All animal procedures were performed according to the Chapman University Animal Care and Use Committee Guidelines. 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 injected intraperitoneally with 5 μg/μL tamoxifen every day for five consecutive days. Frontal cortices were collected. Cells were dissociated with 1× trypsin/Ethylenediaminetetraacetic acid (EDTA) solution through a 1-cm³ 25 G⅝ needle and plated in endothelia-selecting media Dulbecco's modified eagle medium (DMEM) containing 2% Fetal bovine serum (FBS), 0.75 μg/L interferon-γ, 1.0 g/L insulin, 0.67 mg/L sodium selenite, 0.55 g/L transferrin, 0.2 g/L ethanolamine, 36 ng/mL hydrocortisone, 0.10 μmol/L 2,3,5-triido-l-thyronine, 100 U penicillin-G (base) combined with 0.30 mg/mL additional glutamine, 100 μg streptomycin sulfate, and 0.1 mmol/L citrate to maintain penicillin potency. All cell-culture supplements were obtained from Sigma-Aldrich(St Louis, Mo). After 2–3 weeks of growth, endothelial cells were further sorted by incubating the cells with 10 mg/mL of the endothelial marker intracellular adhesion molecule-2 (ICAM-2; Santa Cruz Biotechnology, Santa Cruz, Calif). Fluorescein isothiocyanate (FITC)-conjugated ICAM-2 antibody was applied for 1 h at room temperature at a dilution of 1:100 in Phosphate buffered saline (PBS) containing 1% fetal bovine serum to prevent any non-specific binding of the antibody. After the cells were washed three times to avoid non-specific binding, they were analyzed with FACScan (Becton Dickinson, Franklin Lakes, NJ) at a wavelength of 525 nm (FITC, FL-1) (Supplementary Figure S1). Following cell sorting and as needed to avoid bacteria contamination, 100 U penicillin-G (base) was added to the DMEM with 0.30 mg/mL additional glutamine, 100 μg streptomycin sulfate, and 0.1 mmol/L citrate to maintain penicillin potency. Cells were grown for an additional 4–7 days. For staining cilia structures, acetylated-α-tubulin (1:10,000, Sigma) and the secondary antibodies were also diluted in 10% FBS to decrease the background fluorescence; FITC fluorescence secondary antibody (1:1000; Pierce, Inc.) was used. Cells were then washed three times for 5 min each with cacodylate buffer and mounted with 4',6-diamidino-2-phenylindole (DAPI) (Vector laboratories). Confocal microscopic images were obtained using an inverted Nikon Eclipse Ti confocal microscope.

Human brain microvascular ECs (HBMECs) were purchased from Cell Systems Corporation (Cat # ACBRI 376) and maintained at 37°C in 5% CO₂ in endothelial cell complete media (Promocell, Cat #C22010). All experiments were performed with cells grown between passage 4–6. These cells were extensively characterized for EC markers in our previous publication (Thirugnanam et al., 2022). Cells were grown and maintained at 50% confluence state. IF experiments were performed at 60%–70% confluence to distinguish and avoid the overlapping of cells and cilia.

Proteins were isolated from HBMECs using Radioimmunoprecipitation Assay buffer (RIPA) buffer (Sigma Cat#R2078) with a complete mini EDTA-free protease inhibitor cocktail (Roche Cat#11836170001) and PhosSTOP phosphatase inhibitor (Roche Cat#4906845001). After isolation, the total protein was quantified. Cell lysates were used for probing the following proteins: ARL13B (Proteintech, Cat#17711-I-AP), IFT88 (Thermofisher, Cat#PA5-18467), Cyclin D1 (Thermofisher, Cat#MA5-16356), Cyclin A (Biolegend, Cat#644004), Cyclin B1 (Biolegend, Cat#647906), Cyclin E1 (Cell signaling, Cat#20808), CDK1 (Biolegend, Cat#626901), CEP164 (Proteintech, Cat#22227-1-AP), CENTRIN2 (Biolegend, Cat#698602), and β-actin (Cell signaling Cat#4970). Anti-rabbit Horseradish peroxidase (HRP) (Cell signaling, Cat#7074), anti-goat HRP (Jackson Immunoresearch, Cat#205-052-176), and anti-mouse HRP (Cell signaling, Cat#7076) were secondary antibodies used for chemiluminescence detection. Quantification was done using ImageJ software and plotted against the housekeeping control protein (β-actin) using GraphPad software as described previously (Thirugnanam et al., 2022).

