Skip to main content
. 2018 Nov 26;7:e40325. doi: 10.7554/eLife.40325

Figure 1. RNAi-mediated depletion of Sf3A2 or Prp31 inhibits sister chromatid separation during mitosis.

(A) Western blots of S2 cell extracts and quantitation of relative band intensities showing that RNAi against Sf3A2 or Prp31 strongly reduces the levels of these proteins; tubulin (tub) and actin are loading controls (the full blot is shown in (Figure 1—figure supplement 1). (B) Mitotic figures observed in mock-treated control cells and in Sf3A2- and Prp31-depleted cells stained for DNA (blue), tubulin (green) and Cyclin B (red). meta, metaphase; ana, anaphase; telo, telophase; PML, prometaphase-like; PMLES prometaphase-like cells with elongated spindles. Note that PMLES exhibit a high level of Cyclin B. Scale bar, 5 μm. (C) Mitotic figures observed in control and RNAi cells stained for DNA (blue), tubulin (green) and BubR1 (red). BubR1 is enriched at the kinetochores in PMLES but not in control ana-telophases. Scale bar, 5 μm. (D) Frequencies of mitotic figures observed after RNAi against the indicated Sf3A2 and Prp31 sequences, or against both the Sf3A2 and Zw10 coding sequences; R are rescue constructs expressing either the Sf3A2 or the Prp31 coding sequence devoid of the UTRs. Lag, lagging chromosomes between the ana-telophase nuclei. M.I., mitotic index (i.e. percent of cells undergoing mitosis).

Figure 1—source data 1. Source data for Figure 1A and Figure 1—figure supplement 4.
elife-40325-fig1-data1.xlsx (40.9KB, xlsx)
DOI: 10.7554/eLife.40325.007

Figure 1.

Figure 1—figure supplement 1. Specificity of the anti-Sf3A2 and anti-Prp31 antibodies.

Figure 1—figure supplement 1.

(A, B) Western blots from mock-treated, Sf3A2 RNAi and Prp31 RNAi S2 cell extracts stained with affinity-purified anti-Sf3A2 and anti-Prp31 antibodies; the blots in (B) are the same as those of Figure 1A. Lamin, actin and tubulin are loading controls. (C) Western blots from untreated S2 cell and embryo extracts stained with affinity-purified anti-Sf3A2 and anti-Prp31 antibodies. Actin is a loading control.
Figure 1—figure supplement 2. Examples of monopolar spindles and PMLES observed in Sf3A2 and Prp31 RNAi cells.

Figure 1—figure supplement 2.

(A) Monopolar spindles showing two centrosomes at the pole; cells were stained for tubulin (green), the centrosomal marker Spd2 (Giansanti et al., 2008) (red), and DNA (DAPI, blue). (B) PMLES stained for tubulin, Cid/Cenp-A and DNA; note that the chromosomes comprise both sister chromatids. (C) PMLES stained for tubulin (green), DNA (blue), and the central spindle marker Fascetto (Feo; red), the Drosophila orthologue of PRC1, required for central spindle formation and cytokinesis (Vernì et al., 2004). Note that Feo accumulates in the central spindle midzone of both PMLES and mock-treated cells. (D) Control anaphases and telophases and PMLES stained for tubulin (green), DNA (blue) and the contractile ring marker anillin (red) required for S2 cell cytokinesis (Somma et al., 2002). Note that PMLES showing anaphase B-like and telophase-like spindles exhibit an anillin signal.
Figure 1—figure supplement 3. RNAi against the coding sequences and the UTRs of Sf3A2 and Prp31 rules out off-target effects (see also Figure 1D).

Figure 1—figure supplement 3.

RNAi against the 5' UTR of Sf3A2 or the 3' UTR of Prp31 leads to depletion of the endogenous Sf3A2 and Prp31 proteins but not of Sf3A2-RFP and Prp31-GFP that lack both the 5' and the 3’ UTRs. Sf3A2 dsRNA and Prp31 dsRNA are complementary to the coding sequences of these genes. The asterisks indicate non-specific bands. Actin was used as loading control.
Figure 1—figure supplement 4. RNAi-mediated depletion of Sf3A2 or Prp31 affects spindle structure of larval brain cells and inhibits chromosome segregation.

Figure 1—figure supplement 4.

(A–C) Metaphase (meta), prometaphase-like (PML, monopolar and bipolar), anaphase (ana) and telophase (telo) figures of neuroblasts stained for DNA (blue), the centrosomal marker Spd2 (red) and tubulin (green) from wild type (A), Sf3A2 RNAi (B) and Prp31 RNAi (C) larval brains. Scale bars, 5 μm. (D) Western blots and quantitation of relative band intensities showing that in brain extracts from tub-GAL80ts tub-GAL4 >UAS-Sf3A2RNAi and tub-GAL80ts tub-GAL4 >UAS-Prp31RNAi larvae exposed for 72 hr at 29°C the levels of Sf3A2 and Prp31 are strongly reduced. Tubulin and actin were used as loading controls. (E) Frequencies of mitotic figures observed after RNAi against the indicated genes. UAS-Sf3A2RNAi and UAS-Prp31RNAi are control brains containing the RNAi constructs alone. Lag, lagging chromosomes between the ana-telophase nuclei.