Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. B ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. C ; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). D ; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. E ; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. F ; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Western Blot, Molecular Weight, Expressing, In-Cell ELISA, Transfection, Fluorescence, Labeling, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A , B ; fMLP activates ERK or phosphorylation of ERK ( A ) and Akt or phosphorylation of Akt ( B ) in a dose- and time-dependent manner in monocytes from normotensive control subjects. 10 nmol/L open bars, 100 nmol/L filled bars. Data are mean ± SEM, n = 3. *p<0.05 compared to lower concentration conditions. C , D ; Increased fMLP-induced phosphorylation of ERK ( C ) and Akt ( D ) in monocytes from patients with essential hypertension. The proteins were measured using immunoblotting with specific antibodies. Data are mean ± SEM from three independent experiments. *p<0.05 compared to normotensive control subjects. E ; fMLP activates monocytes by an ERK-dependent and Akt-dependent pathway. Akt, ERK, or pERK and pAkt were measured using immunoblotting with specific antibodies. In the presence of 2-APB or after administration of specific siRNA against TRPC3, the fMLP-induced ERK, pERK; Akt and pAkt were significantly reduced when compared with control conditions. Data are mean ± SEM from six independent experiments. *p<0.05; **p<0.01 compared to control.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Concentration Assay, Western Blot

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Detecting Purinosome Metabolon Formation with Fluorescence Microscopy

doi: 10.1007/978-1-4939-7759-8_17

Figure Lengend Snippet: Colocalization of endogenous FGAMS and GART for visualization of purinosomes by immunofluorescence. Purine-depleted HeLa cells were fixed and permeabilized prior to being probed for with FGAMS rabbit polyclonal antibody and GART mouse monoclonal antibody. Fluorescently labeled secondary antibodies CF488A-conjugated donkey anti-rabbit and CF568-conjugated donkey anti-mouse were used to visualize the expression and localization of FGAMS and GART, respectively. A representative image of an individual cell was captured using a 100× oil objective on an Olympus Fluoview 1000 confocal laser scanning microscope. Sequential imaging of CF488A and CF568 showed colocalization of (a) FGAMS with (b) GART as represented by the yellow puncta present in (c) the merged image. Scale bar: 10 μm

Article Snippet: Primary antibody solution: 1:500 dilution of PFAS rabbit polyclonal antibody (Bethyl Laboratories) and 1:1000 dilution of GART mouse monoclonal antibody (Novus Biologicals) prepared in blocking buffer.

Techniques: Immunofluorescence, Labeling, Expressing, Laser-Scanning Microscopy, Imaging

Journal: Heliyon

Article Title: Di (2-ethylhexyl) phthalate effects on the growth, development, and reproduction of Moina macrocopa (Crustacea: Cladocera)

doi: 10.1016/j.heliyon.2024.e28377

Figure Lengend Snippet: Cellular pathology of ovarian tissue in maternal M. macrocopa (P0). The sections were obtained from a normal adult ovarian cortex showing normal round or oval yolk bodies. (A) : A photomicrograph (40x) of a normal ovarian cortex. Ultrastructures of ovarian tissue were studied under the transmission electron microscope (TEM) after M. macrocopa was exposed for 5 days continuously to a blank control (B) , a solvent control (C) , and 1000 μg/L DEHP (D) . Yolk bodies with irregular shapes and unclear membranes were found in the 1000 μg/L DEHP treatment (E) . Scale bar: 500 nm.

Article Snippet: The average size of the YB in ovarian cortex tissue of P0 M. macrocopa exposed to 1000 μg/L DEHP was determined by analyzing TEM images (Hitachi HT-7700) with ToupView 3.7 software.

Techniques: Transmission Assay, Microscopy, Solvent