were preincubated for 30 min prior to histamine application (incubation time not shown). For experiments showed in (A,C) , arachidonic acid (AA 50 μM) was applied after histamine application to corroborate cell viability. (E) Mean ± SE of the peak amplitude of the Ca 2+ transient evoked by histamine (300 μM) in the absence (Ctrl) and presence of the HR antagonists: pyrilamine 100 μM (H1), ranitidine 50 μM (H2), clobenpropit 50 μM (H3) and JNJ-7777120 10 μM (H4). The numbers in the figure represents the number of cells studied. Comparison between groups was performed using the Kruskal–Wallis test (* = p ≤ 0.05) (ns = no statistically relevant differences between groups).

    Techniques Used: Dissection, Incubation

    Involvement of InsP 3 R in histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of the Ca 2+ response to histamine (300 µM) in fibroblasts pre-incubated with 2-APB (50 μM) for 20 min [orange trace, 2-APB (+)] and its control [dark green trace, 2-APB (-)]. (B) Typical recording of histamine-evoked Ca 2+ signal in absence of extracellular Ca 2+ (0Ca 2+ ) after pre-incubation of fibroblasts for 20 min with 2-APB (50 μM) [gray trace, 2-APB (+)] and its control [blue trace, 2-APB (-)]. In (A , B ) basal Ca 2+ levels were aligned for comparative purposes. (C) Mean ± SE of the peak amplitude of the Ca 2+ response to histamine (300 µM) in normal extracellular Ca 2+ and in absence (dark green bar) or presence of 50 μM 2-APB (orange bar). Mean ± SE of the peak amplitude of the Ca 2+ response to histamine (300 µM) in absence of extracellular Ca 2+ (0Ca 2+ ) and in absence (blue bar) or presence of 50 μM 2-APB (gray bar). Statistic comparison between groups was performed using Mann-Whitney and t-Student test respectively (* = p ≤ 0.05). (D) Mean ± SE of the number of oscillations evoked by histamine (300 µM) in normal extracellular Ca 2+ and in absence (dark green bar) or presence of 50 μM 2-APB (orange bar). Mean ± SE of the number of oscillations evoked by histamine (300 µM) in the absence of extracellular Ca 2+ (0Ca 2+ ) and in absence (blue bar) or presence of 50 μM 2-APB (gray bar, gray bar not visible, marked with a red arrow). Statistical comparison between groups was performed using Mann-Whitney and t -Student test respectively (* = p ≤ 0.05). The numbers in the figure represents the number of cells studied. (E) Typical recording of the Ca 2+ signal evoked by histamine and the effect of 2-APB application.
    Figure Legend Snippet: Involvement of InsP 3 R in histamine-evoked Ca 2+ signals in WI-38 human lung fibroblasts. (A) Typical recording of the Ca 2+ response to histamine (300 µM) in fibroblasts pre-incubated with 2-APB (50 μM) for 20 min [orange trace, 2-APB (+)] and its control [dark green trace, 2-APB (-)]. (B) Typical recording of histamine-evoked Ca 2+ signal in absence of extracellular Ca 2+ (0Ca 2+ ) after pre-incubation of fibroblasts for 20 min with 2-APB (50 μM) [gray trace, 2-APB (+)] and its control [blue trace, 2-APB (-)]. In (A , B ) basal Ca 2+ levels were aligned for comparative purposes. (C) Mean ± SE of the peak amplitude of the Ca 2+ response to histamine (300 µM) in normal extracellular Ca 2+ and in absence (dark green bar) or presence of 50 μM 2-APB (orange bar). Mean ± SE of the peak amplitude of the Ca 2+ response to histamine (300 µM) in absence of extracellular Ca 2+ (0Ca 2+ ) and in absence (blue bar) or presence of 50 μM 2-APB (gray bar). Statistic comparison between groups was performed using Mann-Whitney and t-Student test respectively (* = p ≤ 0.05). (D) Mean ± SE of the number of oscillations evoked by histamine (300 µM) in normal extracellular Ca 2+ and in absence (dark green bar) or presence of 50 μM 2-APB (orange bar). Mean ± SE of the number of oscillations evoked by histamine (300 µM) in the absence of extracellular Ca 2+ (0Ca 2+ ) and in absence (blue bar) or presence of 50 μM 2-APB (gray bar, gray bar not visible, marked with a red arrow). Statistical comparison between groups was performed using Mann-Whitney and t -Student test respectively (* = p ≤ 0.05). The numbers in the figure represents the number of cells studied. (E) Typical recording of the Ca 2+ signal evoked by histamine and the effect of 2-APB application.

