Fig 2: Genotoxic stress induces ciliogenesis.ETO treatment (50 and 100 µM, 24 h) induced DNA damage in RPE1 cells. A Extracts of RPE1 cells treated with or without ETO were analyzed by immunoblotting with antibodies against ?-H2AX and tubulin (Tub.). B ?-H2AX was detected by immunostaining with a specific antibody (green) in scramble control (CTL) or ETO-treated RPE1 cells. DNA was stained with DAPI (blue). Scale bar, 10 µm. ETO induced primary cilia formation in RPE1 cells. Double staining of ETO-treated cells with antibodies against (C) acetylated tubulin (Ac-tub, red) and CEP164 (green); D Ac-tub (red) and Arl13b (green); or E Ac-tub (red) and IFT88 (green). DNA was stained with DAPI (blue). Scale bar, 5 µm. F Quantitative results of the frequency of ciliated cells after treatment with different concentrations of ETO for 24 h. G Quantitative results of the frequency of ciliated cells after treatment with 100 µM ETO for different time periods. The results are presented as the mean ± SD of three independent experiments; more than 100 cells were counted in each individual group. H Ac-tub levels were increased in ETO-treated RPE1 cells. Extracts of RPE1 cells treated with or without ETO were analyzed by immunoblotting with antibodies against Ac-tub and Tub. Serum starvation (Starv) and ETO treatment induce ciliogenesis in RPE1 (I), HTR8 (J), and TM3 (K). **P < 0.01 and ***P < 0.001.

Fig 3: Abrogation of primary cilia has distinct effects in proliferating and quiescent myoblasts. C2C12 myoblasts were transfected with siRNAs targeting IFT88 to block ciliogenesis, and were analyzed for effects of knockdown on proliferation and quiescence. Cells transfected with non-targeting siRNA were used as control. a qRT-PCR analysis was used to detect levels of IFT88 mRNA in control and siRNA treated samples 48 h after transfection. Knockdown myoblasts show reduced levels of IFT88 mRNA. Values represented are mean ± s.e.m., N = 3, **p-value = 0.0062. b Western blotting analysis demonstrates efficient knockdown of IFT88 protein levels 48 h after transfection. c Primary cilia were visualized by immunofluorescence labeling of Acetylated tubulin (Ac. tubulin) in IFT88 knockdown myoblasts cultured in quiescence-inducing conditions. Representative immunofluorescence images are shown. A single cilium is present per cell in control cultures, whereas knockdown cells show greatly reduced cilia (accompanied by increased tubulin staining in the cell body). d Quantification of the reduction of IFT88 protein level shown in (b). Values indicated are mean ± s.e.m., N = 3, ***p-value = 0.0004. e Knockdown of IFT88 caused reduction in frequency of ciliated cells. Values represent mean ± s.e.m., N = 4, ** p-value = 0.034.f Proliferation of cells was analyzed by quantification of Ki67+ cells, detected by immuno-staining. Targeting ciliogenesis had little effect in proliferating conditions (MB). Values represent mean ± s.e.m., N = 3, ns = not significant (p-value = 0.959). g When induced to enter quiescence (G0), knockdown cells displayed increased frequency of Ki67+ compared with control siRNA-transfected cells. Values represent mean ± s.e.m., N = 5, ** p-value = 0.0015. h Ablation of ciliogenesis suppresses expression of quiescence marker p27. C2C12 myoblasts stably expressing the p27 sensor (mVenus-p27K-) were transfected with siRNA targeting IFT88or control siRNA and then cultured in quiescence-inducing conditions. The frequency of p27 (mVenus) positive cells was measured by flow cytometry. Values represent mean ± s.e.m., N = 3, * p-value = 0.0234. i Western blotting revealed the reduction of the quiescence marker, p27 in IFT88KD cells under conditions of G0 arrest. The values are relative protein levels compared to Control, and represent mean of quantification from 2 experiments. j Flow cytometric analysis of IFT88 siRNA-transfected cells cultured in conditions that allow proliferation (MB) does not show a significant shift in cell cycle profile. When placed in conditions that induce quiescence (G0) in control cells, knockdown cells display an increased proportion of cells in G2/M. Representative profiles are shown where the X axis shows the DNA content estimated by florescence intensity of the DNA dye DRAQ5, and the Y axis represents cell numbers as a percentage of maximum (normalized to mode). k Quantification of experiment described in (j) showing shifts in proportion of cells in different cell cycle stages (G1, S, G2/M). Values represent mean ± s.e.m., N = 3, * p-value = 0.043. l Flow cytometric analysis of IFT20 and KIF3A siRNA-transfected cells cultured in conditions that induce quiescence (G0). Cells lacking cilia show an altered cell cycle profile with an increase in proportion of cells in G2/M. Representative profiles are shown where the X axis shows the DNA content estimated by florescence intensity of the DNA dye DRAQ5, and the Y axis represents cell numbers as a percentage of maximum (normalized to mode). m Ablation of ciliogenesis using other IFT targets suppresses expression of quiescence marker p27. C2C12 myoblasts stably expressing the p27 sensor (mVenus-p27K-) were transfected with siRNA targeting IFT20 or KIF3A, or control siRNA and then cultured in quiescence-inducing conditions. The frequency of p27 (mVenus) positive cells was measured by flow cytometry. Values represent mean ± s.e.m., N = 3, ** p-value = 0.0001, n.s (p-value = 0.38).

