hfab rhodamine anti actin primary antibody  (Bio-Rad)

Journal: Biomolecules

Article Title: Cigarette Smoke Induces Canonical Stress Granule Formation in Human Bronchial Epithelial Cells in Reactive Oxygen Species- and PERK-Dependent Manners

doi: 10.3390/biom16040615

Figure Lengend Snippet: SG formation is dependent on the PERK/eIF2α signaling pathway. CFBE cells were pretreated with DMSO, ISRIB or GSK and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging or immunoblotting were then performed. ( A , B ) IF imaging showed that endogenous G3BP1 ( A ) and p-eIF2α ( B ) are homogenously distributed in the cell cytoplasm under control conditions (DMSO + HBSS). ( C ) Smoke exposure induced robust SG formation (white arrows) and ( D ) a significant increase in cytoplasmic eIF2α phosphorylation (p-eIF2α). ( E , F ) Pretreating cells with ISRIB, an inhibitor of p-eIF2α function, at 200 nM for 1 h prior to smoke extract exposure ( E ) completely abolished SG formation, establishing causality ( F ) without reducing p-eIF2α abundance. ( G , H ) Inhibiting p-PERK by pretreating cells with 2 µM GSK for 1 h prior to smoke extract exposure ( G ) completely blocked SG formation and ( H ) abrogated the increase in p-eIF2α levels, indicating that SG formation is PERK/eIF2α-dependent. ( I ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels demonstrated a significant increase in cells’ response to smoke extract exposure as demonstrated by the 3-fold increase in cytoplasmic p-eIF2α fluorescence intensity (IF DMSO+HBSS = 724 ± 25, N DMSO+HBSS = 4, n DMSO+HBSS = 24; IF DMSO+Smoke = 1919 ± 48, N DMSO+Smoke = 16, n DMSO+Smoke = 120 (4 outliers were identified and excluded)). The analysis also showed that ISRIB pretreatment induced further accumulation and a significant increase in cytoplasmic p-eIF2α levels in response to smoke exposure (IF ISRIB+Smoke = 2464 ± 57, N ISRIB+Smoke = 7, n ISRIB+Smoke = 54). GSK pretreatment, on the other hand, significantly attenuated p-eIF2α increase in response to smoke exposure (IF GSK+Smoke = 1187 ± 34, N GSK+Smoke = 11, n GSK+Smoke = 82) without fully returning to the DMSO + HBSS baseline. ( J ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels showed that pretreating cells with DMSO (drug vehicle control) did not induce significant change in cytoplasmic p-eIF2α levels under HBSS or smoke exposure conditions (IF HBSS = 725 ± 13, N HBSS = 18, n HBSS = 132 (1 outlier was identified and excluded); IF Smoke = 2249 ± 62, N Smoke = 19, n Smoke = 142). The nonparametric Kruskal–Wallis test was used to calculate significance in ( I , J ). ns: not significant, **: p = 0.01 and ****: p < 0.0001. Each ROI is an independent biological sample. ( K – N ) immunoblotting demonstrated a significant 5- to 7-fold increase in cellular p-eIF2α level in response to smoke exposure, with ISRIB pretreatment increasing this level non-significantly to 10-fold (N = 4, IB DMSO+HBSS = 1, IB DMSO+Smoke = 7 ± 1, IB ISRIB+Smoke = 10 ± 2). GSK pretreatment reduced p-eIF2α levels significantly but not to the DMSO + HBSS basal level (N = 5, IB DMSO+HBSS = 1, IB DMSO+Smoke = 5.1 ± 0.5, IB GSK+Smoke = 2.8 ± 0.5). Immunoblotting data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . Brown–Forsythe and Welch ANOVA tests were used to calculate significance in ( M , N ), with nd = no discovery and *: p < 0.03. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI = region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments and n = total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

Article Snippet: The hFABTM Rhodamine β-Actin and GAPDH primary antibodies were used for immunoblotting experiments (Bio-Rad).

Techniques: Imaging, Western Blot, Control, Phospho-proteomics, Fluorescence, Immunofluorescence

Journal: Biomolecules

Article Title: Cigarette Smoke Induces Canonical Stress Granule Formation in Human Bronchial Epithelial Cells in Reactive Oxygen Species- and PERK-Dependent Manners

doi: 10.3390/biom16040615

Figure Lengend Snippet: Reactive oxygen species activate the PERK/eIF2α signaling pathway and induce SG formation in response to smoke exposure. CFBE cells were pretreated with H 2 O or 5 mM NAC for 15 min and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging and immunoblotting analyses were then performed. ( A – C ) Immunoblotting (IB) demonstrated a significant 7-fold increase in cellular p-eIF2α level in response to smoke exposure which was fully abolished upon pretreating cells with NAC (IB H2O+HBSS = 1, IB H2O+Smoke = 7.1 ± 0.9, IB NAC+Smoke = 1.4 ± 0.3; N H2O+HBSS = N H2O+Smoke = 36, N NAC+Smoke = 10). Additionally, immunoblotting showed that smoke exposure increased p-PERK level by 2.8-fold, which is also completely abolished by NAC pretreatment (IB H2O+HBSS = 1, IB H2O+Smoke = 2.8 ± 0.3, IB NAC+Smoke = 1.3 ± 0.2; N H2O+HBSS = 41, N H2O+Smoke = 40, N NAC+Smoke = 14). IB data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . ( D , E ) Confocal IF imaging showed that ( D ) endogenous G3BP1 and ( E ) p-eIF2α were homogenously distributed in the cell cytoplasm under control conditions (H 2 O + HBSS). ( F , G ) IF imaging showed that smoke extract exposure induced SG formation (white arrows) and a marked increase in cytoplasmic p-eIF2α abundance. ( H , I ) Pretreating cells with the ROS scavenger NAC prior to smoke extract exposure completely abolished SG formation and inhibited the increase in p-eIF2α levels, establishing that SG formation is ROS-driven in p-eIF2α-dependent manner. ( J ) Quantitative nucleus-based image analysis showed that NAC pretreatment significantly attenuated the significant 3-fold increase in cytoplasmic p-eIF2α level in response to smoke extract exposure (IF H2O+HBSS = 700 ± 9, IF H2O+Smoke = 2485 ± 75, IF NAC+Smoke = 899 ± 28; N H2O+HBSS = 5, n H2O+HBSS = 30; N H2O+Smoke = 8, n H2O+Smoke = 47 (1 outlier was identified and excluded); N NAC+Smoke = 9, n NAC+Smoke = 54). Each ROI is an independent biological sample. The nonparametric Kruskal–Wallis test was used to calculate significance in ( B , C , J ). ns: not significant, *: p < 0.03, **: p = 0.0095, ***: p = 0.0002, and ****: p < 0.0001. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI for region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments, and n = the total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

Article Snippet: The hFABTM Rhodamine β-Actin and GAPDH primary antibodies were used for immunoblotting experiments (Bio-Rad).

Techniques: Imaging, Western Blot, Control, Immunofluorescence