Journal: PLoS ONE

Article Title: Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts

doi: 10.1371/journal.pone.0215490

Figure Lengend Snippet: (A) Comparison of the GFP positive fibroblasts in different groups with (w/) and without (w/o) applying centrifugation. A paired-sample t-test was conducted to compare the percentage of transduced GFP positive cells that either underwent centrifugation or not (*** p <0.0001; ** p <0.05)(n = 3). (B) GFP expression in nhF and ahF fibroblasts 48 hours after transduction. (C) GFP expression in chF1 and chF2 48 hours after transduction. (D) GFP expression in nhF and ahF 48h after transduction and centrifugation. (E) GFP positive cells in chF1 and chF2 48h after transduction and centrifugation. DAPI staining pictures of the right side of each field were shown.

Article Snippet: Human neonatal foreskin fibroblasts (nhF; ATCC CRL-2522) and adult dermal fibroblast (ahF; Thermo Fisher C0135C) were used as control samples.

Techniques: Comparison, Centrifugation, Expressing, Transduction, Staining

Journal: PLoS ONE

Article Title: Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts

doi: 10.1371/journal.pone.0215490

Figure Lengend Snippet: Expression was normalized to iPSC-Ctrl1. Parental fibroblasts were used as a negative control.

Article Snippet: Human neonatal foreskin fibroblasts (nhF; ATCC CRL-2522) and adult dermal fibroblast (ahF; Thermo Fisher C0135C) were used as control samples.

Techniques: Expressing, Negative Control

Journal: Biomaterials

Article Title: 3D Biomaterial Matrix to Support Long Term, Full Thickness, Immunocompetent Human Skin Equivalents with Nervous System Components

doi: 10.1016/j.biomaterials.2018.04.044

Figure Lengend Snippet: Experimental nomenclature and descriptions.

Article Snippet: 2.3 Cell maintenance Primary neonatal foreskin fibroblasts (Lonza, Portsmouth, NH) were maintained in fibroblast media (S1).

Techniques:

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A AG01523c and primary human dermal fibroblasts from an adult donor (HDFs) were irradiated under a UVB lamp for durations corresponding to irradiation doses ranging from 35 to 3100 mJ/cm 2 . After incubation at 37 o C for 72 h, cells were detached by trypsinization and red-colored metabollicaly active cells after staining with Neutral Red were measured using a haemocytometer. In addition, AG01523c fibroblasts were irradiated with a UVB dose of 700 mJ/cm 2 before staining with Neutral Red and cell counting 24, 48, and 72 h post-irradiation. Untreated cells served as the reference sample for the estimation of cell viability. Representative graphs out of at least three similar ones from independent experiments are shown here. Asterisks denote statistically significant differences compared to the untreated control at the respective time-point ( p < 0.05, Student’s t test). B Cells exposed to a 700 mJ/cm 2 UVB irradiation dose were observed under a light or fluorescence [after staining with 2 μg/mL 4 ´,6-diamidino-2-phenylindole (DAPI)] optical microscope for the presence of typical apoptotic features; fixed with 50% (v/v) ice-cold ethanol and stained with PI before cell cycle analysis to reveal the incidence of characteristic sub-G1 peaks; subjected to RNA and protein extraction to assess bcl-2 and bax mRNA levels by RT-qPCR and caspase-3 activation by western blot analysis, respectively. Representative microscopic images, histogram plots, relative gene expression graphs, and western blots of three independent experiments are shown. Arrows in the fluorescence microscopic pictures depict regions of condensed/fragmented chromatin in the DAPI stained nuclei (scale bar = 20 μm). M1 marker designates the sub-G1 peak corresponding to apoptotic cells with fragmented DNA preceding the characteristic G1 and G2/M peaks in the histogram plots of PI-stained cells. Asterisks denote statistically significant differences in the gene expression of UVB-exposed fibroblasts in comparison to the untreated control ( p < 0.05, Student’s t test). Staurosporine was used as a known apoptosis inducer leading to caspase-3 cleavage, while western blot analysis against actin served as the loading control. C SKH-1 hairless mice were exposed to 350 mJ/cm 2 of UVB radiation and the dorsal skin was surgically removed 24 and 48 h post-irradiation for the assessment of cleaved caspase-3 in situ expression in the skin sections after immunohistochemical staining. Pictures shown here are representative of three independent experiments. Scale bar = 50 μm.

