Fig 2: hnRNP A0 maintained the alignment of chromosomes through the stabilization of RAB3GAP1 mRNA in cancer cells.Immunocytochemistry showed that abnormal spindle formation was detected in hnRNP A0 or RAB3GAP1-downregulated cells. Scale bar: 10 µm a. Western blotting revealed that Cyclin B1 and Securin were highly expressed in the cells treated with siRNA of HNRNP A0 or RAB3GAP1 (n = 3) b. RT-PCR confirmed the overexpression of ZWINT-1 in a colorectal cancer line (HCT116 cells c). A GEPIA analysis revealed the overexpression of mRNA of ZWINT-1 in colorectal cancer tissues d. Western blotting showed that the protein expression of RAB3GAP1 and ZWINT-1 was decreased in HNRNP A0-knockdown cells e (n = 3). RT-PCR showed that the mRNA expression of ZWINT-1 was not decreased in hnRNP A0 or RAB3GAP1-knockdown cells f. Western blotting revealed that the ZWINT-1 expression was decreased by the downregulation of hnRNP A0 and RAB3GAP1, and the degree of this decrease was reduced by treatment with MG132 g (n = 3). Immunocytochemistry showed that ZWINT-1 was co-localized with active rab3. Scale bar: 10 µm h. The number of tumors decreased by the downregulation of HNRNP A0 and RAB3GAP1 in AOM-DSS carcinogenesis model mice is shown i. The error bars and numbers show the S.D. *p < 0.05 by Student’s t-test and an ANOVA.

Fig 3: A schematic illustration of the hnRNP A0 function.Phospho-hnRNP A0 rather than non-phospho-hnRNP A0 works dominantly and stabilizes tumor-related RAB3GAP1 mRNA in cancer cells. RAB3GAP1 inactivates Rab3. ZWINT-1 facilitates chromosomal alignment in cancer cells, resulting in tumor cell progression. When hnRNP A0 is dephosphorylated in cancer cells, Rab3 is activated, thereby inducing the proteasomal degradation of ZWINT-1. This results in chromosomal misalignment, subsequently leading to the apoptosis of cancer cells.

Fig 4: Role of Ndc80 complex in kMT capture during early mitosis examined using fixed and live-cell imaging under physiological conditions.A, cells were subjected to the indicated perturbations treated with the indicated drugs/chemicals and fixed according to the scheme. B, immunofluorescence staining of early prometaphase HeLa cells depleted of Ndc80 (top two panels) or knocked out of Nuf2 (bottom two panels) in comparison to controlRNAi or parental cells, respectively, and stained for α-tubulin (green) and a kinetochore marker, Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs in the indicated conditions. Bar (applies to all insets), 1 μm. C and D, quantification of the status of kMT attachments in cells from B. Error bars represent. S.D. from three independent experiments. For each experiment, on average ∼20 kinetochore pairs from ten early prometaphase cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). E, the distance measured between kinetochore and nearest microtubule in cells from B. Error bars represent S.D. from three independent experiments. For each experiment, on average 20 kinetochore pairs from five early prometaphase cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). F, immunofluorescence staining of early prometaphase RPE1 cells depleted of Nuf2 in comparison to controlRNAi cells (top panel) and stained for α-tubulin (green), a kinetochore marker Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs. Bar (applies to all insets), 1 μm. G, quantification of the status of kMT attachments in cells from F. Error bars represent S.D. from three independent experiments. For each experiment, on average ∼20 kinetochore pairs from ten early prometaphase cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). H, selected frames from live imaging of double thymidine–synchronized HeLa cells stably expressing mCherry-Histone H2B to visualize the chromosomes, in addition to GFP-α-tubulin and GFP-CENPA to visualize kMT attachment and treated with controlRNAi (top two panels) or Ndc80RNAi cells (bottom two panels). Images were captured every 1 min interval for 15 to 20 min until chromosome is aligned in controlRNAi. Bars, 5 μm. Yellow arrowheads indicate the kinetochores unattached to microtubule in Ndc80RNAi cells. The images from the videos were cropped and image intensity adjusted as required for better visualization of kinetochores and microtubules. I, selected frames from live imaging of parental (left panel) and Nuf2KO (right panel) HeLa cells treated with Hoechst for 30 min prior to imaging to visualize the chromosomes. Images were captured every 2 min interval for around 30 min until all the chromosomes were aligned in parental cells. Bars, 5 μm. kMT, kinetochore-microtubule; Ndc80, nuclear division cycle 80.

Fig 5: Characterization of the kinetochore-null phenotype induced by Ndc80 inhibition.A, cells were subjected to the indicated perturbations, treated with the indicated drugs/chemicals, and fixed according to the scheme. B and C, mitotic HeLa cells depleted of Ndc80 (B, panel) or knocked out of Nuf2 (C bottom panel) as compared to controlRNAi (B, top panel) or parental control cells (B, bottom panel), and followed by the indicated drug treatments, were immunostained for α-tubulin (green), a kinetochore marker Zwint1 or CENPA (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. D, quantification of the frequency of mitotic cells with null kinetochores (see main text for more details) in samples from B and D. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). E, immunofluorescence staining of mitotic RPE1 cells depleted of Nuf2 in comparison to control cells (top panel) and stained for α-tubulin (green), a kinetochore marker Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. F, quantification of the status of kMT attachments in cells from E. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). G, selected frames from live imaging of HeLa cells stably expressing mCherry-Histone H2B to visualize the chromosomes in addition to GFP-α-tubulin and GFP-CENPA to visualize kMT attachment in controlRNAi (top two panels) and Ndc80RNAi cells (bottom two panels). Images were captured at every 1 min interval starting from the point of nuclear envelope breakdown for around 30 min until a proper mitotic spindle was formed during prometaphase in controlRNAi cells. Bars, 5 μm. H, quantification of mitotic cells with null kinetochores from G. Error bars represent S.D. from three independent experiments. For each experiment, 50 mitotic cells were examined. I, immunofluorescence staining of MG132 + ZM treated mitotic HeLa cells depleted of endogenous Ndc80 and transfected with different Hec1-GFP constructs as indicated and stained for GFP (green), α-tubulin (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. J, quantification of mitotic cells with null kinetochores from A. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ∗∗∗∗p < 0.0001 (Student’s t test). kMT, kinetochore-microtubule; Ndc80, nuclear division cycle 80; ZM, ZM447439.