HBMECs were grown to confluence in six-well plates on coverslips. All the IF experiments were performed by seeding cells on the same day with similar seeding density and synchronized. Cells were washed with 1X PBS (Gibco, Cat#10010023) thrice and fixed with 4% PFA(Electron Microscopy Sciences, Cat#15710) for 15 mins. Fixed cells were washed again with 1X PBS before permeabilization with 0.1% Triton X-100 (BioRad, Cat#1610407). 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). Cells were again washed with 1X PBS and incubated with Alexa fluor-488 anti-rabbit (Invitrogen, Cat#A21206) (1:500), Alexa fluor-488 anti-mouse (Invitrogen, Cat#A32766), Alexa fluor-568 anti-rabbit (Invitrogen, Cat#A10042), and Alexa fluor-568 anti-rat (Invitrogen, Cat#A11077) for 90 min at RT and washed before mounting with DAPI (LifeSpan Biosciences, Cat#LS-J1033-10) and imaged using a Zeiss confocal microscope at a magnification of ×63. We present images of ECs containing cilia in similar confluence areas on the plate. Primary cilia quantification for cilia size or length was measured using ACDC v0.93 cilia-specific software as described in our previous publication (Thirugnanam et al., 2022). For increasing cilia length in HBMECs, we incubated the cells with PDGF-BB ligand (10 ng/mL) for 60 min before staining. Briefly, cells were seeded in a six-well plate with a coverslip on it. After 24 h, the cells were changed to serum-free medium for contact inhibition and then changed to complete medium and treated with MNK2 inhibitor IV (Sigma, Cat#531206001) (2 μM for 24 h) to arrest the cells at the G0/G1 phase of the cell cycle. PDGF-BB ligand stimulation was performed, and then cells were subjected to staining with ARL13B cilia and CENTRIN 2 centrioles antibodies and imaged at ×100 oil immersion objective using a Keyence BZ-×700 fluorescent microscope (Japan). Texas Red filter cube (OP-87765, Keyence), a GFP filter cube (OP-87763, Keyence), and a 4′,6-diamidino-2-phenylindole (DAPI) filter cube (OP-87762, Keyence) were used to image the DAPI-stained samples.

HBMECs were seeded in six-well culture dishes with (for IF) and without coverslip (for Western blot) approximately 24 h prior to transfection. Control and CEP164 siRNA (Horizon inspired cell solutions, Cat#D-001320–10–05, and Cat#J-020351-17-0005) were transfected using Lipofectamine 2000 reagent (Gibco, Cat#11668019) and incubated for 48 h. Cells were washed twice with PBS, replaced with a complete growth medium, and incubated at 37°C until further experimentation such as primary cilia immunostaining or Western blot.