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: Irradiation, Incubation, Staining, Cell Counting, Control, Fluorescence, Microscopy, Cell Cycle Assay, Protein Extraction, Quantitative RT-PCR, Activation Assay, Western Blot, Gene Expression, Marker, Comparison, In Situ, Expressing, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A Cells were exposed to 700 mJ/cm 2 of UVB radiation before protein extraction at the designated time-points post-irradiation and western blot analysis for phopsho-ATM, phospho-p53, and total p53. Western blot analysis for actin was performed to verify equal loading. Experiments were repeated three times and representative blots are presented. B Skin fibroblasts were cultured on glass coverslips before UVB treatment with 700 mJ/cm 2 and further incubated at 37 o C for 24 h. They were then fixed with 4% (w/v) formaldehyde followed by labeling using an antibody against phospho-H2A.X at Ser139 and a FITC-conjugated IgG. Labeled cells were visualized using a confocal laser scanning microscope. Cells exposed to 10 Gy of ionizing radiation served as the positive control. Representative images of three independent experiments are shown here. Scale bar = 20 μm. C SKH-1 hairless mice were exposed to 140 and 350 mJ/cm 2 of UVB radiation before sacrifice and removal of dorsal skin 6 h post-irradiation. Non-irradiated (control) vs. irradiated tissue sections were subjected to immunohistochemical staining for the phosphorylated form of H2A.X at Ser139. Pictures shown here are representative of three independent experiments and arrows depict γH2A.X-positive cells in the epidermis and dermis of SKH-1 hairless mouse skin. Scale bar = 50 μm. D Cell cultures were pre-incubated with 1 μΜ of the Akt inhibitor MK2206 and 5 μΜ of the JNKs inhibitor SP600125 for 1 h before exposure to 700 mJ/cm 2 of UVB radiation, protein extraction and western blot analysis for phopsho-p53 at Ser15. Western blot analysis using an anti-actin antibody was performed for the validation of the equal loading. A representative experiment of three similar ones is depicted here. E Human skin fibroblasts were cultured in 96-well plates. When confluent, cells were pre-incubated with 10 μM 2 ´,7 ´-dichlorfluorescein-diacetate (DCFH-DA) for 1 h at 37 o C and then exposed to 140, 350, and 700 mJ/cm 2 of UVB radiation. Intracellular levels of ROS were measured at the designated time-points post-irradiation by recording fluorescence (excitation wavelength: 485 nm, emission wavelength: 520 nm). ROS production was expressed as a % ratio of the untreated control. F Cells were exposed to an irradiation dose of 700 mJ/cm 2 and further incubated at 37 o C for 2, 4, 6, and 10 h before RNA extraction and RT-qPCR analysis using specific primers for HO-1 and NQO1. The 2 −ΔΔCt method was applied to quantify mRNA expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. A representative RT-qPCR analysis performed in triplicates is depicted here. The asterisk denotes statistically significant differences in comparison to the untreated control ( p < 0.05, Student’s t-test). G Cells were pre-incubated with 20 μΜ of the known anti-oxidant Trolox before UVB treatment with 700 mJ/cm 2 and total protein extraction at 1 and 12 h for western blot analysis for phospho-Akt (Ser473)/JNKs and phospho-p53, respectively. Representative images of three independent experiments with similar results are shown. The non-phosphorylated forms of the kinases, as well as actin served as the loading controls. H Cells pre-incubated with 20 μΜ Trolox overnight were treated with 10 μM DCFH-DA for 1 h at 37 o C before measurement of intracellular ROS levels. ROS production was expressed as a % ratio of the untreated control ( N = 3). In parallel, cells pre-treated with 20 μΜ Trolox overnight were exposed to 700 mJ/cm 2 of UVB radiation. Cultures were further incubated at 37 o C for 72 h before trypsinization and measurement of cell number after staining with Neutral Red using a haemocytometer ( N = 5, conducted in duplicates). Differences in comparison to the untreated control are considered statistically significant when p < 0.05 (Student’s t test).

