Journal: JCI Insight

Article Title: FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium

doi: 10.1172/jci.insight.174888

Figure Lengend Snippet: ( A and B ) Immunofluorescence staining for p16 and colabeled with either Krt5 (KRT5; secretory cells) or uteroglobin (UTG; basal cells) in Cftr –/– rat lung tissue and nuclear staining with DAPI. Boxes indicate bronchial epithelium that shows the double labeling. The scale bar is 20 µm, and it is a ×40 original magnification.

Article Snippet: The specimen was then washed and blocked with 1% BSA for 60 minutes prior to primary incubation overnight with the following antibodies: Krt5 (1:50, Invitrogen, Thermo Fisher Scientific, MA5-15348), uteroglobin (1:50, R&D Systems, Bio-Techne, MAB4218), FoxJ1 (1:50, Invitrogen, Thermo Fisher Scientific, PA5-36210 and 14-9965-82), p16 (1:100, Proteintech 10883-1-AP), and p21 (1:100, Cell Signaling Technology 29478).

Techniques: Immunofluorescence, Staining, Labeling

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Establishment of basal cell (BC) and endothelial cell (EC) co-culture system to study BC differentiation into a mucociliated epithelium. A. Immunofluorescence assessment of a normal human airway biopsy with staining for BC (KRT5, green), EC (CD31, red) and nuclei (DAPI, blue). Scale bar 50 μm. B. Schematic representation of the BC and EC co-culture system to study the impact of EC on BC differentiation into a mucociliated epithelium. C. Immunofluorescence assessment of BC and EC co-culture with staining for BC (KRT5, red), EC (VE-cadherin, green) and nuclei (DAPI, blue). Upper panel, BC cultured alone. Lower panel, BC and EC co-culture. White dashed line outlines the membrane of the Transwell insert. Scale bar 20 μm. D. Immunofluorescence staining of the epithelial layer of ALI day 28 cultures. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio and then stained for ciliated cells (β-tubulin IV, green), secretory cells (SCGB1A1, red) and nuclei (DAPI, blue). Scale bar 20 μm. E. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 7 days to assess the impact of EC on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell (FOXJ1) markers in BC cultured alone or co-cultured with EC cells at 10:1 and 2:1 (BC:EC) ratio. Bars indicate the mean for n=6 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC alone. The experiments for E were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Cell Culture, Expressing

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: Endothelial cell (EC) co-culture increases differentiation of basal cells (BC) into ciliated cells. Basal cells were cultured alone or co-cultured with EC at a 10:1 and 2:1 (BC:EC) ratio on air-liquid interface (ALI) culture for 28 days to assess the impact of EC on BC differentiation into a mucociliated epithelium at the histological level via immunofluorescence staining with cell type specific markers. A. KRT5+ BC. Sections of cells were stained for KRT5 (red) and DAPI (nuclei, blue). B. KRT8+ intermediate cells. Sections of cells were stained for KRT8 (red) and DAPI (nuclei, blue). C. SCGB1A1+ secretory cells. Sections of cells were stained for SCGB1A1 (red) and DAPI (nuclei, blue). D. MUC5B+ secretory cells. Sections of cells were stained for MUC5B (red) and DAPI (nuclei, blue). E β-tubulin IV+ cells. Sections of cells were stained for β-tubulin IV (ciliated, green) and DAPI (nuclei, blue). A–E. Scale bar 20 μm. H. Quantification of KRT5+, KRT8+, SCGB1A1+, MUC5B+ and β-tubulin IV+ cells. The bars indicate the mean for n=4 independent experiments and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC cultured alone. The experiments for A–H were performed with n=4 independent donors of BC and EC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Staining

Journal: Stem cell reviews

Article Title: Endothelial Cell Mediated Promotion of Ciliated Cell Differentiation of Human Airway Basal Cells via Insulin and Insulin-like Growth Factor 1 Receptor Mediated Signaling

doi: 10.1007/s12015-016-9707-z

Figure Lengend Snippet: siRNA mediated knockdown of INSR and IGF1R in basal cells (BC) suppresses ciliated cell differentiation. Basal cells with transfected with either control, INSR or IGF1R specific siRNA and cultured on ALI for 7 days to assess the impact of each receptor on the early stages of BC differentiation into a mucociliated epithelium at the molecular level. A. TaqMan PCR analysis to assess INSR mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. B. TaqMan PCR analysis to assess IGF1R mRNA expression at ALI day 0. Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. C. TaqMan PCR analysis to assess mRNA expression of BC (KRT5), intermediate (KRT8), secretory (non-mucus producing, SCGB1A1 and mucus producing, MUC5B) and ciliated cell markers (FOXJ1, MYB and DNAI1) in BC at ALI day 7 following siRNA mediated knockdown of INSR (black bars) and IGF1R (grey bars). Bars indicate the mean for n=3 independent experiments each performed in triplicate and error bars indicate standard error of the mean. Asterisks (*) indicate p<0.05 compared to BC transfected with control siRNA. The experiments for A–C were performed with n=3 independent donors of BC.