    Techniques Used: Incubation, MANN-WHITNEY

    Heterogeneity in the Ca 2+ response elicited by histamine in WI-38 human lung fibroblasts. Application of histamine (300 μM) elicited different Ca 2+ signal patterns in FURA-2 AM-loaded WI-38 human lung fibroblasts. The intracellular Ca 2+ signal consisted of (A) a rapid Ca 2+ peak (spike) (19.28% of cells analyzed) which could be followed by (B) Ca 2+ oscillations (64.70%) (peak-oscillations), (C) sustained plateau, superimposed by Ca 2+ oscillations (13.39%) (peak-plateau-oscillations) or (D) only a plateau (0.65%) (peak-plateau). In this and the following figures, histamine was added at the time indicated by the horizontal bar drawn over the Ca 2+ signal recording.
    Figure Legend Snippet: Heterogeneity in the Ca 2+ response elicited by histamine in WI-38 human lung fibroblasts. Application of histamine (300 μM) elicited different Ca 2+ signal patterns in FURA-2 AM-loaded WI-38 human lung fibroblasts. The intracellular Ca 2+ signal consisted of (A) a rapid Ca 2+ peak (spike) (19.28% of cells analyzed) which could be followed by (B) Ca 2+ oscillations (64.70%) (peak-oscillations), (C) sustained plateau, superimposed by Ca 2+ oscillations (13.39%) (peak-plateau-oscillations) or (D) only a plateau (0.65%) (peak-plateau). In this and the following figures, histamine was added at the time indicated by the horizontal bar drawn over the Ca 2+ signal recording.

    Techniques Used:

    Transcriptomic characterization of the Ca 2+ handling machinery in WI-38 fibroblasts. Gel electrophoresis of the PCR products are shown. Electrophoresis was performed as indicated in Materials and Methods. The PCR products were of the expected size: InsP 3 R1, 180 bp; InsP 3 R 2 , 158 bp; InsP 3 R 3 , 173 bp; RyR 1 , 75 bp; TRPC1, 307 bp; TRPC3, 336 bp; TRPC4, 300 bp; TRPC5, 221 bp; TRPC6, 341 bp; STIM2, 186 bp; and Orai3, 159 bp. No signal was observed for STIM1 and Orai1. MW, molecular weight markers. B, reaction without the template.
    Figure Legend Snippet: Transcriptomic characterization of the Ca 2+ handling machinery in WI-38 fibroblasts. Gel electrophoresis of the PCR products are shown. Electrophoresis was performed as indicated in Materials and Methods. The PCR products were of the expected size: InsP 3 R1, 180 bp; InsP 3 R 2 , 158 bp; InsP 3 R 3 , 173 bp; RyR 1 , 75 bp; TRPC1, 307 bp; TRPC3, 336 bp; TRPC4, 300 bp; TRPC5, 221 bp; TRPC6, 341 bp; STIM2, 186 bp; and Orai3, 159 bp. No signal was observed for STIM1 and Orai1. MW, molecular weight markers. B, reaction without the template.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Electrophoresis, Molecular Weight

    Effect of extracellular Ca 2+ removal on histamine-evoked Ca 2+ signal in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals in the absence of extracellular Ca 2+ (0Ca 2+ ). (B) Mean ± SE of basal [Ca 2+ ] i in fibroblasts exposed to an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the t-Student test (* = p ≤ 0.05). (C) Mean ± SE of peak and plateau amplitudes of the Ca 2+ signal evoked by histamine in fibroblasts expose to an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the t -Student test for peak amplitude data and Mann-Whitney test for plateau amplitude data (* = p ≤ 0.05). (D) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the Mann-Whitney test (* = p ≤ 0.05). The numbers in the figures represents the number of cells studied.
    Figure Legend Snippet: Effect of extracellular Ca 2+ removal on histamine-evoked Ca 2+ signal in WI-38 human lung fibroblasts. (A) Typical recording of histamine-evoked Ca 2+ signals in the absence of extracellular Ca 2+ (0Ca 2+ ). (B) Mean ± SE of basal [Ca 2+ ] i in fibroblasts exposed to an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the t-Student test (* = p ≤ 0.05). (C) Mean ± SE of peak and plateau amplitudes of the Ca 2+ signal evoked by histamine in fibroblasts expose to an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the t -Student test for peak amplitude data and Mann-Whitney test for plateau amplitude data (* = p ≤ 0.05). (D) Mean ± SE of the number of oscillations recorded during the first 400 s after histamine application in an extracellular solution with (dark green bar) or without extracellular Ca 2+ (blue bar). Comparison between groups was performed using the Mann-Whitney test (* = p ≤ 0.05). The numbers in the figures represents the number of cells studied.