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: Protein Extraction, Irradiation, Western Blot, Cell Culture, Incubation, Labeling, Laser-Scanning Microscopy, Positive Control, Control, Immunohistochemical staining, Staining, Biomarker Discovery, Fluorescence, RNA Extraction, Quantitative RT-PCR, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A Human dermal fibroblasts were pre-incubated with 100 μM suramin for 1 h before exposure to 700 mJ/cm 2 of UVB radiation. Then, cells were further incubated at 37 o C for 72 h and cell number was counted after staining with Neutral Red. A representative experiment out of three similar ones is shown. p for statistically significant differences in comparison to the respective samples in the absence of suramin (Student’s t test) is presented in the graph. B Cells were pre-treated with 100 μM suramin for 1 h before exposure to 700 mJ/cm 2 of UVB radiation. Protein extracts after 1 and 12 h were subjected to western blot analysis for phospho-Akt (Ser473)/JNKs and phospho-p53, respectively. Representative blots of three independent experiments with similar results are depicted here. The non-phosphorylated forms of the kinases and actin were analyzed to verify equal loading. C A 100 μΜ suramin solution was analyzed spectrophotometrically in a range from 200 to 600 nm in order to assess its absorption spectrum. PBS served as the negative control. Gray shading marks the wavelength range corresponding to the UVB band (280–315 nm). D Cells were pre-incubated for 1 h with the growth factor receptor inhibitors [STI571 for PDGFR (2 μΜ); SU5402 for FGFR and VEGFR (20 μΜ); SB431542 for TGFβR1 (10 μΜ); I-OMe-AG538 for IGFIR (12 μΜ); AG1478 for EGFR (10 μΜ)] and then exposed to 700 mJ/cm 2 UVB radiation. Cultures were further incubated at 37 o C for 72 h, cells were detached by trypsinization, stained with Neutral Red and counted in a haemocytometer. p for statistically significant differences in comparison to the respective samples with no inhibitor (Student’s t test) is provided. E Cells were incubated with 10 μΜ of the EGFR inhibitor AG1478 for 1 h, exposed to 700 mJ/cm 2 of UVB radiation and further incubated at 37 o C for 1 or 12 h before protein extraction and western blot analysis for phospho-Akt (Ser473)/JNKs or phospho-p53, respectively. Representative blots of three independent experiments are shown here, while total Akt and JNKs, as well as actin were analyzed to validate equal loading. F Cell cultures were treated with 100 ng/ml EGF, exposed to 700 mJ/cm 2 of UVB radiation and further incubated at 37 o C for 72 h. Neutral Red-positive cells were counted in a haemocytometer. A representative graph from three independent similar experiments is presented. p for statistically significant differences in comparison to the respective samples in the absence of the growth factor (Student’s t test) is shown.

