Journal: Biology Open

Article Title: The snRNA-processing complex, Integrator, is required for ciliogenesis and dynein recruitment to the nuclear envelope via distinct mechanisms

doi: 10.1242/bio.20136981

Figure Lengend Snippet: RPE cells were transfected with siRNA, serum-starved, fixed, and stained for acetylated tubulin, γ-tubulin, and DNA. (A–J) Representative images show decreased PC formation after knockdown of most INT subunits tested. Scale bars, 10 (A,B) or 5 (C–J) µm. (K) Quantification of PC formation (normalized to NT-siRNA) in INT-depleted cells. Gray, P <0.0001; black, not significant (both relative to CETN2-siRNA, red). (L) Comparison of INT subunit requirements in snRNA processing , dynein localization, and ciliogenesis (presented herein). (+), required; (−), not required; (N.D.), not determined.

Article Snippet: Primary antibodies were used as follows: acetylated tubulin (6-11B-1, 1:500, Sigma-Aldrich), γ-Tubulin (ab16504, 1:500, Abcam, Cambridge, MA), CEP164 (NBP-77006, 1:100, Novus Biologicals, Littleton, CO), CEP89 (HPA040056, 1:100, Sigma), FBF-1 (HPA023677; 1:100, Sigma), CETN2 (N-17-R, 1:200, Santa Cruz Biotechnology, Dallas, TX), PCTN (ab4448, 1:2000, Abcam), Ninein (ab4447, 1:500, Abcam), dynein intermediate chain (74.1, 1:500, Abcam), and PH3 (Mitosis Marker, 1:1000, Millipore, Billerica, MA).

Techniques: Transfection, Staining, Knockdown, Comparison

Journal: Cell Proliferation

Article Title: LncRNA H19 ‐Encoded Micropeptide altH19 Promotes DNA Replication and Mitosis in Myeloma Cells by Enhancing the Phosphorylation of CDK2 at Threonine 160

doi: 10.1111/cpr.70089

Figure Lengend Snippet: altH19 induces mitosis in myeloma cells. (A) Western blot analysis of Aurora B, Centrin 2, phospho‐H3 and altH19 expression in altH19‐overexpressing cells. (B) Western blot analysis of Aurora B, Centrin 2 phospho‐H3 and altH19 expression in altH19‐knockout cells. (C, D) Immunofluorescence staining was conducted using α‐tubulin, phalloidin and DAPI, followed by confocal microscopy analysis. The histogram indicates the number of dividing cells. (E, F) Confocal microscopy analysis of multipolar cell division, with a histogram showing the ratio of multipolar dividing cells. Data were presented as mean ± SD from three independent experiments. ** p < 0.01; *** p < 0.001.

Article Snippet: The following antibodies were used in this study: GAPDH (60004‐1‐Ig), CDK2 (10122‐1‐AP), RB (10048‐2‐Ig), Centrin 2 (15877‐1‐AP), Aurora B (39261), Cyclin B1 (55004‐1‐AP) and CDK1 (19532‐1‐AP) were obtained from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Knock-Out, Immunofluorescence, Staining, Confocal 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: 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: This was followed by blocking in 4% BSA in PBS and overnight incubation with primary antibodies of ARL13B (1:500), IFT88 (1:100), CEP164 (1:100), CENTRIN2 (1:200), NINEIN (Novus biologicals, Cat#NBP2-13657) (1:100), and HsSAS (Santa Cruz Biotechnology, Cat#SC-81431) (1:100).

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: 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: This was followed by blocking in 4% BSA in PBS and overnight incubation with primary antibodies of ARL13B (1:500), IFT88 (1:100), CEP164 (1:100), CENTRIN2 (1:200), NINEIN (Novus biologicals, Cat#NBP2-13657) (1:100), and HsSAS (Santa Cruz Biotechnology, Cat#SC-81431) (1:100).

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