    Techniques Used: MANN-WHITNEY

    13) Product Images from "Senescent cells develop PDK4-dependent hypercatabolism and form an acidic microenvironment to drive cancer resistance"

    Article Title: Senescent cells develop PDK4-dependent hypercatabolism and form an acidic microenvironment to drive cancer resistance

    Journal: bioRxiv

    doi: 10.1101/2022.08.29.505761

    Genotoxicity induces senescence of human stromal cells which display upregulation of PDK4 and a full spectrum SASP. a. Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line PSC27 by microarray. Cell lysates were collected for analysis 7 d after treatment. CTRL, control. RAD, radiation. BLEO, bleomycin. HP, hydrogen peroxide. Red highlighted, typical soluble factors of the SASP. Microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature 23 . b. Quantitative RT-PCR to detect PDK4 expression after PSC27 cells were subject to individual treatment as indicated. Cell lysates were collected for measurement 7 d after establishment of stable cell sublines or completion of in vitro treatment. Signals normalized to CTRL. RS, replicative senescence. p16, lentiviral transduction of human tumor suppressor p16 INK4a . RAS, lentiviral transduction of human oncogene HRAS G12V . c. Immunoblot analysis of PDK4 expression in stromal cells as delineated in ( b ). GAPDH, loading control. d. Comparative RT-PCR assay of PDK4 expression after treatment of PSC27 or prostate epithelial cells by agents as indicated. Cell lysates were collected for measurement 7 d after treatment. Signals normalized to CTRL. BPH1, M12, PC3, DU145, LNCaP and VCaP, human epithelial lines of prostate origin. e. Comparative RT-PCR assay of PDK4 expression in human stromal cells 7 d after treatments performed as indicated. WI38, HFL1, HBF1203 and BJ, human stromal lines of different origins. f. A time course RT-PCR assessment of the expression of PDK4 and a set of typical SASP factors (MMP1, WNT16B, SFRP2, SPINK1, MMP3, CXCL8, EREG, ANGPTL4 and AREG) after drug treatment of PSC27 cells in vitro . Numeric numbers indicate the individual days after treatment (indexed at the top line). g. Immunoblot measurement of PDK4 expression at protein level at the individual timepoints as indicated. β-actin, loading control. h. Comparative appraisal of human PDK family expression at transcript level in PSC27 cells after BLEO treatment. Signals normalized to untreated sample per gene. CXCL8, experimental control as a hallmark SASP factor. i. Immunoblot assessment of the expression of PDK4 family members at protein level after BLEO treatment. β-actin, loading control. Data are representative of 3 independent experiments. P values were calculated by Student’s t -test ( b , e , f , h ) and one-way ANOVA ( d ). ^, P > 0.05. *, P
    Figure Legend Snippet: Genotoxicity induces senescence of human stromal cells which display upregulation of PDK4 and a full spectrum SASP. a. Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line PSC27 by microarray. Cell lysates were collected for analysis 7 d after treatment. CTRL, control. RAD, radiation. BLEO, bleomycin. HP, hydrogen peroxide. Red highlighted, typical soluble factors of the SASP. Microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature 23 . b. Quantitative RT-PCR to detect PDK4 expression after PSC27 cells were subject to individual treatment as indicated. Cell lysates were collected for measurement 7 d after establishment of stable cell sublines or completion of in vitro treatment. Signals normalized to CTRL. RS, replicative senescence. p16, lentiviral transduction of human tumor suppressor p16 INK4a . RAS, lentiviral transduction of human oncogene HRAS G12V . c. Immunoblot analysis of PDK4 expression in stromal cells as delineated in ( b ). GAPDH, loading control. d. Comparative RT-PCR assay of PDK4 expression after treatment of PSC27 or prostate epithelial cells by agents as indicated. Cell lysates were collected for measurement 7 d after treatment. Signals normalized to CTRL. BPH1, M12, PC3, DU145, LNCaP and VCaP, human epithelial lines of prostate origin. e. Comparative RT-PCR assay of PDK4 expression in human stromal cells 7 d after treatments performed as indicated. WI38, HFL1, HBF1203 and BJ, human stromal lines of different origins. f. A time course RT-PCR assessment of the expression of PDK4 and a set of typical SASP factors (MMP1, WNT16B, SFRP2, SPINK1, MMP3, CXCL8, EREG, ANGPTL4 and AREG) after drug treatment of PSC27 cells in vitro . Numeric numbers indicate the individual days after treatment (indexed at the top line). g. Immunoblot measurement of PDK4 expression at protein level at the individual timepoints as indicated. β-actin, loading control. h. Comparative appraisal of human PDK family expression at transcript level in PSC27 cells after BLEO treatment. Signals normalized to untreated sample per gene. CXCL8, experimental control as a hallmark SASP factor. i. Immunoblot assessment of the expression of PDK4 family members at protein level after BLEO treatment. β-actin, loading control. Data are representative of 3 independent experiments. P values were calculated by Student’s t -test ( b , e , f , h ) and one-way ANOVA ( d ). ^, P > 0.05. *, P

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Stable Transfection, In Vitro, Transduction, Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "Silica-Coated Magnetic Nanoparticles for Vancomycin Conjugation"

