Fig. 2: OFD1 is dynamically regulated upon actin filament reorganization.

a hTERT-RPE1 cells were co-stained with antibodies against OFD1 (red) and PCM1 (green). Cells were subjected to DMSO, serum starvation, 120 μM CK-689, 120 μM CK-666, 100 nM Cyto D, 20 μM SMIFH2 treatment for 96 h, si-Control (control siRNA), si-OFD1 (siRNA targeted OFD1) treatment for 72 h or 15 ng/mL nocodazole treatment for 48 h. Scale bars: 10 μm, 3 μm (zoom in). b Live cell images of the fusion and fission events of centrosomal OFD1 in Tet-inducible EGFP-OFD1-expressing RPE1 cells treated with 0.1 ng/mL Doxycycline and DMSO or 120 μM CK-666 for 72 h. c FRAP analysis of EGFP-OFD1 condensates upon DMSO or CK-666 treatment (120 μM, 96 h) in Tet-inducible EGFP-OFD1-expressing RPE1 cells treated with 0.1 ng/mL Doxycycline, and the fluorescence recovery was recorded every 1 s for ∼10 min. The dashed green squares indicate the bleached sites. d Plots of fluorescence intensity before and after photobleaching. Data shown represent mean ± SD, n = 3, and shaded areas show the standard deviation of the means. 50 cells examined over three independent experiments. e Immunoblot analysis of the protein levels of OFD1 and β-tubulin in hTERT-RPE1 cells that were subjected to DMSO, 120 μM CK-689, 120 μM CK-666 treatment for 96 h, or serum starvation (SS) for 72 h. The numbers under the gel lanes represent the ratio of OFD1 band intensity to β-tubulin band intensity, which were normalized relative to the line 1 sample. f Immunoblot analysis of OFD1 protein expression in exponentially growing RPE1 cells (control), 72 h serum-starved (SS) cells, and contact-inhibited cells induced by high density (HD) confluence. The numbers under the gel lanes represent the ratio of OFD1 band intensity to β-tubulin band intensity, which were normalized relative to the line 1 sample. g Immunoblot analysis of OFD1 protein levels in RPE1 cells cultured within indicated conditions. Cells transfected with control siRNA or OFD1 siRNA were starved by serum deprivation for 72 h (Lane 1 and 2), or were plated at high density for 96 h (Lane 5 and 6). Starved cells were stimulated by serum addition for 30 h (Lane 3 and 4). Cells at high density for 96 h were passaged at a regular density for 30 h (Lane 7 and 8). The numbers under the gel lanes represent the ratio of OFD1 band intensity to β-tubulin band intensity, which were normalized relative to the line 1 sample. h Proliferation curves of serum-starved cells for 72 h, followed by 30 h of serum stimulation. Data shown represented as mean values ± SD, error bar was defined as SD. i Proliferation curves of cells cultured at high density for 96 h, and then passaged at regular density for 30 h. Data shown represented as mean values ± SD, error bar was defined as SD. j Representative images of immuno-staining for ARL13B (green) and Ki67 (red). RPE1 cells were transfected with siRNAs as indicated. In the Left Panel, cells were starved by serum deprivation for 72 h, followed by 30 h of 10% serum stimulation. In the Right Panel, cells were seeded at a high density to induce contact inhibition for 96 h, followed by passage at regular density for 30 h. k Quantification of ARL13B-positive cells described in (j). 300 cells examined over three independent experiments, P  = 0.000012, P  = 0.00023, two-tailed unpaired student’s t-test. Data shown represented as mean values ± SD, error bar was defined as SD. l Quantification of Ki67-positive cells described in (j). 300 cells examined over three independent experiments, P  = 0.000041; P  = 0.000092, two-tailed unpaired student’s t-test. All data shown represented as mean values ± SD, error bar was defined as SD.