Article Snippet: The samples were then stained with the following primary antibodies: rabbit monoclonal CD31 (5 μg/ml; ab76533; Abcam, Cambridge, UK); goat polyclonal VE-Cadherin (1 μg/ml; AF938; R&D Systems Inc., Minneapolis, MN), mouse monoclonal KRT5 (4 μg/ml; MA5-12596; ThermoFisher Scientific); rabbit polyclonal KRT5 (2 μg/ml; PA1-37974; ThermoFisher Scientific); rabbit polyclonal KRT8 (10 μg/ml; NBP2-16094; Novus Biological, Littleton, CO); rabbit polyclonal SCGB1A1 (5 μg/ml; RD181022220; BioVendor LLC, Candler, NC); rabbit polyclonal MUC5B (4 μg/ml; sc-20119; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal β-tubulin IV (10 μg/ml; MU178-UC; Biogenex, Fremont, CA) at 4°C overnight.

Techniques: Cell Differentiation, Transfection, Cell Culture, Expressing

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 1. eEF1A1 regulates HSP70 expression and thermotolerance. (A) Knock down of eEF1A1 decreases HSP70 protein expression upon heat shock. Western blots of HSP70, eEF1A1, HSF1, and GAPDH from total cell lysates of MDA-MB231 cells transfected with siRNA (pair A) against eEF1A1 or HSF1, or mock-transfected (Si:NT with no target). Control (C)—unstressed cells kept at 37°C. Heat shock (HS)—cells kept for 1 hr at 43°C followed by 6 hr of recovery at 37°C. (B) HSF1- or eEF1A1-knocked down cells are less thermo-tolerant. Cell death was quantified by FACS analysis after propidium iodide staining. Thermotolerance was induced in mock-transfected (Si:NT) or eEF1A1/HSF1-depleted cells by two heat treatments (1 hr at 43°C, 12 hr at 37°C, and 1 hr at 45°C), followed by 12 hr of recovery. Data from three independent experiments are presented as the mean ± SEM. *p < 0.05. (C) Knock down of eEF1A1 decreases HSP70 mRNA expression during heat shock. Total RNA from heat shocked MDA-MB231 cells was reverse-transcribed with random hexamer primers followed by quantification of GAPDH and HSP70 mRNAs by QPCR. HSP70 mRNA values were normalized to that of GAPDH. Bars represent the amount of HSP70 mRNA in cells depleted of eEF1A1 relative to that obtained for mock-transfected cells (Si:NT). 1 is the value of HSP70 mRNA in Si:NT cells at 1 hr of heat shock. Data from three independent experiments are presented as the mean ± SEM. *p < 0.05. (D) eEF1A1 controls RNAPII occupancy at the HSP70 gene after stress as determined by ChIP-QPCR. Schematic of the HSPA1A locus is shown at the top. Arrowheads indicate the regions amplified by QPCR. Panels show the effect of eEF1A1 knock down (si:eEF1A1) on RNAPII occupancy relative to input Ct value under non-heat shock conditions (control) and after 30 min of heat shock. The relative value for the IgG is indicated for each of the PCR fragments on top of the plot. Values below that of the IgG mean non-specific binding. Mock-transfected cells (Si:NT). By comparison, GAPDH showed no change with si;eEF1A1. Data from three independent experiments are presented as the mean ± SEM. (*p < 0.05). (E) eEF1A1 mediates HSP70 transcription upon HS. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. eEF1A1 expression was knocked down by siRNA. At 30 min after HS, cells were fixed and HSP70 mRNA detected by FISH. Nuclei were stained with DAPI. Merged images show Cherry-eEF1A1 in red, HSP70 TS in gray and nucleus in blue. White arrows indicate cells knocked down of eEF1A1. Bar = 10 microns. (F) eEF1A1 mediates HSP70 transcription upon HS. Plots are the quantification of the intensity of HSP70 transcription, per cell or per TS, detected by FISH and quantified by airlocalize. A total of seventy cells per condition were analyzed from three independent experiments. NT = non-transfected cells. eEF1A1 = cells transfected with siRNA for eEF1A1. DOI: 10.7554/eLife.03164.003 The following figure supplements are available for figure 1:

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Expressing, Knockdown, Western Blot, Transfection, Control, Staining, Reverse Transcription, Random Hexamer, ChIP-qPCR, Amplification, Binding Assay, Comparison, Infection