    Article Title: Silica-Coated Magnetic Nanoparticles for Vancomycin Conjugation

    Journal: ACS Omega

    doi: 10.1021/acsomega.2c03226

    In vitro applications: (a) MTT cytotoxicity assay on WI-38 cells (fibroblasts human cell line from lung tissue) for VM, APTMS–TEOS-coated MNPs (MNPs–TEOS–APTMS), VM-conjugated MNPs (MNPs–TEOS–APTMS–DSS–VM). (b) Antibacterial activity using the agar diffusion method for VM and VMNPs against S. aureus and MRSA. (c) MIC for VM and VMNPs against S. aureus and MRSA. Image of the representative agar plate of MNPs–TEOS–APTMS against (d) MRSA and (e) S. aureus , VM at 25 μg/mL against (f) MRSA and (g) S. aureus , VMNPs at an equivalent VM concentration of 25 μg/mL against (h) MRSA and (i) S. aureus .
    Figure Legend Snippet: In vitro applications: (a) MTT cytotoxicity assay on WI-38 cells (fibroblasts human cell line from lung tissue) for VM, APTMS–TEOS-coated MNPs (MNPs–TEOS–APTMS), VM-conjugated MNPs (MNPs–TEOS–APTMS–DSS–VM). (b) Antibacterial activity using the agar diffusion method for VM and VMNPs against S. aureus and MRSA. (c) MIC for VM and VMNPs against S. aureus and MRSA. Image of the representative agar plate of MNPs–TEOS–APTMS against (d) MRSA and (e) S. aureus , VM at 25 μg/mL against (f) MRSA and (g) S. aureus , VMNPs at an equivalent VM concentration of 25 μg/mL against (h) MRSA and (i) S. aureus .

    Techniques Used: In Vitro, MTT Assay, Cytotoxicity Assay, Activity Assay, Diffusion-based Assay, Concentration Assay

    15) Product Images from "Tumor suppressor Hypermethylated in Cancer 1 represses expression of cell cycle regulator E2F7 in human primary cells"

    Article Title: Tumor suppressor Hypermethylated in Cancer 1 represses expression of cell cycle regulator E2F7 in human primary cells

    Journal: bioRxiv

    doi: 10.1101/2022.07.25.501405

    Expression profiles of human WI38 cells after knockdown of the HIC1 gene. (A) Down-regulation of HIC1 mRNA in WI38 cells at 24, 48 and 72 h after transfection with HIC1-specific siRNAs from Ambion (Amb HIC1 siRNA) or Dharmacon (Dhar HIC1 siRNA); transfection with non-coding siRNA was used as a control. Graphs show the results of RT-qPCR analysis of WI38 cells transfected with the indicated siRNAs; the obtained quantification cycle (Cq) values were normalized to the UBB mRNA levels; the HIC1 expression level in cells treated with non-silencing control siRNA was arbitrarily set to 1. Error bars indicate standard deviations (SDs); * p-value
    Figure Legend Snippet: Expression profiles of human WI38 cells after knockdown of the HIC1 gene. (A) Down-regulation of HIC1 mRNA in WI38 cells at 24, 48 and 72 h after transfection with HIC1-specific siRNAs from Ambion (Amb HIC1 siRNA) or Dharmacon (Dhar HIC1 siRNA); transfection with non-coding siRNA was used as a control. Graphs show the results of RT-qPCR analysis of WI38 cells transfected with the indicated siRNAs; the obtained quantification cycle (Cq) values were normalized to the UBB mRNA levels; the HIC1 expression level in cells treated with non-silencing control siRNA was arbitrarily set to 1. Error bars indicate standard deviations (SDs); * p-value

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    16) Product Images from "JAK2-CHK2 signaling safeguards the integrity of the mitotic spindle assembly checkpoint and genome stability"

    Article Title: JAK2-CHK2 signaling safeguards the integrity of the mitotic spindle assembly checkpoint and genome stability

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-05077-0

    CHK2 is phosphorylated at Tyr156 during mitosis. A Endogenous CHK2 is tyrosine-phosphorylated upon nocodazole treatment. HEK293T cells were treated with nocodazole (50 ng/ml) for 16 h, and lysates were prepared for immunoprecipitation and western blotting using the indicated antibodies. B , C Tyrosine phosphorylation in CHK2 in nocodazole-arrested cells ( B ) or G2/M phase cells ( C ) was diminished by the Y156F mutation. HEK293T cells were transfected with either myc-tagged WT or Y156F CHK2, and lysates were subjected to immunoprecipitation using the anti-myc antibody. Cells in different phases of the cell cycle in ( C ) were collected using double thymidine block and release. D Phosphorylation of endogenous CHK2 at Y156 was increased by nocodazole treatment in HEK293T. E CHK2 Y156 is phosphorylated in the G2/M normal human fibroblasts MRC5. Cells were synchronized by serum starvation. F Phosphorylation of endogenous CHK2 at Y156 was increased by nocodazole treatment in normal human fibroblasts WI38 and MRC5. For the detection of tyrosine phosphorylation, cells were treated with 1 mM pervanadate for 10 min prior to collection. EV, empty vector. “*” indicates a non-specific band.
    Figure Legend Snippet: CHK2 is phosphorylated at Tyr156 during mitosis. A Endogenous CHK2 is tyrosine-phosphorylated upon nocodazole treatment. HEK293T cells were treated with nocodazole (50 ng/ml) for 16 h, and lysates were prepared for immunoprecipitation and western blotting using the indicated antibodies. B , C Tyrosine phosphorylation in CHK2 in nocodazole-arrested cells ( B ) or G2/M phase cells ( C ) was diminished by the Y156F mutation. HEK293T cells were transfected with either myc-tagged WT or Y156F CHK2, and lysates were subjected to immunoprecipitation using the anti-myc antibody. Cells in different phases of the cell cycle in ( C ) were collected using double thymidine block and release. D Phosphorylation of endogenous CHK2 at Y156 was increased by nocodazole treatment in HEK293T. E CHK2 Y156 is phosphorylated in the G2/M normal human fibroblasts MRC5. Cells were synchronized by serum starvation. F Phosphorylation of endogenous CHK2 at Y156 was increased by nocodazole treatment in normal human fibroblasts WI38 and MRC5. For the detection of tyrosine phosphorylation, cells were treated with 1 mM pervanadate for 10 min prior to collection. EV, empty vector. “*” indicates a non-specific band.