Cells were harvested from the six-well plates using TrypLE Express (Gibco, Cat#12604021). Single-cell suspensions were washed twice with FACS buffer (1× PBS with 5% FBS and 0.1% NaN₃) at 300 g for 5 min. Then, cells were fixed and permeabilized using TF fix/perm buffer of a transcription factor buffer set (BD biosciences, Cat#562574) per the manufacturer’s protocol. Cells were stained for the following proteins: ARL13B, cyclin B1, cyclin A, cdc2 (CDK1), and ki67 (eBioscience, cat#25-5698-80). Suitable secondary reagents were used. Primary antibodies were diluted at 1:50 and secondary antibodies at 1:500. Following each staining, cells were washed thrice in 500 μL buffer using BD biosciences transcription factor (BD TF) perm wash buffer (component from Cat#562574). Primary antibodies were incubated for 45 min and secondaries for 30 min at 4°C with appropriate secondary controls. After staining, cells were resuspended in FACS buffer. For cell cycle analysis, per the manufacturer’s protocol, FxCycle Violet stain (Invitrogen, Cat#F10347) was added to cell suspensions just before running the samples in the flow cytometer (BD LSRFortessa). Sample acquisition was done using FACSDiva software (BD) with subsequent analysis on FlowJo software.

Data were presented as the mean and standard error of the mean (SEM). A t-test, Welch’s t-test, or analysis of variance (ANOVA) was performed to compare the outcome measures between different groups or phases of the cell cycle. Cilia length from the mother and daughter centrioles of HBMECs was compared using the paired t-test. Count data were compared using a generalized linear model with a negative binomial distribution. For some analyses, data were log-transformed to improve fit. p < 0.05 was considered significant. Dunnett’s test was used to adjust for multiple comparisons. Statistical analysis was performed using SAS V9.4 (SAS Institute Inc., Cary, NC) and GraphPad Prism software.

Previously, we reported that HBMECs and human embryonic stem cell-derived brain microvascular ECs showed a two-cilia phenotype (Thirugnanam et al., 2022) in vitro. To investigate whether the two-cilia phenotype is observed in other conditions, we chose to investigate cilia in mouse primary brain ECs from polycystic kidney disease (Pkd2) knockout mouse, a ciliopathic condition associated with an abnormal primary ciliary function that causes phenotype of polyploidy and a defective cell cycle (AbouAlaiwi et al., 2011). We isolated brain ECs from the frontal cortices of Tie2Cre·Pkd2 ^WT/WT (wild type; WT) and Tie2Cre·Pkd2 ^flox/flox (Pkd2) mice. Isolated brain ECs were stained for ciliary marker acetylated-α-tubulin and nuclear marker DAPI by IF (Figure 1A). IF staining for ciliary markers suggested brain ECs isolated from Pkd2 knockout mice possess more than one cilium. Approximately 1% of Pkd2 null ECs possess two cilia (Figure 1B). To determine the origin of second cilium in brain ECs, we used HBMECs, and immunostained for co-localization of the primary cilia and centrioles. HBMECs were characterized extensively in our previous work (Thirugnanam et al., 2022). The centriole markers were chosen with the rationale to distinguish mother and daughter centrioles (Figure 1F). CEP164 and NINEIN are specific markers for distal and subdistal appendage regions of the mother centriole, respectively (Hall and Hehnly, 2021). Immunostaining mother centriole markers with ciliary axonemal markers ARL13B (Figure 1C) or IFT88 (Supplementary Figure S2) suggests only one cilium is co-localized with mother centriole markers CEP164 and NINEIN. We quantified the size of the cilia using an automated cilia-specific software ACDC v0.93 as described in our previous publication (Thirugnanam et al., 2022). The cilium arising from the mother centriole is bigger in size than the cilium from the daughter centriole (Figure 1E). Because the cilia appeared as dots without clear definition of the axoneme and basal body structure, we rationalized that increasing cilia length will offer better resolution of the two-cilia structure. Based on a past publication (Thirugnanam et al., 2022), we treated HBMECs with Platelet-Derived Growth Factor-BB (PDGF-BB) ligand for 60 min to increase the length of the cilia and stained for CENTRIN2, DAPI, and ARL13B markers. A single brain EC with two cilia that has the classical basal body (Figure 1G, red stain) and axoneme (Figure 1G, green stain) was visible. Additionally, cells with single cilium were also observed in the field. These results collectively suggest that the second cilium arises from the daughter centriole. Collectively, these results suggest brain microvascular ECs possess two cilia, and the second cilium arises from the daughter centriole. We also used a second set of centriole markers: CENTRIN2 and HsSAS markers, which label distal lumen and procentriole respectively of both mother and daughter centrioles (Hall and Hehnly, 2021). Indeed, ARL13B co-stained with ciliary markers CENTRIN2 and HsSAS (Figure 1D). CENTRIN2 was also confirmed with a second ciliary Intraflagellar Transport 88 (IFT88) (Supplementary Figure S2).