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 2. eEF1A1 mediates HSF1 recruitment to HSP70 promoter. (A) eEF1A1 enhances HSF1 DNA binding upon heat shock. Mock-transfected, eEF1A1 or HSF1-knocked down MDA-MB231 cells were heat-shocked for 30 min at 43°C and analyzed by HSF1-HSE EMSA (top panel). 10 μg of total protein were loaded in the EMSA. HSF1, eEF1A1, and GAPDH levels were determined by immunoblotting (lower panel). (B) eEF1A1 is required for HSF1 promoter binding in vivo. ChIP-QPCR was performed on mock-transfected (Si:NT) or eEF1A1-knocked down (Si:eEF1A1) cells. Panel shows the effect of eEF1A1 depletion on HSF1 occupancy at the HSP70 promoter (relative to the input Ct value) under non-heat shock conditions (C) and after 30 min of heat shock (HS). The reference value for IgG control is indicated on top of the plot. Values below those numbers mean non-specific binding. Data from three independent experiments are represented as the mean ± SEM. (*p < 0.05). (C) Stress-induced formation of the eEF1A1-HSF1 complex in vivo. Extracts from unstressed (C) or heat-shocked (HS) MDA-MB231 cells IPed with eEF1A1 antibody or IgG. IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting. (D) eEF1A1-HSF1 complex formation at HSE. Panels show the super-shift of HSF1-HSE EMSA caused by specified antibodies. MDA-MB231 cells were heat-shocked for 20 min at 43°C (HS). Extracts were incubated with antibodies to HSF1, HSF2 (positive control), or eEF1A1 antibodies, or IgG (mock). HSE-HSF1 indicates specific binding of HSF1 to labeled HSE; NS—a non-specific band. (E) Direct binding of eEF1A1 to HSP70 promoter DNA. Radiolabeled fragment of the region −141 to −91 of the human HSP70 promoter was incubated with purified eEF1A1 (lanes 1–3), chased with fivefold molar excess of cold oligonucleotide (lanes 4 and 5), or mutant fragment of the same region (lane 6). Arrows mark the specific eEF1A1 shift. (F) eEF1A1 binds the HSP70 promoter before and after stress. Mock transfected or eEF1A1-knocked down (Si:eEF1A) MDA-MB231 cells were kept at 37°C or heat-shocked (HS) for 20 min at 43°C. Chromatin IP with eEF1A1 or IgG antibodies and amplified by PCR for the HSP70 (HSPA1A) and GAPDH promoters. Relative value of the control IgG vs input for HSPA1A is 0.04 and for GAPDH is 0.05. Data from three independ ent experiments are presented as the mean ± SEM. *p < 0.05. DOI: 10.7554/eLife.03164.010 The following figure supplement is available for figure 2:

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Binding Assay, Transfection, Western Blot, In Vivo, ChIP-qPCR, Control, SDS Page, Incubation, Positive Control, Labeling, Purification, Mutagenesis, Chromatin Immunoprecipitation, Amplification

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 3. eEF1A1 localizes at HSP70 TS and interacts with RNAPII upon HS. (A) eEF1A1 localizes to HSP70 TS upon HS. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. At the indicated times after HS, cells were fixed and HSP70 mRNA detected by FISH. Nucleus stained with DAPI. Merged images show HSP70 FISH in green and cherry-eEF1A1 in red. The nascent mRNA signal is much brighter than the Cherry-eEF1A1 because there were many nascent chains each detected with 48 probes (48 fluors), compared to a single fluorescent protein for eEF1A1, further diminished by fixation. Yellow arrows indicate TS for HSP70 where Cherry-eEF1A1 was also detected. Inset location indicated by white arrowheads. n = total number of cells analyzed from three independent experiments. (%) = percentage of TS with co-localization for eEF1A1. Bar = 10 microns. (B) eEF1A1 localizes in nuclear dots. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. At 1 hr of HS cells were fixed and FISH was carried out to detect β-actin mRNA. Nucleus DAPI stained. Merged images show HSP70 FISH in green and Cherry-eEF1A1 in red. Arrowhead = inset location. Note that nuclear localization of Cherry-eEF1A1 does not coincide with the β-actin mRNA TS. (C) Quantification of co-localization between eEF1A1 and HSP70 TS. Percentages of co-localization were quantified by airlocalize software at the indicated HS times. Average of three different experiments. Total n = (80–90) cells per time point. (D) DRB decreases RNAPIIS2 and eEF1A1 occupancy within the HSP70 gene in HS cells. Data are the mean ± SEM from three independent experiments. MEF cells expressing eEF1A1 tagged with Cherry and Flag were kept under normal growth conditions (control) or heat-shocked for 40 min at 43°C (HS) or treated with 100 µM DRB for 15 min followed by HS (HS+DRB). ChIP was performed using antibodies for RNAPII phosphorylated at Ser2 (RNAPS2) and Flag eEF1A1 followed by QPCR with the indicated primers. (E) eEF1A1 binds RNAPII during heat shock. Extracts from unstressed (C) or heat-shocked (HS) MDA-MB231 cells were IP with an eEF1A1 antibody or IgG (mock). IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting with RNAPII and eEF1A1 antibodies. (*) Indicates the hyperphosphorylated form of RNAPII. DOI: 10.7554/eLife.03164.012 The following figure supplements are available for figure 3:

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Infection, Expressing, Staining, Software, Control, SDS Page, Western Blot

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 4. eEF1A1 binds the 3′UTR of HSP70 mRNA and stabilizes it. (A) eEF1A1 binds chromatin-associated HSP70 mRNA. Native ChIP samples were IPed using anti-eEF1A1 antibodies and reverse transcribed followed by HSP70 mRNA quantification. Data are represented as mean ± SEM from three independent experiments. (B) eEF1A1 interacts with the HSP70 mRNA in vivo as detected by RNA-IP. The panel shows the RT-PCR products of HSP70 and GAPDH mRNA from control (C) and heat-shocked (HS) cells after IP with indicated antibodies. IgG indicates the mock control. Input is total RNA. (C) Interaction between the HSP70 mRNA 3′UTR and eEF1A1 as detected by RNA-EMSA. 4 μg of purified eEF1A1 were incubated with 105 cpm of the 3′UTR or 5′UTR of HSP70 mRNA or a 200 nt fragment of the β-actin ORF radiolabeled by in vitro transcription. Only the 3′UTR of HSP70 mRNA was shifted by eEF1A1 (red line) and further super-shifted by 4 μg of antibody against eEF1A1 (red star). (D) Knock down of eEF1A1 diminishes HSP70 mRNA stability. Actinomycin D (Ac) was added 30 min after the onset of heat shock. The level of HSP70 and GAPDH mRNA at this time was taken as 100%. Data are represented as mean ± SEM from three experiments. *p < 0.05. (E) eEF1A1-mediates luciferase expression cloned in a HSP70 backbone plasmid. Mock transfected (si:NT) or eEF1A1-knocked down (si:EF) MDA-MB231 cells were transfected with plasmids expressing the SV40- or HSP70- driven luciferase gene fused to the HSP70 3′UTR, and a SV40-renilla luciferase plasmid used as a control. Luciferase activity was measured in cells heat-shocked for 1 hr at 43°C followed by 4 hr of recovery at 37°C. The values were normalized to those of renilla in the same cellular extracts. Bars represent luciferase activity in eEF1A1-deficient cells relative to that obtained for mock-transfected cells. Data are represented as mean ± SEM from three experiments. *p < 0.05. DOI: 10.7554/eLife.03164.017 The following figure supplement is available for figure 4:

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Reverse Transcription, In Vivo, Reverse Transcription Polymerase Chain Reaction, Control, Purification, Incubation, In Vitro, Knockdown, Luciferase, Expressing, Clone Assay, Plasmid Preparation, Transfection, Activity Assay

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 5. eEF1A1 is required for HSP70 mRNA export from the nucleus. (A) eEF1A1 mediates HSP70 mRNA export upon HS. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. eEF1A1 expression was knocked down by siRNA. At 120 min after HS cells were fixed and HSP70 mRNA detected by FISH. Nucleus stained with DAPI. Merged images show cherry-eEF1A1 in red and nucleus in blue. Gray image shows HSP70 mRNA FISH. Bar = 10 microns. (B) HSP70 mRNA retention in the nucleus of eEF1A1-knocked down cells. The plot shows total (T) and nuclear (N) HSP70 mRNA in control (Si:NT) or eEF1A1-knocked down cells after heat shock. RNA from HeLa cells was RT with random primers followed by quantification of GAPDH and HSP70 mRNAs by QPCR. Total or nuclear HSP70 mRNA was normalized to that of total GAPDH. Data are represented as the mean ± SEM from three independent experiments. *p < 0.05. (C) Knock down of eEF1A1 suppresses binding of TPR1 to HSP70 mRNA. HSP70 mRNA was co-IPed with antibodies against eEF1A1 or TPR1 from heat-shocked HeLa cells knocked down of eEF1A1 or mock transfected. Total and IP RNA was RT with random primers and GAPDH and HSP70 mRNAs were quantified by QPCR. Total and IP HSP70 mRNA was normalized against total GAPDH mRNA. Data are represented as the mean ± SEM from three experiments. (D) MEFs were infected with a lentivirus expressing Cherry-eEF1A1. At 120 min of HS, cells were fixed and HSP70 mRNA detected by FISH. Nucleus stained with DAPI. Merged images show HSP70 FISH in green and cherry-eEF1A1 in red. Yellow arrowheads indicate areas with high density of HSP70 mRNA and brighter signal for cherry-eEF1A1. (E) eEF1A1 contributes to loading of HSP70 mRNA into polysomes. RNA collected from light and heavy polysome fractions was reverse-transcribed with random primers followed by quantification of GAPDH and HSP70 mRNAs by QPCR. Values relate to those obtained from total RNA (Input). Data are presented as the mean ± SEM from three independent experiments. DOI: 10.7554/eLife.03164.019 The following figure supplement is available for figure 5:

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Infection, Expressing, Staining, Control, Knockdown, Binding Assay, Transfection, Reverse Transcription

Journal: eLife

Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

doi: 10.7554/elife.03164

Figure Lengend Snippet: Figure 6. eEF1A1 synchronizes the expression of HSP70 mRNA from transcription to translation. The cartoon summarizes the results of this study, implicating eEF1A1 at each stage of HSPs induction. Prior to stress, eEF1A1 resides mostly in the cytoplasm where it is an essential component of the translation machinery (top left quadrant). Upon heat shock (bottom left quadrant), a fraction of eEF1A1 is detected in the HSP70 locus where it recruits HSF1 to an HSP70 promoter, thus activating transcription. eEF1A1 interacts with elongating RNAP II and binds the 3′UTR of HSP70 mRNA to stabilize the transcript and to export it to cytoplasm for efficient translation. By synchronizing all the major steps of HSP70 gene expression, eEF1A1 renders the process of HSR exceptionally robust and coordinated. In cells knocked down for eEF1A1 HSP70 remain at low levels even when cells are stressed (right hand quadrants). DOI: 10.7554/eLife.03164.021

Article Snippet: DOI: 10.7554/eLife.03164 16 of 19 Research article chromatin A260) (Santa Cruz, Dallas, Texas, USA), RNAPII S2 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Abcam, Cambridge, MA, USA), eEF1A (4 μg of Antibody for 80 μg of relative amount of chromatin A260) (Designed in Nudler lab and produced in rabbits and purified by Prosci incorporated), HSF1 (2 μg of Antibody for 20 μg of relative amount of chromatin A260) (Enzo life sciences, Farmigdale, NY, USA), or Flag (2 μg of Antibody for 80 μg of relative amount of chromatin A260) (Sigma, Sigma, Grand Island, NY, USA).

Techniques: Expressing, Gene Expression

Journal: Scientific reports

Article Title: Synergistic inhibition effects of andrographolide and baicalin on coronavirus mechanisms by downregulation of ACE2 protein level.

doi: 10.1038/s41598-024-54722-5

Figure Lengend Snippet: Figure 1. Staining of pathological tissues of mouse lung tissues ACE2 and CC10. SARS-COV-2: mice infection with a pseudovirus; Drug intervention group: Andrographolide and baicalein intervene in infected mice. The scale bars of 100 × and 400 × magnification in the panels represent 200 μm and 50 μm, respectively. *P < 0.05, **P < 0.01 SARS-COV-2 compared with Drug intervention group.

Article Snippet: Immunohistochemical analysis of paraffin-embedded mouse lung tissue slides was performed using ACE2 (Affinty, AF5165) and CC10 antibody (Proteintech, 10,490–1-AP) under 20 × and 40 × lenses.

Techniques: Staining, Infection

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Cluster identification. SAE cell types were identified from Dropseq single cell RNA sequencing datasets from six individuals (three healthy nonsmokers and three healthy smokers) using established cell specific markers including KRT5 for basal cells, SCGB1A1 for club cells, MUC5AC for goblet cells, and FOXJ1/DNAI1 for ciliated cells . Club cells were identified by the presence of SCGB1A1, low levels of the basal marker KRT5, and the absence of the differentiated goblet cell marker MUC5AC. Club cells underwent Seurat unsupervised clustering which identified three unique subsets of club cells: club cells 1–3 (CC1, CC2, CC3, respectively). One nonsmoker sample was enriched in pseudogenes ( arrow ); the pseudogene expression data was not used in subsequent analyses. A three-dimensional representation helps to discern the distinct localization of the three clusters in the tSNE plot. The distribution of CC1, 2 and 3 within the original low resolution cluster map of the airway cells is shown in Supplementary Fig. . PNEC pulmonary neuroendocrine cells, APC antigen-presenting cells; NCL high represents a distinct cluster of cells identified in Zuo et al. 2020 . The relative size of the populations shown on the tSNE plot is not representative of the proportions of cells found in the tissue in vivo. The single-cell RNA-seq process involves separation of the epithelium into single, live cells, a process that incurs greater loss and cell death among larger cells such as ciliated cells and favors preservation of smaller cells such as basal cells. It is potentially possible for a cell type or subcluster to be under- or over-represented in this analysis. b Quantification of the average percent of club cells in each club cell subset relative to all club cells. c Heatmap of the top ten differentially expressed genes in each subcluster highlighting the differences among the three subclusters. Yellow represents increased expression levels. For the identity of genes contributing to this heatmap, see Supplementary Fig. . d Representation of each of the club cell subsets in each of the six study subjects. Note the presence of the pseudogene expression in the first nonsmoker subject ( arrow ) that was marked in panel a and excluded from subsequent analysis .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: RNA Sequencing, Marker, Expressing, In Vivo, Preserving

