Journal: bioRxiv

Article Title: Myosin-II mediated traction forces evoke localized Piezo1 Ca 2+ flickers

doi: 10.1101/294611

Figure Lengend Snippet: A. Schematic of the Molecular Tension Sensor (MTS) used for traction force imaging. The N-terminal region of the sensor (blue) is tethered to a PEG-functionalized glass coverslip. A fibronectin domain at the C-terminal end (brown) binds to the cell’s integrins (purple), allowing cells to attach to the glass coverslip. An elastic spring domain bridges the two ends of the sensor and separates a FRET donor (green) and acceptor (red). Cell-generated traction forces pull the FRET pair apart, resulting in reduced FRET efficiency. The FRET index (the ratio of acceptor intensity over summed donor and acceptor intensities), serves as a measure of force; with a low FRET index indicating high force, and a high FRET index indicating a low force. B. Imaging of Piezo1 Ca 2+ flickers. Panels show resting fluorescence of HFF cells loaded with Ca 2+ indicator Cal-520, with overlaid red dots marking the centroid locations of Ca 2+ flickers. C. Corresponding force maps from the same cells, overlaid with red dots marking the Ca 2+ flicker locations. Blue denotes low FRET (high force) and red denotes high FRET (low force). The color bar in C represents FRET efficiency (top) and the average force per MTS per pixel in pN obtained from calibrated FRET-Force curves of the MTS as described in Methods. D. Box and whisker plots with red boxes showing distances from Piezo1 flicker localizations to the nearest traction force region for HFFs (top: 515 flickers from 9 cells) and hNSPCs (bottom: 66 flickers from 18 cells). Grey boxes show corresponding mean distances derived from simulations of 9000 random intracellular locations for each cell. Box range is 25 th to 75 th percentile; whiskers denote 10 th and 90 th percentile, horizontal lines represent the median and filled black squares represent mean. *** denotes p < 0.001 by Kolmogorov-Smirnov test.

Article Snippet: Mat-tek dishes of shRNA-transfected cells loaded with Cal-520 AM were first scanned using an Olympus UPLSAPO 10× objective to identify cells expressing TurboRFP.

Techniques: Imaging, Generated, Fluorescence, Whisker Assay, Derivative Assay

Journal: Brain Pathology

Article Title: Early growth response‐1 regulates acetylcholinesterase and its relation with the course of Alzheimer’s disease

doi: 10.1111/bpa.12688

Figure Lengend Snippet: Expression of mRNA of early growth response 1 (EGR1) and acetylcholinesterase (AChE) in the prefrontal cortex (Brodmann area 46) during the course of Alzheimer’s disease (AD). A. EGR1‐mRNA levels showed significant changes (ANOVA, F(6,42) = 4.651, P = 0.001) among Braak stages. EGR1‐mRNA is especially decreased in Braak IV compared with Braak I and II, and further decreased in Braak V–VI (Bonferroni test, P ≤ 0.043); B. EGR1‐mRNA levels also showed a significant change among Reisberg groups (ANOVA, F(2,22) = 4.189, P = 0.0287), and in particular they significantly declined in the mild cognitive decline (MCD) group compared with the no cognitive decline (NCD) group (Bonferroni test, P = 0.033). C. AChE‐mRNA did not show a significant decrease along the Braak stages (ANOVA, F(6,42) = 1.877, P = 0.107). D. AChE‐mRNA also did not show significant changes among Reisberg groups (ANOVA, F(2,22) = 2.176, P = 0.137). The normalized data (log2‐transformation) are represented as mean (± standard deviation). (*) compared with (A) Braak II and (B) NCD, (#) compared with Braak I. *P < 0.05, **P < 0.01, # P < 0.05.

Article Snippet: EGR1‐pcDNA3.1 or EGR1‐shRNA, as well as their corresponding controls were transfected into SY5Y cells, by Viromer Red kit (Weinbergweg, Halle, Germany).

Techniques: Expressing, Transformation Assay, Standard Deviation

Journal: Brain Pathology

Article Title: Early growth response‐1 regulates acetylcholinesterase and its relation with the course of Alzheimer’s disease