    Techniques Used: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Blocking Assay, Plasmid Preparation

    17) Product Images from "Effect of Decellularized Extracellular Matrix Bioscaffolds Derived from Fibroblasts on Skin Wound Healing and Remodeling"

    Article Title: Effect of Decellularized Extracellular Matrix Bioscaffolds Derived from Fibroblasts on Skin Wound Healing and Remodeling

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2022.865545

    Preparation and characterization of fdECM. (A) The extraction method of WI-38 fibroblast-derived decellularized extracellular matrix (fdECM). (B) Western blot analysis of ECM proteins before and after decellularization with β-actin as an internal control. (C) Analysis of angiogenic factors by a semi-quantitative method. Dot blot of the matrix before and after decellularization. The graph of some of the 55 principal angiogenic factors detected (box denotes factors). Results are expressed as mean ± standard deviation (SD). *** p
    Figure Legend Snippet: Preparation and characterization of fdECM. (A) The extraction method of WI-38 fibroblast-derived decellularized extracellular matrix (fdECM). (B) Western blot analysis of ECM proteins before and after decellularization with β-actin as an internal control. (C) Analysis of angiogenic factors by a semi-quantitative method. Dot blot of the matrix before and after decellularization. The graph of some of the 55 principal angiogenic factors detected (box denotes factors). Results are expressed as mean ± standard deviation (SD). *** p

    Techniques Used: Derivative Assay, Western Blot, Dot Blot, Standard Deviation

    18) Product Images from "Effect of Decellularized Extracellular Matrix Bioscaffolds Derived from Fibroblasts on Skin Wound Healing and Remodeling"

    Article Title: Effect of Decellularized Extracellular Matrix Bioscaffolds Derived from Fibroblasts on Skin Wound Healing and Remodeling

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2022.865545

    Preparation and characterization of fdECM. (A) The extraction method of WI-38 fibroblast-derived decellularized extracellular matrix (fdECM). (B) Western blot analysis of ECM proteins before and after decellularization with β-actin as an internal control. (C) Analysis of angiogenic factors by a semi-quantitative method. Dot blot of the matrix before and after decellularization. The graph of some of the 55 principal angiogenic factors detected (box denotes factors). Results are expressed as mean ± standard deviation (SD). *** p
    Figure Legend Snippet: Preparation and characterization of fdECM. (A) The extraction method of WI-38 fibroblast-derived decellularized extracellular matrix (fdECM). (B) Western blot analysis of ECM proteins before and after decellularization with β-actin as an internal control. (C) Analysis of angiogenic factors by a semi-quantitative method. Dot blot of the matrix before and after decellularization. The graph of some of the 55 principal angiogenic factors detected (box denotes factors). Results are expressed as mean ± standard deviation (SD). *** p

    Techniques Used: Derivative Assay, Western Blot, Dot Blot, Standard Deviation

    19) Product Images from "Methyl Viologens of Bis‐(4’‐Pyridylethynyl)Arenes – Structures, Photophysical and Electrochemical Studies, and their Potential Application in Biology"

    Article Title: Methyl Viologens of Bis‐(4’‐Pyridylethynyl)Arenes – Structures, Photophysical and Electrochemical Studies, and their Potential Application in Biology

    Journal: Chemistry (Weinheim an Der Bergstrasse, Germany)

    doi: 10.1002/chem.202200753

    Cell survival of A549 and WI38 cells exposed to: a) 1M and b) 3M (with or without exposure to visible light by irradiation in the photoreactor Luzchem, LZC‐ICH2, equipped with eight overhead‐mounted LZC‐vis lamps, 400–700 nm, in total dose 50.6 mW m −2 , ∼18 cm distance lamp to cell‐plate). Data from 4 replicates are presented as mean±SD, relative to the control samples. Control samples are cells treated with DMSO at the same concentration as the tested compound. Representative data from three independent experiments, which yielded similar results, are shown.
    Figure Legend Snippet: Cell survival of A549 and WI38 cells exposed to: a) 1M and b) 3M (with or without exposure to visible light by irradiation in the photoreactor Luzchem, LZC‐ICH2, equipped with eight overhead‐mounted LZC‐vis lamps, 400–700 nm, in total dose 50.6 mW m −2 , ∼18 cm distance lamp to cell‐plate). Data from 4 replicates are presented as mean±SD, relative to the control samples. Control samples are cells treated with DMSO at the same concentration as the tested compound. Representative data from three independent experiments, which yielded similar results, are shown.