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Airway in adult normal nonsmoker human lung was analyzed for club cell subtypes. Club cells were identified as SCGB1A1 + cells (green) and subsets were identified using various markers in the single cell sequencing. a Secretoglobin family 3A member 1 (SCGB3A1) (red) and secretory leukocyte protease inhibitor (SLPI) (purple). b SCGB1A1 (green), mucin 5B (MUC5B; purple). Club cells that only express SCGB1A1 but no secondary markers are denoted with a white arrow. Club cells expressing multiple markers are denoted with a white arrowhead. Scale bar = 20 μm.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Sequencing, Protease Inhibitor, Expressing

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Differentially expressed genes in progenitor vs. effector club cells a .

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Most commonly enriched genes in each club cell subcluster.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Expressing, Binding Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Assessment of epithelial cell differentiation using Slingshot pseudotime value. Shown are epithelial cells differentiating in the secretory pathway including basal cells (BC, pink), intermediate cells (IC, orange), the progenitor club cell cluster (CC1, red), the effector club cell cluster (CC3, yellow), the proliferating club cell cluster (CC2, blue) and mucous cells (MC, green) for nonsmokers (NS) and smokers (S) plotted separately. Superimposed on each tSNE plot, the Slingshot pseudotime analysis shows the path of differentiation assuming basal cells as a starting point. b Pseudotime analysis. The extent of differentiation from the starting point was evaluated using a pseudotime analysis separately for nonsmokers (NS) and smokers (S). The results for each cluster have been plotted to enable pairwise comparisons. Significance of differences in the differentiation of each cluster is indicated by the p value to the right of the graph. p values were determined using a t test applied to the pseudotime values extracted by Slingshot. c Stem-progenitor activity of CC1 correlates with elevated KRT5 expression. Expression levels measured by the number of unique molecular identifiers (UMI) detected per cell during single cell RNA sequencing were plotted for KRT5 and KRT7 as a function of cluster for NS and S. Significant differences between NS and S are indicated by p values. Note that KRT5 levels in CC1 in NS have a mean that is greater than KRT5 expression in IC. KRT5 levels are considered a marker of stem-progenitor cells and are commonly thought to steadily fall as differentiation proceeds (see Supplementary Fig. ). Expression of high levels of KRT5 in CC1 support CC1 as a branch point in the trajectory. At the same time, KRT7, a marker of club cells (see Supplementary Fig. ), is clearly elevated in CC1 confirming the identity of CC1 as club cells rather than basal cells. d Change in branch point in smokers correlates with elevated SPDEF activity. Significant changes in expression levels of genes known to be up- or down-regulated by SPDEF were assessed in CC1 and CC3. Up-regulated genes included CEACAM5 and GCNT3. Down-regulated genes included SCGB1A1, SCGB3A1, LTF, and AQP5. Note that AQP5 is also up-regulated by oxidant stress such as exposure to cigarette smoke, potentially explaining the lack of down-regulation by smoking in CC1 and CC3. Significance of difference for panels c and d was determined based on the combined conditions of an adjusted p value < 0.05 (Bonferroni corrected) and a log fold change > 0.25. Violin plots in panels b – d include box-and-whisker plots showing the mean, 2nd and 3rd quartiles (box), range from minimum to maximum (whiskers), and outliers (dots).

