Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 1. Effect of in vitro (A and B) and in vivo (C and D) aging on fibroblast motility (A and C) and proliferation (B and D). A and B, Hs68 were passaged and tested at different passages with PDR back-calculated. Cell migration assays and cell proliferation assays were performed in the absence (E and open bars) and presence (G and black bars) of EGF (1 nM) as described under “Experimental Procedures.” The data are the mean 6 S.E. of more than three independent studies, each performed in triplicate. Statistical analysis was performed by Student’s t test as compared with P5 (PDR42) of Hs68: *, p , 0.05, **, p , 0.01 C, fibroblasts from fetus male (‚, basal; Œ, with EGF), 1-month-old male (CRL-1489) (M, basal; f, with EGF), 17-year-old male (CRL-7315) (L, basal; l, with EGF), and 83-year-old male (CRL-7815) (E, basal; G, with EGF) were assessed by cell migration assay. Fibroblasts from 1-month-old male (CRL-1489) and 83-year-old male (CRL-7815) were passaged and assessed at indicated passages. Basal and EGF-induced cell motility were measured as described under “Experimental Procedures.” The data are the mean of more than three independent studies, each performed in triplicate. Statistical analysis was preformed by Student’s t test as compared with P8 (PDR24) of CRL-1489 or P3 (PDR12) of CRL-7815: *, p , 0.05, **, p , 0.01 D, fibroblasts from different aged donor were obtained. Basal (clear bars) and EGF-induced (black bars) thymidine incorporation were measured as described under “Experimental Procedures.” The data are the mean 6 S.E. of more than three independent studies performed in triplicate. Statistical analysis was performed by Student’s t test as compared with fetal cells: *, p , 0.05, **, p , 0.01

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: In Vitro, In Vivo, Migration, Cell Migration Assay

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 4. Ligand-induced internalization of EGFR in in vitro aged Hs68 fibroblasts (A) and fibroblasts from male donors (B). Internalized and surface-bound EGF were determined using 125I-EGF as described under “Experimental Procedures.” The endocytic rate con- stants were calculated by the time course of loss of surface-bound EGF and accumulation of internalized EGF. The data are the mean 6 S.E. of at least two experiments at each point except for CRL-7815. Statistical analysis was performed by Student’s t test as compared with early passage of cells: **, p , 0.01.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: In Vitro

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 8. Expression of exogenously encoded EGFR in near senescent Hs68 fibroblasts (C and D) and effects on cell motility (A) and mitogenesis (B). Eukaryotic expression plasmids for EGFR or GFP (control) were introduced into P18 (PDR3) of Hs68 using electropo- ration technology (Gene Pulser, Bio-Rad). The cells were incubated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under “Experimen- tal Procedures.” A, cell migration assay; B, thymidine incorporation; C, immuno- blot analysis with anti-phosphotyrosine antibody phosphotyrosine (PY-20, Trans- duction Laboratories); D, immunoblot analysis and densitometry using anti- EGFR (#05–104, Upstate Biotechnology Inc.) or anti-a-actin (A-2066, Sigma) anti- bodies. The data in graphs (A) and (B) are the mean 6 S.E. of three independent electroporations, with each experiment performed in triplicate. The data in (C) and (D) are representative of the electro- poration experiments. Statistical analysis was performed by Student’s t test as com- pared with control cells: *, p , 0.05, **, p , 0.01.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: Expressing, Control, Incubation, Cell Migration Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 9. Effect of increased EGFR signaling capacity on early passage of Hs68 fibroblasts. A and B. EGFR (10 mg/107 cells) or GFP (20 mg/107 cells as control) plasmid were introduced into P5 (PDR42) Hs68 cells. The cells were incu- bated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under “Experimental Procedures.” A, cell migration assay; B, immunoblot analysis with anti-phosphotyrosine antibody phos- photyrosine (PY-20, Transduction Labo- ratories), anti-EGFR (Upstate Biotech- nology Inc.) or anti-a-actin (Sigma) antibodies. The data are the mean 6 S.E. of two independent electroporations, with each experiment performed in triplicate. The blots shown are representative of the electroporation experiments. C, in vitro wound healing assays were performed with P5 of Hs68 in the absence and pres- ence of EGF (1 or 10 nM). The data are the mean 6 S.E. of more than two independ- ent studies, each performed in triplicate. There were no statistical differences in motility responses between the EGF treatments in A and C.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: Control, Plasmid Preparation, Cell Migration Assay, Western Blot, Transduction, Electroporation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: LEM domain–containing protein 3 antagonizes TGFβ–SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol

doi: 10.1074/jbc.ra118.003658

Figure Lengend Snippet: Figure 2: LEMD3-Smad2/3 interactions are inversely correlated to substrate stiffness and occur in the nucleus and cytoplasm. A, micrographs of LEMD3-Smad2/3 PLA interactions on soft (top row, 1 kPa) and stiff(bottom row, 25 kPa) matrices (PLA in green, f-Actin in blue, nucleus in white). B, quantification of PLA interactions grouped by substrate stiffness and by TGFβ dose. Total LEMD3-Smad2/3 interactions were negatively correlated to substrate stiffness (p<0.0001 for 1 kPa vs 25 kPa for both 0 pg/mL and 50 pg/mL TGFβ) but not correlated to TGFβ dose. Cytoplasmic (p=0.0164 for 0 pg/mL TGFβ, p=0.0087 for 50 pg/mL TGFβ) and nuclear compartment (p = 0.0428 for 0 pg/mL TGFβ, p=0.1228 for 50 pg/mL TGFβ) interactions were also negatively correlated to substrate stiffness. C, total HFFs and CCL210s LEMD3- Smad2/3 interactions by PLA normalized to 0.5 kPa on surfaces with stiffness of 0.5, 1, 4, 8, 25 kPa and glass. Each fibroblast population showed a biphasic trend, centered around a peak of LEMD3-Smad2/3 interactions at 1kPa. CCL210 demonstrated greater dynamic range in interaction frequency and a slower loss of interactions on stiffer substrates than HFFs. D, subcellular location of LEMD3-Smad2/3 PLA interactions in HFFs and CCL210s from (C). Each cell line demonstrated a cytoplasmic shift in location with increasing substrate stiffness (p<0.0001 and p=0.0199 for HFFs and CCL210s, respectively; ANOVA - Test for trend). E, micrographs of V5-Smad2/3 PLA interactions with pFLAG-LEMD3-V5 on soft (top row) and stiff(bottom row) matrices (PLA in green, f-actin in blue, FLAG in red, nucleus in white). Area above dashed line in 25kPa image indicates filler space. F, V5-Smad2/3 PLA interactions were also negatively correlated with substrate stiffness (for 1 kPa vs 25 kPa: Total PLA - p<0.0001, Cytoplasmic PLA - p<0.0001, Nuclear PLA - p=0.0018) and also occurred in the cytoplasm. G, V5-Smad2/3 PLA interactions were a significantly higher on soft substrates independent of the degree of pFLAG-LEMD3-V5 expression (difference in linear regression slopes - p<0.0001). All PLA groups were statistically compared using a 2-way ANOVA with Tukey post-test unless noted. All scale bars are 10 μm. All data represented by the mean with SEM except Panel (G) where individual data points are plotted. 26

Article Snippet: Primary Fibroblast Cultures and Transfection, Human Lung Biopsies, and Other Reagents — Primary human foreskin fibroblasts (HFFs) were procured from ATCC (ATCC SCRC-1041, ATCC, 1SCV000332734.2, SCV000341129.2, SCV000380866.2, SCV000380877.2, SCV000380879.2, SCV000380878.2 by guest on A ugust 16, 2018 http://w w w .jbc.org/ D ow nloaded from Manassas, VA) and routinely cultured in DMEM (ThermoFisher, Waltham, MA) supplemented with 15% FBS and 1% penicillin/streptomycin (ThermoFisher, Waltham, MA) in 5% CO2 at 37C in a humified incubator to passage 13.