    Techniques Used: Irradiation, Concentration Assay

    20) Product Images from "Methyl Viologens of Bis‐(4’‐Pyridylethynyl)Arenes – Structures, Photophysical and Electrochemical Studies, and their Potential Application in Biology"

    Article Title: Methyl Viologens of Bis‐(4’‐Pyridylethynyl)Arenes – Structures, Photophysical and Electrochemical Studies, and their Potential Application in Biology

    Journal: Chemistry (Weinheim an Der Bergstrasse, Germany)

    doi: 10.1002/chem.202200753

    Cell survival of A549 and WI38 cells exposed to: a) 1M and b) 3M (with or without exposure to visible light by irradiation in the photoreactor Luzchem, LZC‐ICH2, equipped with eight overhead‐mounted LZC‐vis lamps, 400–700 nm, in total dose 50.6 mW m −2 , ∼18 cm distance lamp to cell‐plate). Data from 4 replicates are presented as mean±SD, relative to the control samples. Control samples are cells treated with DMSO at the same concentration as the tested compound. Representative data from three independent experiments, which yielded similar results, are shown.
    Figure Legend Snippet: Cell survival of A549 and WI38 cells exposed to: a) 1M and b) 3M (with or without exposure to visible light by irradiation in the photoreactor Luzchem, LZC‐ICH2, equipped with eight overhead‐mounted LZC‐vis lamps, 400–700 nm, in total dose 50.6 mW m −2 , ∼18 cm distance lamp to cell‐plate). Data from 4 replicates are presented as mean±SD, relative to the control samples. Control samples are cells treated with DMSO at the same concentration as the tested compound. Representative data from three independent experiments, which yielded similar results, are shown.

    Techniques Used: Irradiation, Concentration Assay

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    ATCC normal lung wi 38 va 13 subclone 2ra cells
    Effects of Tempol and/or Z-VAD on intracellular GSH depletion in Calu-6, A549, and <t>WI-38</t> <t>VA-13</t> <t>cells.</t> Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. The intracellular CMF (GSH) levels in <t>lung</t> cells were measured using a FACStar flow cytometer. The graphs indicate the percentages of (-) CMF (GSH-depleted) Calu-6 cells ( A ), A549 cells ( B ), and WI-38 VA-13 cells ( C ). ANOVA test was used. * p
    Normal Lung Wi 38 Va 13 Subclone 2ra Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC normal vero e6 cell lines
    Anti -SARS-CoV-2 activity and cytotoxicity against <t>normal</t> <t>Vero-E6</t> <t>cells</t> for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).
    Normal Vero E6 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal vero e6 cell lines/product/ATCC
    Average 94 stars, based on 1 article reviews
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    Effects of Tempol and/or Z-VAD on intracellular GSH depletion in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. The intracellular CMF (GSH) levels in lung cells were measured using a FACStar flow cytometer. The graphs indicate the percentages of (-) CMF (GSH-depleted) Calu-6 cells ( A ), A549 cells ( B ), and WI-38 VA-13 cells ( C ). ANOVA test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol and/or Z-VAD on intracellular GSH depletion in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. The intracellular CMF (GSH) levels in lung cells were measured using a FACStar flow cytometer. The graphs indicate the percentages of (-) CMF (GSH-depleted) Calu-6 cells ( A ), A549 cells ( B ), and WI-38 VA-13 cells ( C ). ANOVA test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Flow Cytometry

    Effects of Tempol on sub-G1 cells of normal and lung cancer cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Cells in sub-G1 phase were measured with a FACStar flow cytometer. Graphs show proportions of sub-G1 cells in Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol on sub-G1 cells of normal and lung cancer cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Cells in sub-G1 phase were measured with a FACStar flow cytometer. Graphs show proportions of sub-G1 cells in Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Incubation, Flow Cytometry

    Effects of Tempol on the growth of normal and lung cancer cells. Exponentially growing cells were incubated with Tempol at indicated concentrations for 48 h. Cell growth was evaluated by MTT assays. Graphs show the growth of Calu-6 cancer cells ( A ), A549 cancer cells ( B ), WI-38 VA-13 normal cells ( C ), and primary HPF normal cells ( D ). Student’s t -test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol on the growth of normal and lung cancer cells. Exponentially growing cells were incubated with Tempol at indicated concentrations for 48 h. Cell growth was evaluated by MTT assays. Graphs show the growth of Calu-6 cancer cells ( A ), A549 cancer cells ( B ), WI-38 VA-13 normal cells ( C ), and primary HPF normal cells ( D ). Student’s t -test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Incubation, MTT Assay