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Differentiation, Activity Assay, Expressing, RNA Sequencing, Marker, Whisker Assay

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Nonsmoker primary small airway epithelium (SAE) basal cells were cultured in air–liquid interface (ALI) culture for 28 days. a Morphology of the cultures at day 7 and 28. Shown are cross sections of the ALI culture, hematoxylin and eosin stain. b , c Multicolor immunofluorescence assessment of club cell subtypes. All club cells were identified using SCGB1A1 marker (green); effector club cells were further identified by the co-expression of MUC5B (panel b red) or SCGB3A1 (panel c red). DAPI identifies the nucleus of all cells. The number of days after establishment of the air liquid interface is noted. Green arrowheads mark cells that are SCGB1A1 + club cells that lack other markers. Orange arrowheads mark cells that are positive for SCGB1A1 and either MUC5B or SCGB3A1. Red arrowheads mark cells that are positive for MUC5B or SCGB3A1, but express little if any SCGB1A1. Scale bars are 50 μm. d Gene expression on ALI over time. TaqMan probes for genes enriched in effector club cells were assessed by qPCR and normalized to an 18S rRNA control. Probes were tested at days 0, 7, 14, and 28 of SAE nonsmoker cells differentiated in ALI. Error bars represent standard deviation among three different ALI cultures. Genes assessed for expression include MUC5B, SCGB1A1, SLPI, PIGR, and LYZ.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Cell Culture, H&E Stain, Immunofluorescence, Marker, Expressing, Gene Expression, Control, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: Smoking-related reprogramming of small airway epithelium club cells.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques:

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Numbers of club cells in smokers vs. nonsmokers. Club cells from nonsmoker and smoker human small airway epithelium (SAE) cytopreps, identified by SCGB1A1 + immunostaining and absence of KRT5, were quantified and compared against total cells, quantified by DAPI staining, per cytoprep. Three samples of each phenotype were evaluated by a blinded observer, with over 500 total cells per sample; plots show mean ± standard error. b Proportion of club cells exhibiting characteristics of subclusters 1 (progenitor), 2 (proliferative), and 3 (effector) in the population of club cells derived from nonsmokers and smokers. c Effect of cigarette smoke extract (CSE) exposure on differentiation of small airway epithelial club cells. Exposure of basal cells differentiated on air liquid interface (ALI) to cigarette smoke extract (3% Marlboro Red) led to a decrease in defense-related transcript in cells: CYP1A1, positive control demonstrating exposure to cigarette smoke extract; MUC5B; PIGR; SLPI; LYZ; and MUC1. Values are expressed as normalized expression relative to 18S rRNA. Each point represents one well of an experiment done in triplicate; plot shows mean ± standard deviation; p values are from a two-sided unequal variance Student’s t -test. All data is from ALI day 28.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunostaining, Staining, Derivative Assay, Positive Control, Expressing, Standard Deviation

Journal: NPJ Genomic Medicine

Article Title: Smoking shifts human small airway epithelium club cells toward a lesser differentiated population

doi: 10.1038/s41525-021-00237-1

Figure Lengend Snippet: a Immunofluorescence analysis of cytopreps containing club cells (SCGB1A1 + ) expressing the effector club cell genes PIGR or MUC5B. White arrowheads indicate dual-labeled cells. White arrows indicate SCGB1A1 + club cells that lack significant expression of a PIGR or MUC5B. Black arrows mark examples of SCGB1A1-negative non-club cells in the cytoprep. Bar = 20 µm. b Total number of dual labeled (SCGB1A1 + PIGR + or SCGB1A1 + MUC5B + ) cells, or SCGB1A1 single labeled cells were quantified by a blinded observer in nonsmokers and compared with smokers. Plot shows mean of three experiments ± standard error; p values are from a two-sided unequal variance Student’s t -test.

Article Snippet: The following primary antibodies were applied to samples overnight at 4 °C: rabbit polyclonal anti-human MUC5B (sc-20119; Santa Cruz; Santa Cruz, CA; 1:50 dilution); rabbit polyclonal anti-human PIGR (HPA012012; Sigma; affinity purified against the immunogen: 1:100 dilution); rabbit polycloncal anti-human SLPI (NBP1-89139; Novus Biologicals, Centennial, CO; affinity purified against immunogen; 1:100 dilution); mouse monoclonal anti-human SCGB3A1 (MAB27901; R&D, Minneapolis, MN; purified monoclonal antibody; purified from hybridoma; 1:50 dilution from 0.5 mg/mL solution); and rat monoclonal anti-human SCGB1A1 (MAB4218; R&D Systems; purified from hybridoma; 1:100 dilution from 0.5 mg/mL solution).