Techniques: Expressing

Journal: Journal of Biological Chemistry

Article Title: LEM domain–containing protein 3 antagonizes TGFβ–SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol

doi: 10.1074/jbc.ra118.003658

Figure Lengend Snippet: Figure 5: C-Terminal fragments of LEMD3 bind Smad2/3 and antagonize Smad2/3-Smad4 complexes. A, Representative images of V5-Smad2/3 PLA reactions imaged in fibroblasts transfected with pFLAG- LEMD3p.Δ21-669-V5 (top two rows) and electroporation control cells (bottom row, “Neon Only”) on 1 kPa and 25 kPa hydrogel surfaces (PLA in green, f-actin in blue, V5 in red, nucleus in white). B, PLA interactions from (A) between the V5 tag of a C-terminal fragment (“CTF”) of LEMD3 (pFLAG-LEMD3p.Δ21-669-V5) and Smad2/3, normalized to the total interactions observed on 1 kPa hydrogels. HFFs on 1 kPa hydrogels have significantly more interactions overall (p<0.0001), in the cytosol (p<0.0001), and in the nucleus (p=0.0082) relative to fibroblasts on 25 kPa hydrogels. All transfected cells had more PLA interactions than electroporation-only ("Neon") populations (p<0.0001 and p<0.0227 for 1 kPa and 25 kPa hydrogels, respectively). Scale bars are 11 μm. All statistical testing done with 2-way ANOVA with Tukey’s post-test. C, Representative images from HFFs on 1 kPa hydrogels assayed for Smad2/3-Smad4 interactions. Cells in the 2nd and 3rd rows were treated with 500pg/mL TGFβ for 1 hour before fixation, while cells in the first row were untreated. Cells in the 3rd row were electroporated with pFLAG-LEMD3p.Δ21-669-V5 while cells in the top two rows were electroporation control cells (PLA in green, f-actin in blue, nucleus in white). All scale bars are 17 μm. D, Normalized quantification of data from (C). Fibroblasts treated with 500pg/mL TGFβ and overexpressing the CTF of LEMD3 had significantly less Smad2/3-Smad4 complexes than untransfected cells treated with 500pg/mL TGFβ (p=0.0081). Untransfected TGFβ fibroblasts treated with 500pg/mL also had significantly more Smad2/3-Smad4 complexes than untransfected fibroblasts (p=0.001). All statistical testing done with a 2-way ANOVA with Tukey’s post-test. E, Interactions between PPM1α and endogenous LEMD3 or its CTF assayed by PLA on glass surfaces. The first and 2nd rows used antibody pairs between PPM1α and endogenous LEMD3. The 3rd and 4th rows used antibody pairs between PPM1α and V5 tag on cells transfected with pFLAG-LEMD3p.Δ21-669-V5 (PLA in green, f-actin in blue, nucleus in white). All scale bars are 17 μm. F, Normalized quantification of the interaction rates between LEMD3 or its CTF by subcellular compartment. Both endogenous LEMD3 and its CTF interacted with PPM1α as demonstrated by PLA frequencies above their respective negative controls (for “Total” interactions: p<0.0001 for both LEMD3 vs. No Primary and for CTF vs. Neon Only, respectively). Both LEMD3 and CTF transfected cells also had significantly higher PPM1α interaction rates than their respective controls in the cytoplasm (for “Cytoplasmic” rates: p<0.0001 and p=0.0081 for LEMD3 and CTF pairs, respectively), but only endogenous LEMD3 had statistically significant higher interaction rate in the nucleus (for “Nuclear” rates: p<0.0001 and p=0.2569 for endogenous LEMD3 and CTF-expressing fibroblasts, respectively). All statistical testing was performed by a 2-way ANOVA with Sidak’s post-test. All data represented by the mean with SEM. 29

Article Snippet: Primary Fibroblast Cultures and Transfection, Human Lung Biopsies, and Other Reagents — Primary human foreskin fibroblasts (HFFs) were procured from ATCC (ATCC SCRC-1041, ATCC, 1SCV000332734.2, SCV000341129.2, SCV000380866.2, SCV000380877.2, SCV000380879.2, SCV000380878.2 by guest on A ugust 16, 2018 http://w w w .jbc.org/ D ow nloaded from Manassas, VA) and routinely cultured in DMEM (ThermoFisher, Waltham, MA) supplemented with 15% FBS and 1% penicillin/streptomycin (ThermoFisher, Waltham, MA) in 5% CO2 at 37C in a humified incubator to passage 13.

Techniques: Transfection, Electroporation, Control, Expressing