    Effects of Tempol on annexin V-positive lung cancer and normal cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Annexin V-FITC positive cells were evaluated with a FACStar flow cytometer. Graphs show the proportion of annexin V-positive Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol on annexin V-positive lung cancer and normal cells. Cells in exponential growth phase were incubated with Tempol at indicated concentrations for 48 h. Annexin V-FITC positive cells were evaluated with a FACStar flow cytometer. Graphs show the proportion of annexin V-positive Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Incubation, Flow Cytometry

    Effects of Tempol on MMP (∆Ψm) levels in lung cancer and normal cells. Exponentially growth of cells incubated with Tempol at indicated concentrations for 48 h. MMP (∆Ψm) in lung cells was measured using a FACStar flow cytometer. Graphs show the proportion of rhodamine 123-negative [MMP (∆Ψm) loss] Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol on MMP (∆Ψm) levels in lung cancer and normal cells. Exponentially growth of cells incubated with Tempol at indicated concentrations for 48 h. MMP (∆Ψm) in lung cells was measured using a FACStar flow cytometer. Graphs show the proportion of rhodamine 123-negative [MMP (∆Ψm) loss] Calu-6 cells ( A ), A549 cells ( B ), WI-38 VA-13 cells ( C ), and primary HPF cells ( D ). Student’s t -test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Incubation, Flow Cytometry

    Effects of Tempol and/or Z-VAD on caspase-3 activity and cell death in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. ( A ): Graph shows the activities of caspase-3 in Calu-6, A549, and Wi-38 VA-13 cells, measured via Colorimetric Assay. ( B – D ): Graphs show the proportion of annexin V-positive Calu-6 cells ( B ), A549 cells ( C ), and WI-38 VA-13 cells ( D ), measured with a FACStar flow cytometer. ANOVA test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol and/or Z-VAD on caspase-3 activity and cell death in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. ( A ): Graph shows the activities of caspase-3 in Calu-6, A549, and Wi-38 VA-13 cells, measured via Colorimetric Assay. ( B – D ): Graphs show the proportion of annexin V-positive Calu-6 cells ( B ), A549 cells ( C ), and WI-38 VA-13 cells ( D ), measured with a FACStar flow cytometer. ANOVA test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Activity Assay, Colorimetric Assay, Flow Cytometry

    Effects of Tempol and/or Z-VAD on ROS levels in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. Intracellular DCF (ROS) and DHE (O 2 •− ) levels in lung cells were measured using a FACStar flow cytometer. ( A – C ): The graphs indicate the mean DCF (ROS) levels (%) in Calu-6 ( A ), A549 cells ( B ), and WI-38 VA-13 cells ( C ). ( D – F ): The graphs indicate the mean DHE (O 2 •− ) levels (%) in Calu-6 ( D ), A549 cells ( E ), and WI-38 VA-13 cells ( F ). ANOVA test was used. * p

    Journal: Molecules

    Article Title: Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•− Levels and Glutathione Depletion

    doi: 10.3390/molecules27217341

    Figure Lengend Snippet: Effects of Tempol and/or Z-VAD on ROS levels in Calu-6, A549, and WI-38 VA-13 cells. Exponentially growing cells were pretreated with Z-VAD for 1 h and then treated with 2 mM Tempol for 48 h. Intracellular DCF (ROS) and DHE (O 2 •− ) levels in lung cells were measured using a FACStar flow cytometer. ( A – C ): The graphs indicate the mean DCF (ROS) levels (%) in Calu-6 ( A ), A549 cells ( B ), and WI-38 VA-13 cells ( C ). ( D – F ): The graphs indicate the mean DHE (O 2 •− ) levels (%) in Calu-6 ( D ), A549 cells ( E ), and WI-38 VA-13 cells ( F ). ANOVA test was used. * p

    Article Snippet: Human SCLC Calu-6 cells, NSCLC A549 adenocarcinoma cells, and normal lung WI-38 VA-13 subclone 2RA cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Flow Cytometry

    Anti -SARS-CoV-2 activity and cytotoxicity against normal Vero-E6 cells for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).

    Journal: Journal of Drug Delivery Science and Technology

    Article Title: Quercitrin loaded cyclodextrin based nanosponge as a promising approach for management of lung cancer and COVID-19

    doi: 10.1016/j.jddst.2022.103921

    Figure Lengend Snippet: Anti -SARS-CoV-2 activity and cytotoxicity against normal Vero-E6 cells for free QCT, plain CD-NS and QCT loaded CD-NS formulations. Each data point represents mean ± SD (n = 3).

    Article Snippet: Human lung cancer A549 and normal Vero-E6 cell lines were purchased from ATCC (American Type Culture Collection, CCL-75™).