During the cell cycle, ciliogenesis occurs at the G0/G1 phase. To assess cilia protein expression and regulation, we investigated the expression pattern of ciliary proteins ARL13B and IFT88 in different cell cycle phases. Briefly, HBMECs were arrested at different phases of the cell cycle using specific cell cycle inhibitory molecules: mitogen-activated protein kinase-interacting kinase 2 (MNK2) inhibitor (Yen et al., 2022) at 2 μM for G0/G1, CI-994 (an inhibitor of histone deacetylase 1) at 10 μM for the S phase, Murine Double Minute-2 (MDM2) inhibitor (which blocks p53 transcriptional activation) at 100 nM for the G2 phase, and vinblastine sulfate (a microtubule inhibitor) at 2 nM for the M phase. To confirm that the cells were arrested at different phases of the cell cycle, the expression profile of cyclin and CDK proteins were assessed using Western blot protein expression (Figures 2A, B). Cells arrested at different phases of the cell cycle revealed that ciliary proteins ARL13B and IFT88 expression were high only in the G0/G1 and S phases of the cell cycle (Figure 2C), suggesting that brain EC ciliogenesis occurs at the G0/G1 phase and extends into the S phase. The Western blot data were supported by flow cytometry analysis of the cell cycle (Supplementary Figure S3) and ciliary proteins. The net fluorescence intensity of ARL13B was highest in the G0/G1 phase (Supplementary Figure S3). Interestingly, in flow cytometry, we observed a fraction of cells distinctly high in ARL13B (ARL13B^Hi) expression in the G0 and Sub-G0 cell cycle phases (Figure 2D). The number of ARL13B^Hi cells was even higher following G0/G1 arrest. At G0, ∼30% control cells displayed the ARL13B^Hi phenotype, while it was >40% following G0/G1 arrest (Figure 2D). These data collectively suggest that the ciliary proteins responsible for cilia growth and ciliary membrane trafficking (ARL13B) are synthesized during the G0/G1 phase of cell cycle.

To assess when in the cell cycle brain microvascular ECs exhibit the two-cilia phenotype, HBMECs were arrested at specific stages of the cell cycle using the cell cycle inhibitors mentioned above and analyzed using immunofluorescence (IF) with cilia-specific ARL13B antibody (Figure 3). In total, 30.6% of cells arrested at the G0/G1 phase exhibited the two-cilia phenotype and about 56.5% of the cells exhibited one cilium. Further, approximately 21.6% of the cells in the S phase exhibited one cilium, and about 1.3% of cells showed the two-cilia phenotype. We did not observe any one- or two-cilia phenotype in the G2/M phase. Taken together, these data suggest that brain ECs exhibiting the two-cilia phenotype are restricted to the G0/G1 phase of the cell cycle.

To assess whether ciliogenesis occurs from the daughter centriole, we knocked down (KD) CEP164. The regulation of CEP164 is essential for the proper recruitment of mother centriole-mediated membrane-associated ciliary proteins and primary ciliogenesis (Kobayashi et al., 2020). IF analysis of efficacy confirmed CEP164 KD brain ECs showed minimal ciliogenesis from both the mother and daughter centriole (Figure 4A). Further, the protein expression of CENTRIN2, and ciliary proteins ARL13B were also depleted in the CEP164 KD cells (Figures 4B, C). Taken together, these results suggest the role of the mother centriole is important in determining primary as well as secondary cilium from the daughter centriole in brain ECs.