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: Incubation, Staining, Comparison, Western Blot, Negative Control, Protein Extraction

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A Skin fibroblasts were cultured on glass coverslips before treatment with 25 μM of SC79 and they were then fixed with 4% (w/v) formaldehyde in PBS. Labeling was performed using an antibody against Nrf2 and a FITC-conjugated IgG. Labeled cells were visualized using a confocal laser scanning microscope (Scale bar = 20 μm). In addition, RNA was extracted and RT-qPCR analysis was performed to estimate ho-1 and nqo1 mRNA levels. Representative microscopic pictures and representative graphs of means ± standard deviations from three independent experiments conducted in triplicates are depicted here. Asterisks show statistically significant differences compared to the untreated control ( p < 0.05, Student’s t-test). B Cells were transfected with 50 nM of either a scramble or an siRNA sequence targeting Nrf2, treated with 25 μΜ of SC79 for 1 h and then exposed to 700 mJ/cm 2 . Cell number was measured after 72 h in Neutral Red-stained cells. A representative experiment is presented out of three independent experiments. p for statistically significant difference in comparison to scramble SC79/UVB-treated cells (Student’s t-test) is provided. C Dermal fibroblasts were transfected with 50 nM of either scramble or Nrf2 siRNA, treated with 25 μΜ of SC79 for 1 h and then exposed to 700 mJ/cm 2 UVB. Total protein extraction was performed 1 h post-irradiation followed by western blot analysis for phospho-Akt (Ser473) and phospho-JNKs. Representative blots of three independent experiments are shown. Western blot analysis of the non-phosphorylated forms of the kinases was done to evidence equal loading. D Cells were pre-treated with 1 μΜ of the Akt inhibitor MK2206 and 5 μM of the JNKs inhibitor SP600125 for 1 h or transfected with 50 nM scramble and SignalSilence ® Akt siRNA I sequences, before the addition of 25 μΜ SC79 for another 1 h. RT-qPCR analysis for ho-1 gene expression was performed in the extracted RNA samples. A representative experiment from three similar ones is presented. Asterisks denote statistically significant differences in comparison to the respective samples without any inhibitor or siRNA ( p < 0.05, Student’s t test). E Cells were treated with 25 μM SC79 or 10 μΜ sulforaphane (SFN) for 1 h and then exposed to an irradiation dose of 700 mJ/cm 2 UVB before protein extraction and western blot analysis for phospho-Akt (Ser473) and phospho-JNKs, as well as their non-phosphorylated forms. Representative blots of three independent experiments are shown. F Cells were transfected with 50 nM of a scramble or a Nrf2 siRNA sequence, treated with 10 μΜ of SFN for 1 h and then exposed to 700 mJ/cm 2 . Cell number was measured after 72 h in Neutral Red-stained cells. A representative experiment out of three similar ones is presented. p for statistically significant difference in comparison to scramble SFN/UVB-treated cells (Student’s t test) is shown. G Cells were cultured in 96-well plates and treated with 20 μΜ Trolox, 25 μΜ SC79, or 10 μΜ SFN overnight. They were then incubated with 10 μM 2 ´,7 ´-dichlorfluorescein-diacetate (DCFH-DA) for 1 h at 37 o C before their exposure to 700 mJ/cm 2 of UVB radiation. Intracellular levels of ROS were estimated by recording fluorescence (excitation wavelength: 485 nm, emission wavelength: 520 nm). ROS production was expressed as a % ratio of the untreated control. Experiment was repeated three independent times and one representative graph is depicted. p for statistically significant differences compared to the respective sample without Trolox, SC79 or SFN (Student’s t test) is presented. H Cells were treated with 25 μΜ SC79, 10 μΜ SFN or both for 1 h and then exposed to 700 mJ/cm 2 . Cell number was measured after 72 h in Neutral Red-stained cells. A representative graph of three independent experiments is shown here. p for statistically significant difference in comparison to the UVB treated cells in the presence of SC79 or SFN alone (Student’s t test) is presented.