Techniques: Immunofluorescence, Expressing, Labeling

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia.

doi: 10.1152/ajplung.00347.2011

Figure Lengend Snippet: Fig. 1. Experimental timeline and effect of bone marrow-derived mesenchymal stromal cell-conditioned media (MSC-CM) on hyperoxic alveolar injury. A: neonatal mouse pups were exposed to hyperoxia on postnatal day (PND) 1, systemically injected with MSCs or MSC-CM on PND 4 and killed on PND 14 as previously reported (1). Control mice were injected with pulmonary artery smooth muscle cells (PASMCs) or PASMC-conditioned media (PASMC-CM). Histological specimens were prepared, and lung sections were stained with immunofluorescent (IF) markers for prosurfactant protein C (SPC) and Clara cell secretory protein (CCSP) followed by microscopic quantification. B: representative lung sections from normoxia-exposed control animals compared with hyperoxia-exposed animals treated with either PASMC-CM or MSC-CM. Treatment with MSC-CM prevented alveolar loss and improved volume density of the alveolar wall tissue compared with PASMC-CM. 100 FITC images are shown.

Article Snippet: Primary antibody staining was performed with rabbit -mouse pro-SPC (1: 750; WRAB-9337, Seven Hills Bioreagents, Cincinnati, OH) and goat -mouse CCSP (1:250; sc-9773; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Derivative Assay, Injection, Control, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Flow chart of methodological steps. The methods are subdivided in a telomere and gamma-H2AX staining phase and a second, postelution phase, which include the IF staining of specific cell markers. Integration of data from phase 1 and 2 was done by scanning coordinates. Abbreviations: IF, immunofluorescence; CC10, club cell-10; FISH, fluorescence in situ hybridization; aSMA, smooth muscle actin.

Article Snippet: Slide 2: mouse anti-human αSMA (1:50; Sigma-Aldrich, Darmstadt, Germany) and rabbit anti-human CC10 antibody (1:100 sc-25554; Santa Cruz Biotechnology).

Techniques: Staining, Immunofluorescence, Fluorescence, In Situ Hybridization

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

doi: 10.1369/0022155418761351

Figure Lengend Snippet: Bronchial club and arterial smooth muscle cells identification using CC10- and aSMA-selective immunofluorescence stains. White arrows point to the (A1–A3) row of bronchus epithelial cells and to (B1–B3) cells of the arterial wall. Z-stacked LSCM pictures of (A1 and B1) telomere (red dots) and (A2 and B2) gamma-H2AX (yellow dots). DAPI DNA staining (blue) and cell-type identification of (A3) CC10-positive club cells (white) and (B3) aSMA-positive smooth muscle cells (purple). Scale bar is valid for all images and represents 10 µm. Abbreviations: CC10, club cell-10; aSMA, smooth muscle actin; LSCM, laser scanning confocal microscopy; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Slide 2: mouse anti-human αSMA (1:50; Sigma-Aldrich, Darmstadt, Germany) and rabbit anti-human CC10 antibody (1:100 sc-25554; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Confocal Microscopy

Journal: The European respiratory journal

Article Title: Club cell 10-kDa protein attenuates airway mucus hypersecretion and inflammation.

doi: 10.1183/09031936.00080913

Figure Lengend Snippet: FIGURE 4 Haematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining. Normal human bronchial epithelial cells were grown for 14 days with club cell 10-kDa protein (CC10) (0, 1, 10 or 20 ng?mL-1) and exposed to lipopolysaccharide (LPS) (5 mg?mL-1) or LPS vehicle (PBS) on day 14. a, f) PBS alone; b, g) LPS without CC10; c, h) LPS and CC10 at 1 ng?mL-1; d, i) LPS and CC10 at 10 ng?mL-1; e, j) LPS and CC10 at 20 ng?mL-1. Scale bars550 mm.

Article Snippet: The following reagents were purchased: recombinant human CC10 (R&D Systems, Inc., Minneapolis, MN, USA); LPS (from Escherichia coli serotype 0111:B4; Sigma-Aldrich Co., St Louis, MO, USA); recombinant human interleukin (IL)-13 (Biotec, Hamburg, Germany); MUC5AC monoclonal antibody (45M1; Lab Vision, Fremont, CA, USA); MUC5B rabbit polyclonal IgG (Santa Cruz Biotechnology, TX, USA); antimouse-IgG horseradish peroxidase (HRP)-linked whole antibody (GE Healthcare, Piscataway, NJ, USA); phospho- and nonphospho-specific ERK1/2, phospho-specific NF-kB p65 (Ser536) and nonphosphospecific NF-kB p65 and anti-rabbit-IgG HRP antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA); DMEM, Ham’s F12 medium and PBS (Gibco, Grand Island, NY, USA); bronchial epithelial cell growth medium supplemented with the SingleQuotR 1 kit and Hanks’ balanced salt solution (HBSS) (Lonza Walkersville Inc., Walkersville, MD, USA); PD98059 (Calbiochem, La Jolla, CA, USA); and PS1145 (SigmaAldrich Co.).

Techniques: Staining