Fig. 4.

Characterization of stable clone 2.43 generated with shRNA-mediated silencing of Katnal2. a Efficiency of Katnal2-specific silencing in stable clone 2.43, assessed by RT-qPCR, using primers specific for all Katnal2s (lane 1) or Katnal2-L1 + L2 (“Large”; lane 2), depicts, respectively, a normalized average knockdown in silenced cells, expressed as a percentage of the normalized average control values (parental NIH 3T3 line). Error bars correspond to SD of three independent experiments. L19 and Pum1 mRNA levels were used for sample normalization. b1, b2 Representative WB to confirm silencing of KATNAL2 isoform proteins (b1). Concurrent detection of calnexin in the same samples was used as loading control for normalization. The quantification of three independent experiments shown in (b2), depicting the average normalized value of KATNAL2 protein levels as a percentage of the control value, reveals reduction of KATNAL2, Nubp1 and Nubp2 protein levels, but not of actin. c Growth curves of clone 2.43 (blue) and parental control (gray) reveal a severely reduced growth rate in Katnal2-silenced cells. Shown are the average values ±SD of three replicate cultures, each seeded with 6680 cells, grown in parallel for 96 h and sampled every 24 h. d1, d2 Analysis of total cellular size (d1) and size of nucleus (d2) in clone 2.43 (blue) and parental control line (gray), revealing large increases of both due to Katnal2 silencing. The total cell surface area and the nuclear area were measured and plotted out as a size distribution in groups ranging from 0 to 70,000 μm² (d1) or 200 to 3,000 μm² (d2). e1, e2 Visualization of the remarkable difference in cell size between control (e1) and Katnal2-silenced clone 2.43 (e2) in two random fields, photographed at the same magnification. Labeling for α-tub in red and for DNA in blue. Scale bars 20 μm. f Cell cycle analysis of clone 2.43 and parental control line by flow cytometry, illustrating reduction of S phase and increase of G2/M phase cells (3 independent experiments, n = 75–200 thousand events each from control and clone 2.43 silenced cells). This indicated that Katnal2 silencing causes stalling in G2/M phase, being consistent with an altered cell cycle progression pattern. g Quantification of the Katnal2 silencing effect on centriole numbers in a bar chart depicting the overall distribution of the number of centrioles per cell in the populations of silenced clone 2.43 (blue) or control parental line (gray). While most cells possess 2 centrioles, in the silenced cells supernumerary centrioles can reach very high numbers (>12 per cell). The overall distribution of centriole numbers per cell is different (p < 0.0001). Shown are the means (±SD values) from three independent experiments (see also Table S6). Sample size corresponded to “dataset 1” (“Materials and methods”)