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: Cell Culture, Labeling, Laser-Scanning Microscopy, Quantitative RT-PCR, Control, Transfection, Sequencing, Staining, Comparison, Protein Extraction, Irradiation, Western Blot, Gene Expression, Incubation, Fluorescence

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A Cells were pre-incubated with 5 μM of the specific ATM inhibitor KU55933 for 1 h, exposed to 700 mJ/cm 2 of UVB radiation and further incubated at 37 o C for the designated time periods before total protein extraction and western blot analysis using antibodies against phospho-ATM at Ser1981, phospho-Akt at Ser473 and phospho-JNKs. Additionally, cells were transfected with 50 nM of either a predesigned scramble or the human p53 siRNA sequence before their exposure to 700 mJ/cm 2 of UVB radiation, protein extraction 12 h post-irradiation and western blot analysis for phospho-Akt at Ser473 and phospho-JNKs. The non-phosphorylated forms of Akt and JNKs, as well as actin were analyzed in order to verify equal loading. Representative blots of three independent experiments are shown. Cells were pre-incubated with 5 μM of the specific ATM inhibitor KU55933 for 1 h ( B ) or transfected with 50 nM of either scramble or p53 siRNA ( C ), exposed to 700 mJ/cm 2 of UVB radiation and further incubated at 37 o C for 72 h before staining with Neutral Red and cell counting. Graphs are representative of three independent experiments and p for statistically significant differences in comparison to the respective sample without the inhibitor ( B ) or to the respective scramble ( C ) is presented. D MDAH041 fibroblasts from a Li-Fraumeni patient were irradiated with UVB irradiation doses ranging from 35 to 3100 mJ/cm 2 . After incubation at 37 o C for 72 h, cells were detached by trypsinization, stained with Neutral Red, and measured using a haemocytometer. Untreated cells served as the reference sample for the estimation of cell viability. A representative graph out of three similar ones from independent experiments is shown here. Asterisks denote statistically significant differences in comparison to the respective untreated control ( p < 0.05, Student’s t test).

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: Incubation, Protein Extraction, Western Blot, Transfection, Sequencing, Irradiation, Staining, Cell Counting, Comparison, Control

Journal: Cell Death & Disease

Article Title: Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

doi: 10.1038/s41419-022-05106-y

Figure Lengend Snippet: A UVB-induced inherent cellular responses. UVB radiation leads human dermal fibroblasts to apoptosis in vitro and in vivo. UVB treatment induces in parallel the EGFR-Akt signaling pathway and the JNKs MAPKs which intervene with the phosphorylation of p53, part of the ROS-independent DNA damage response of the cells towards UVB genotoxicity. EGFR-Akt, JNKs, and ATM-p53 pathways seem to co-operate for the protection towards UVB radiation and the retention of a sufficient percentage of viable cells in the exposed population. This is evidenced by the increased loss of viability after Akt inhibition or siRNA-mediated knocking-down and the unmitigated cell death when directly (or indirectly through JNKs inhibition) impeding the activation of the ATM-p53 pathway. B Interplay of exogenously supplied photoprotective molecules with the intrinsic UVB-induced machinery to amplify the resistance of human skin fibroblasts against UVB radiation. Whereas the known anti-oxidant Trolox does not increase cell survival rate after UVB exposure, extra protection is conferred by other exogenously supplied molecules: suramin acts as a UV shield; EGF activates EGFR; SC79 enhances inherent cellular response (i.e., it activates further Akt and JNKs) and triggers Nrf2 activation downstream of Akt; sulforaphane induces Nrf2 activation independent of the Akt or JNKs signaling pathways. Nrf2-mediated protection by SC79 and sulforaphane is not based on ROS-scavenging and can be attributed to the transcription factor’s ability to safeguard genome integrity or to produce a protective extracellular matrix.

Article Snippet: Neonatal foreskin fibroblasts (AG01523c) used in this study were from the Coriell Institute for Medical Research (Camden, NJ, USA), primary human dermal fibroblasts from an adult donor were from a preexisting cell bank of the Laboratory of Cell Proliferation and Ageing [ ], while MDAH041 fibroblasts from a Li-Fraumeni patient were a generous gift of Dr. M. Agarwal.

Techniques: In Vitro, In Vivo, Phospho-proteomics, Inhibition, Activation Assay, Protein-Protein interactions