Fig. 3: Depletion of MIF results in aberrant ciliogenesis.

The levels of MIF in RCTE cells transfection with MIF and control siRNA were examined with qRT-PCR (a) and Western blot (b) analysis. c Representative images of RCTE cells transfected with MIF and control siRNAs for 48 h and serum starved for 24 h, and then stained with MIF (green) and Ac- αTub (red) and γ-Tub (red) antibodies. We quantified the percentage of cells without MIF at the centrioles in over 100 cells in indicated group, about 68.5% of cells were missing MIF at centrioles in MIF siRNA treated cells, whereas about 10% of control siRNA-treated cells had MIF at centrioles. Scale bars, 10 μm. d Knockdown of MIF in RCTE cells (c) increased the percentage of ciliated cells and cilia length. We quantified the percentage of ciliated cells and cilia length (>6 μm) in cells with missing MIF at centrioles. For each group, over 100 cells were counted. e Western blot analysis of Mif in kidneys from wild-type and Mif knockout mice (n = 5). f Representative images of kidney sections from 21 days old Mif ^+/+ and Mif ^–/– mice (n = 5 mice for each group) stained with Ac-αTub (red) and DBA (dolichos biflorus agglutinin), a collecting duct marker and LTL (lotus tetragonolobus lectin), a proximal tubule marker, and as well as DAPI (blue). White dashed lines represent the border of each renal tubule. Scale bars, 10 μm. g Knockout of Mif in kidneys (f) increased cilia length (top) and the percentage of ciliated cells (bottom) in the lumens of renal tubules compared to the controls. h Representative images of primary renal tubule cells isolated from 21 days old Mif ^+/+ and Mif ^–/– kidneys (n = 5 mice for each group) and stained with Ac- αTub (red) and DAPI (blue), about 50 proximal tubule or distal tubule/collecting duct cells were measured. Scale bars, 5 μm. i Knockout of Mif in primary renal tubular cells (h) increased cilia length (left) and the percentage of ciliated cells (right) compared to the controls, for (g) and (i), data are represented as the mean value ± s.d. p < 0.05 as determined by unpaired two-tailed Student’s t test.