human brain microvascular ecs  (ATCC)

Journal: Frontiers in Molecular Biosciences

Article Title: Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole

doi: 10.3389/fmolb.2023.1250016

Figure Lengend Snippet: Brain ECs possess a second cilium from the daughter centriole. (A) . Brain endothelial cells from frontal cortex of one-week-old Tie2Cre·Pkd2 WT/WT (with Cre activation; control wild-type group) and Tie2Cre·Pkd2 flox/flox (with Cre activation; experimental Pkd2 group) mice were collected and immunostained for primary cilia with acetylated-α-tubulin and nuclei with DAPI using the immunofluorescence method. Scale bar = 20 μm. (B) . The table represents the number of brain endothelial cells with a two-cilia phenotype. A minimum of 50 cells were assessed in triplicates for two-cilia phenotype detection. Scale bar = 10 μm. (C) . Human primary brain microvascular endothelial cells (HBMECs) were immunostained for mother centriole distal and subdistal appendage markers, CEP164 and NINEIN, respectively (red), nuclei stained with DAPI, and primary cilia stained with ARL13B (green). White arrows indicate cilia from the mother centriole and arrowheads represent second cilia from the daughter centriole. (D) . HBMECs stained for Centrin2 and HsSAS, markers for distal lumen and procentriolar markers of the mother and daughter centriole (Red), nuclei stained with DAPI, and primary cilia stained with ARL13B (green). White arrows indicate cilia from the mother and daughter centriole and their respective cilia. Scale bar = 10 μm. (E) . Primary cilia size varies based on ciliogenesis from the mother and daughter centrioles of HBMECs. N = 25 double cilia cells were quantified for their respective size. (F) . Pictorial representation of the centriole markers used in the study. All the experiments were performed in triplicates. Results are presented as mean ± SEM. SEM, standard error to the mean. p < 0.05 was considered significant. Statistics were performed using paired t-tests. (G) . HBMECs were contact inhibited, arrested at the G0/G1 phase of the cell cycle for 24 h, treated with PDGF-BB ligand for 60 min, immunostained for ARL13B cilia (green) and CENTRIN 2 (red) centriole antibodies, and imaged at 100X using a Keyence BZ-X700 fluorescent microscope (Japan). Scale bar = 5 μm.

Article Snippet: Human brain microvascular ECs (HBMECs) were purchased from Cell Systems Corporation (Cat # ACBRI 376) and maintained at 37°C in 5% CO 2 in endothelial cell complete media (Promocell, Cat #C22010).

Techniques: Activation Assay, Control, Immunofluorescence, Staining, Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole

doi: 10.3389/fmolb.2023.1250016

Figure Lengend Snippet: Cell cycle and ciliary protein expression profile. (A) . HBMECs were arrested at their respective cell cycle stages and assessed for cell cycle and ciliary proteins using the Western blotting method using MNK2 inhibitor at 2 μM for the G0/G1 phase, CI-994 at 10 μM for the S phase, MDM2 inhibitor at 100 nM for the G2 phase, and vinblastine sulfate at 2 nM for the M phase and (B) . quantified using Image J software and plotted in GraphPad prism software. (C) . Color-coded peaks represent the high and low ciliary and cell cycle protein expression in their respective cell cycle stage. p < 0.05 was considered significant; n = 3 in each experimental group. Results are presented as mean ± SEM. SEM, standard error to the mean. (D) . Quantification of ARL13B Hi cells in various stages of the cell cycle. p < 0.05 was considered significant; n = 4 in each experimental group. Results are presented as mean ± SEM. SEM, standard error to the mean. ANOVA was used to examine the effects of various conditions on the outcomes.

Article Snippet: Human brain microvascular ECs (HBMECs) were purchased from Cell Systems Corporation (Cat # ACBRI 376) and maintained at 37°C in 5% CO 2 in endothelial cell complete media (Promocell, Cat #C22010).

Techniques: Expressing, Western Blot, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole

doi: 10.3389/fmolb.2023.1250016

Figure Lengend Snippet: Human primary brain microvascular endothelial cells ciliogenesis at different cell cycle stages. (A) . HBMECs were arrested at the respective cell cycle stages using specific inhibitors, MNK2 inhibitor at 2 μM for the G0/G1 phase, CI-994 at 10 μM for the S phase, MDM2 inhibitor at 100 nM for the G2 phase and vinblastine sulfate at 2 nM for the M phase, and imaged using confocal microscopy for immunofluorescence. DAPI, ARL13B and merged images, scale bar = 10 μm and zoomed image, scale bar = 10 μm for zoomed. (B) . Quantification was done as mentioned in the methods section for cilia length and number. G0/G1 group, n = 154 nuclei; S group, n = 144 nuclei; G2 group, n = 143 nuclei; and M group, n = 153 nuclei. Statistical p -value comparisons across groups are shown ( n = 6) for each quantification method. Results are presented as mean ± SEM. SEM, standard error to the mean. p < 0.05 was considered significant. Statistics were performed using paired t-tests.

Article Snippet: Human brain microvascular ECs (HBMECs) were purchased from Cell Systems Corporation (Cat # ACBRI 376) and maintained at 37°C in 5% CO 2 in endothelial cell complete media (Promocell, Cat #C22010).

Techniques: Confocal Microscopy, Immunofluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Brain microvascular endothelial cells possess a second cilium that arises from the daughter centriole

doi: 10.3389/fmolb.2023.1250016

Figure Lengend Snippet: CEP164 knockdown in HBMECs. (A) . HBMECs were knocked down for CEP164 siRNA and control siRNA and immunostained for CENTRIN2, a centriole antibody. (B) . CEP164 knockdown was performed in HBMECs and protein expression of CEP164 for knockdown efficiency and CENTRIN2 and ARL13B was performed. (C) . Quantification of the Western blot was performed using ImageJ software. n = 3 in each experimental group. Results are presented as mean ± SEM. SEM, standard error to the mean. Statistics were performed using paired t-tests.

Article Snippet: Human brain microvascular ECs (HBMECs) were purchased from Cell Systems Corporation (Cat # ACBRI 376) and maintained at 37°C in 5% CO 2 in endothelial cell complete media (Promocell, Cat #C22010).

Techniques: Knockdown, Control, Expressing, Western Blot, Software