doi: 10.1111/bpa.12688

Figure Lengend Snippet: Early growth response 1 (EGR1) initiates acetylcholinesterase (AChE) transcription by binding to the potential EGR1‐binding sites in the gene promoter of AChE. A. Schematic illustration of the deletion constructs of AChE gene promoter. B. Luciferase activity of the deleted constructs in 293T cells. +108/+707 pGL3 and −193/+707 pGL3 were significantly increased compared with vector pGL3, −193/+707 pGL3 was significantly higher than +108/+707 pGL3; −60/+107 pGL3 and −98/+107 pGL3 were both significantly decreased compared with −193/+707 pGL3, while −98/+107 pGL3 was significantly higher than −60/+107 pGL3; mutation 2 and 3 were both significantly decreased compared with −193/+707 pGL3, while mutation 1 did not change. C. Electrophoretic mobility shift assay (EMSA) results of EGR1 overexpressing SY5Y cells. Incubation of nuclear extracts with the 3 oligonucleotides (probe 1–3) revealed bands (lanes 2, 6 and 10), which were abolished by an excess of appropriately related unlabeled oligonucleotide (lanes 3, 7 and 11); the specific bands that represented EGR1‐oligonucleotide complexes super‐shifted with the addition of EGR1 antibody (lane 4, 8 and 12). Student’s t‐test was applied. Luciferase assay was repeated twice in triplicates with a similar outcome and data are represented as mean (± standard deviation) of one of the experiments. Four to six pilot experiments had been performed before the final experiments. EMSA was performed three times with a similar outcome, a representative result was shown in C. (#) compared with pGL3, ($) compared with −108/+707 pGL3, (&) compared with −60/+107 pGL3 and (*) compared with −193/+107 pGL3. ## P < 0.01, ### P < 0.001, $$ P < 0.01, && P < 0.01, *P < 0.05, **P < 0.01.

Article Snippet: EGR1‐pcDNA3.1 or EGR1‐shRNA, as well as their corresponding controls were transfected into SY5Y cells, by Viromer Red kit (Weinbergweg, Halle, Germany).

Techniques: Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Electrophoretic Mobility Shift Assay, Incubation, Standard Deviation

Journal: Brain Pathology

Article Title: Early growth response‐1 regulates acetylcholinesterase and its relation with the course of Alzheimer’s disease

doi: 10.1111/bpa.12688

Figure Lengend Snippet: Expression of mRNA of early growth response 1 (EGR1) and acetylcholinesterase (AChE) in the frontal cortex of triple‐transgenic AD (3xTg‐AD) and wild type (WT) mice. A. EGR1‐mRNA and (C) AChE‐mRNA both significantly decreased in the entire group of 3xTg‐AD mice compared with WT mice (Student’s t‐test, P = 0.031 and P < 0.001). In addition, B. EGR1‐mRNA significantly decreased in the 7–8‐months group (Student’s t‐test, P = 0.025), while (D) AChE‐mRNA significantly decreased in the 7–8‐months group (Student’s t‐test, P = 0.0103) and the 11–12‐months group (Student’s t‐test, P = 0.041) compared with their respective month‐of‐age WT group. The WT mice showed stable (B) EGR1‐mRNA and (D) AChE‐mRNA expression among the different months of ages (ANOVA, F(2,51) = 0.0824, P = 0.921 and F(2,50) = 0.125, P = 0.883). The 3xTg‐AD mice, on the other hand, showed significant changes in (B) EGR1‐mRNA levels among ages (ANOVA, F(2,52) = 4.355, P = 0.018), especially in the 7–8‐months group compared with the 3–4‐months group (Bonferroni test, P = 0.019), while (D) AChE‐mRNA expression was stable among different months of age (ANOVA, F(2,52) = 0.435, P = 0.65). The normalized data (square root transformation) are represented as mean (± standard deviation). (*) compared with corresponding WT, (#) compared with the 3–4‐months group. *P < 0.05, ***P < 0.001, # P < 0.05.

Article Snippet: EGR1‐pcDNA3.1 or EGR1‐shRNA, as well as their corresponding controls were transfected into SY5Y cells, by Viromer Red kit (Weinbergweg, Halle, Germany).

Techniques: Expressing, Transgenic Assay, Transformation Assay, Standard Deviation

Journal: Brain Pathology

Article Title: Early growth response‐1 regulates acetylcholinesterase and its relation with the course of Alzheimer’s disease

doi: 10.1111/bpa.12688

Figure Lengend Snippet: Early growth response 1 (EGR1) upregulates acetylcholinesterase (AChE) expression in the SY5Y cells. AChE expression was increased with the up‐regulation of EGR1 and decreased with down‐regulation of EGR1 in both (A) mRNA and (B–C) protein levels. Transfections of EGR1‐pcDNA3.1 or EGR1‐shRNA plasmids were repeated three times. Student’s t‐test was applied and data are represented as mean (± standard deviation) of the three times of transfection experiments, each of which was performed in triplicates. A representative result of the gel‐running results is presented in B. (*) compared with corresponding control. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: EGR1‐pcDNA3.1 or EGR1‐shRNA, as well as their corresponding controls were transfected into SY5Y cells, by Viromer Red kit (Weinbergweg, Halle, Germany).

Techniques: Expressing, Transfection, shRNA, Standard Deviation