Fig 4: Generation of a murine Cfap161-null allele Cfap161?ex2,3. (A) Schematic drawing showing the structure of the targeted Cfap161 locus and mutated alleles (Cfap161loxP and Cfap161?ex2,3). (B) Western blots of testis and epididymis lysates with monoclonal pI and polyclonal pII antibody show absence of CFAP161 in the mutant (Cfap161?ex2,3/?ex2,3). a-IFT88 was used as loading control. The full-size Western blots are shown in Fig. S9C. (C) Indirect immunofluorescence of wild type (a, a', c, c') and Cfap161?ex2,3 mutant (b, b', d, d') testis sections showing absence of CFAP161 from flagella of mutant sperm. White arrowheads: flagella; asterisk: non-specific staining around seminiferous tubules. Scale bar: (C) = 50 µm.

Fig 5: Ciliogenesis in POMC neurons during the early postnatal period is critical for adulthood energy balance.a Cilia (AC3) staining in the developing hypothalamus of C57 mice (n = 4 for P0 and P28, n = 5 for E12.5, E15.5, P14, and P60, n = 6 for E18.5 and P7). The graphs depict the average length of about 100 ARH cilia and the ciliated cell percentage (the cilia numbers divided by the DAPI numbers) in each mouse. Scale bars: 20 µm. 3V third cerebroventricle. b IFT88 immunoblotting in the developing hypothalamus of C57 mice (n = 4). c AC3 and ß-endorphin double staining in the ARH of C57 mice showing POMC neuron ciliogenesis during development (n = 4 for E18.5, P7, and P60, n = 5 for P1 and P14). Scale bars: 20 µm. d Experimental scheme in mice with POMC-specific IFT88 depletion that was induced by tamoxifen injections during the postnatal period (P1–P14). e Representative cilia images showing ciliary dysgenesis in POMC neurons (n = 4). Arrowheads indicate cilia of POMC neurons. Scale bars: 20 µm. f Body weights in mice with postnatal ciliary dysgenesis in POMC neurons and their IFT88f/f litters on a chow diet (n = 5 for males, n = 6 for females). g Lean mass and fat mass measured at 15 weeks (n = 5 for males, n = 6 for females). h Average values of weekly food intake during 10–15 weeks (n = 5 for males, n = 6 for females). i Energy expenditure measured at 12 weeks (n = 4). Data are presented as the mean ± SEM values. Statistics were performed using one-sided one-way ANOVA (a–c), one-sided two-way ANOVA (f) followed by post hoc LSD test and two-sided Student’s t test (e, g–i). *p < 0.05, **p < 0.01, ***p < 0.001 vs. IFT88f/f controls or between the indicated groups.