    Techniques: Activity Assay

    ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2. a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence ( left ), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels ( right ). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 μM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c ; GSEA associations ( left ), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF ( right ). e WI-38 cells were processed as in b and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA , BCL2L2 , and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b , f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCα/β II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2 , PUMA , and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i . In a , g , h , ACTB was included as loading control. Data in e , f , i were normalized to ACTB mRNA. Graphs ( b , e , f , i , and j ), represent the values ± SD from n = 3 experiments; significance (* p

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Activation Assay, Western Blot, Transfection, Cell Counting, RNA Sequencing Assay, Quantitative RT-PCR, Blocking Assay

    BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: BDNF is a marker of surviving senescent cells. WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) ( a ) and RT-qPCR analysis was used to measure the levels of BDNF , p21 , and IL6 mRNAs ( b ). WI-38 cells were treated with H 2 O 2 and viability ( c ) was measured as in a and BDNF mRNA levels ( d ) were measured as in b . e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 ( left ) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels ( right ) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e ( g ); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a ( h ), and in H 2 O 2 -treated WI-38 cells as described in c ( i ). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j . l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l ; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b , d , e , f were normalized to ACTB mRNA. Values in a – f are the means ± SD; significance (* p

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Marker, Quantitative RT-PCR, Transfection, Western Blot, Expressing, RNA Sequencing Assay, Immunofluorescence, Staining

    Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: Screening of a library of drugs directed at MAPK signaling proteins unveils Trk inhibitors as potential senolytic compounds. a Overview of the experimental strategy of the survey. Briefly, senescent and proliferating WI-38 and BJ fibroblasts were treated for 48 h with a collection of MAPK inhibitors, whereupon cell viability was assessed. b Schematic representation of the protocol used to reach full senescence using etoposide (Etop, 25 and 50 μM for BJ and WI-38 cells, respectively) for 10 days, refreshing etoposide twice, before treating the cell cultures with each of the drugs in the library for an additional 48 h; created using BioRender. c Bar plot displaying the % change of proliferating and senescent WI-38 fibroblasts (top graph) and BJ fibroblasts (bottom graph) after treatment with the drug library for 48 h (day 12) relative to the time before drug addition (day 10). Red line, percentages of cells present at each condition right before starting the treatments at day 10. All the drugs were added at a concentration of 10 μM. d Heat map representing the percentage of senolysis (percentage of cell death) after 48 h when comparing day 12 to day 10 in senescent cells. Drugs were grouped by the target proteins inhibited within the MAPK superfamily. Graphs in c represent the mean values ±SD of n = 3 experiments. See also Supplementary Fig. 1 .

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Concentration Assay

    Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: Pharmacological inhibition of TrkB selectively induces apoptotic death of senescent cells. a Viability curves obtained after testing a range of concentrations of Trk inhibitor drugs (GNF 5837, ANA 12, PF 06273340) for 48 h in WI-38 fibroblasts that were either proliferating or undergoing etoposide-induced senescence (ETIS). b Representative micrographs of select doses for each drug tested in ( a ); bar, 100 µm. Cell viability ( c ) and caspase 3/7 activity ( d ) were assessed 48 h after the indicated treatments and doses in WI-38, BJ, IMR-90, HSAEC, HREC, and HUVEC cultures undergoing ETIS, and WI-38 cultures undergoing oxidative stress-induced senescence (OSIS), ionizing radiation-induced senescence (IRIS), and replicative senescence (RS). Assessments of cell viability ( e ) and caspase 3/7 activity ( f ) measured in WI-38 cells undergoing ETIS that were treated for 48 h with different Trk inhibitors, either alone or in combination with the pan-caspase inhibitor Z-VAD-FMK at the indicated doses. Graphs in a , c – f , represent the mean values ± SD of n = 3 experiments; significance (* p

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Inhibition, Activity Assay

    BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: BDNF is a SASP factor that ensures viability of senescent cells. a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c . b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a . c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) ( d ), % cells with positive BDNF staining ( e ), and BDNF fluorescence intensity measurements ( f ) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF , p21 , IL6 , and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted ( k ) and S cells were assessed by microscopy ( l ) and by caspase 3/7 activity ( m ). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n , but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g , h were normalized to ACTB mRNA. Graphs in b , e – k , m , and o represent the mean values ± SD of n = 3 experiments; significance (* p

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Transfection, Blocking Assay, Microscopy, Activity Assay, Western Blot

    Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p

    Journal: Nature Communications

    Article Title: A BDNF-TrkB autocrine loop enhances senescent cell viability

    doi: 10.1038/s41467-022-33709-8

    Figure Lengend Snippet: Increased TrkB levels in senescent cells promotes resistance to apoptosis. a , b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c ), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB ( a ), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs ( b ). Western blot analysis ( c ) and RT-qPCR analysis ( d ) of the same molecules as in a , b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e . g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1 , p16 , and IL6 mRNAs in the same conditions analyzed in h . j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a , c , g , h , ACTB was included as loading control. Data in b , d , f , i were normalized to ACTB mRNA. Graphs in b , d – f , i display the mean values ± SD of n = 3 experiments; significance (* p

    Article Snippet: To achieve senescence by exposure to ionizing (γ) radiation (IR), WI-38 cells were exposed to 15 Gray (Gy) and cells were cultured for up to 10 days.

    Techniques: Western Blot, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence