Journal: International Journal of General Medicine

Article Title: RCN1 Binds KIF14 and Promotes the Malignant Growth of Cervical Cancer Through the PI3K-AKT Pathway

doi: 10.2147/IJGM.S531003

Figure Lengend Snippet: RCN1 regulates the PI3K-AKT signaling pathway by targeting KIF14. ( A ) Effects of RCN1 knockdown and overexpression on key proteins in KIF14, PI3K/AKT/mTOR pathway in C33A cells. ( B ) Effects of RCN1 knockdown and overexpression on key proteins in KIF14, PI3K/AKT/mTOR pathway in Hela cells. The data are shown as mean ± SEM. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, as compared to the NC group.

Article Snippet: Among them, anti-RCN1 antibody (P0023D) was from the Biyuntian Institute of Biotechnology in China, anti-KIF14 antibody (DF15723), anti-p-PI3K antibody (AF3242), anti-PI3K antibody (AF6241), anti-p-AKT-antibody (AF0016), anti-AKT antibody (AF6261), anti-p-mTOR antibody (AF3308), and anti-mTOR antibody (AF6308) were all from Affinity Biosciences in China.

Techniques: Knockdown, Over Expression

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: The lack of ZO-2 blocked the development of astral and mitotic spindle microtubules, reduced the spindle length, and altered the accumulation at the spindle poles of acetylated tubulin, NuMA, p-Aurora, KIF14, and TPX2. A The lack of ZO-2 inhibits the localization of EB1 in astral microtubules and the mitotic spindle. Subconfluent parental and ZO-2 KD cells treated or not with docetaxel were processed for immunofluorescence with DAPI to stain their DNA and with antibodies against γ-tubulin and EB1, a plus-end microtubule marker. Representative images of two independent experiments. Bars, 10 μm. B The concentration of acetylated tubulin at the spindle poles and the mitotic spindle length are reduced in ZO-2 KD MDCK cells. Sparse cultures of parental and ZO-2 KD MDCK cells that re-express or not ZO-2 were fixed and processed for immunofluorescence with DAPI to stain their DNA, with antibodies against ZO-1 or ZO-2 to stain the cell border, and with antibodies against acetylated tubulin to detect the mitotic spindle poles. (a,b and c) Representative images of two independent experiments. Bars, 5 μm. d) Quantitation of the area of acetylated tubulin present at the spindle poles. Statistical analysis was done using the Mann–Whitney test. Numbers in parentheses indicate the number of spindle poles analyzed per condition. *** p < 0.001. e) Quantification of the spindle length. Statistical analysis was done using the Mann–Whitney test. Numbers in parentheses indicate the number of spindle poles analyzed per condition. *** p < 0.001, **** p < 0.0001. C ZO-2 depletion reduces the expression of kinesin KIF14 at spindle poles. Subconfluent parental and ZO-2 KD cells were processed for immunofluorescence using DAPI to stain their DNA and rabbit antibodies against acetylated tubulin, and a mouse monoclonal anti-KIF14 antibody. A secondary antibody against rabbit IgG, coupled to Alexa594, was employed in conjunction with a mouse IgGκ light chain binding protein, coupled to FITC. a-d Representative images of two independent experiments. e Quantification of KIF14 fluorescent intensity. Statistical analysis was done using Student’s t test. *** p < 0.001. Bars, 5 μm. f KIF14 is detected by Western blot in a ZO-2 immunoprecipitate derived from suconfluent parental cells. D The absence of ZO-2 triggers the accumulation of NuMA condensates in the spindle poles. Subconfluent parental (a) and ZO-2 KD (b, c) MDCK cells were incubated or not for 60 s with 10% 1,6-hexanediol. They were then fixed and processed for immunofluorescence with DAPI to stain their DNA, along with antibodies against γ-tubulin and NuMA, a minus-end microtubule marker. Representative images of two independent experiments. Bars, 10 μm. Statistical analysis was done with the Mann–Whitney test (d). Numbers in parenthesis indicate the number of spindle poles analyzed in parental and ZO-2 KD cells ** p < 0.01, **** p < 0.0001. E In ZO-2 KD cells, active Aurora exhibits a more diffuse distribution around the spindle pole than in parental cells. Sparse parental and ZO-2 KD MDCK cells were fixed and processed for immunofluorescence with DAPI to stain their DNA, with antibodies against γ-tubulin to stain the centriole in the mitotic spindle poles, and with antibodies against phosphorylated Aurora (p-Aurora) to detect the active form of the kinase. Upper panel, representative images of three independent experiments. Bars, 10 μm. In the lower panel, linescan analysis with fluorescence intensities of the two channels is performed as a function of distance from the midline γ-tubulin labeling

Article Snippet: We also employed the IgG 1 kappa light chain of mouse monoclonal antibodies against: TPX2 (Cat. sc-390183, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), IFT57 (Cat. sc-390120, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), KIF14 (Cat. sc-365553, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX) and Aurora-A (Cat.Sc-373856, dilution 1:500, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Immunofluorescence, Staining, Marker, Concentration Assay, Quantitation Assay, MANN-WHITNEY, Expressing, Binding Assay, Western Blot, Derivative Assay, Incubation, Fluorescence, Labeling

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: The amount of CEP64, IFT-B protein IFT57, and kinesin KIF14 diminishes at the base of the procilia in ZO-2 KD cells. Parental and ZO-2 KD MDCK cells were plated to confluence and incubated in CDMEM for 24 h. The monolayers were then transferred to DMEM without serum to stimulate cilium development. After 48 h, the monolayers were fixed and processed for immunofluorescence using antibodies against acetylated tubulin and CEP164 ( A ), IFT57 ( B ), or KIF14 ( C ). A The concentration of CEP164 at the ciliary base diminishes in ZO-2-depleted cells in comparison to parental cells. Panels a and b, representative immunofluorescence images of CEP164 present in the base of cilia and procilia in parental and ZO-2 KD MDCK cells. Bars 1 μm. Panel c, quantification of CEP164 immunofluorescent intensity in the ciliary base. The statistical analysis was done using the Mann–Whitney test. *** p < 0.001. B In parental cells, IFT57 concentrates at the ciliary base and the ciliary tip. The accumulation of IFT57 at the ciliary base diminishes in ZO-2KD cells. Panels a,b,e, and f, representative immunofluorescence images of IFT57 present in the base of procilia in parental (a,b) and ZO-2 KD (e,f) MDCK cells. Bars, 1 μm. Panels c and d, representative immunofluorescence images showing the concentration of IFT57 in the base and tip of cilia present in parental MDCK cells. Bars, 5 μm. Panel g, quantification of IFT57 immunofluorescent intensity in the ciliary base of parental and ZO-2 KD cells. The statistical analysis was done using the Mann–Whitney test. *** p < 0.001. h) IFT57 is detected by Western blot in a ZO-2 immunoprecipitate derived from confluent parental MDCK cells. C In parental cells, KIF14 is present at the ciliary base and along the ciliary axoneme. The accumulation of KIF14 at the ciliary base diminishes in ZO-2KD cells. Panels a,b,e, and f, representative immunofluorescence images of KIF14 present in the base of procilia in parental (a,b) and ZO-2 KD (e,f) MDCK cells. Bars, one μμm. Panels c and d, representative immunofluorescence images of KIF14 present in the ciliary base and along the axoneme of elongated cilia in parental MDCK cells. Bars, 2 μm. Panel g, quantification of KIF14 immunofluorescent intensity in the ciliary base of parental and ZO-2 KD cells. The statistical analysis was done using the Mann–Whitney test. ** p < 0.01

Article Snippet: We also employed the IgG 1 kappa light chain of mouse monoclonal antibodies against: TPX2 (Cat. sc-390183, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), IFT57 (Cat. sc-390120, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), KIF14 (Cat. sc-365553, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX) and Aurora-A (Cat.Sc-373856, dilution 1:500, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Incubation, Immunofluorescence, Concentration Assay, Comparison, MANN-WHITNEY, Western Blot, Derivative Assay

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: Schematic representation of the changes triggered in epithelial cells by ZO-2 depletion on the mitotic spindle and primary cilia. A ZO-2 is present at the distal appendages of the mother centriole. DA, distal appendage; SDA, subdistal appendage; MC, mother centriole; DC, daughter centriole. B ZO-2 depletion concentrates NuMA at the spindle pole and reduces the spindle pole accumulation of active Aurora (p-Aurora), TPX2, and KIF14. This suggests an increase in poleward spindle microtubule flux, which might explain the poor development of astral and mitotic spindle microtubules and the reduction in mitotic spindle length. The accumulation of NuMA at the spindle pole may also explain the previously reported spindle pole misorientation in ZO-2 KD cells (Raya-Sandino, et al. ), as illustrated by the dashed line. C In parental cells, ZO-2 functions as a scaffold at the ciliary basal body that promotes the concentration of CEP164, IFT57, and KIF14. Instead, ZO-2 depletion stabilizes the presence of p-Aurora and p-AKT at the basal body and reduces the accumulation of CEP164. Consequently, the development of primary cilia and Gli1 signaling through the SHH pathway is impaired

Article Snippet: We also employed the IgG 1 kappa light chain of mouse monoclonal antibodies against: TPX2 (Cat. sc-390183, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), IFT57 (Cat. sc-390120, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX), KIF14 (Cat. sc-365553, dilution 1:250, Santa Cruz Biotechnology, Dallas, TX) and Aurora-A (Cat.Sc-373856, dilution 1:500, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Concentration Assay

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: The lack of ZO-2 blocked the development of astral and mitotic spindle microtubules, reduced the spindle length, and altered the accumulation at the spindle poles of acetylated tubulin, NuMA, p-Aurora, KIF14, and TPX2. A The lack of ZO-2 inhibits the localization of EB1 in astral microtubules and the mitotic spindle. Subconfluent parental and ZO-2 KD cells treated or not with docetaxel were processed for immunofluorescence with DAPI to stain their DNA and with antibodies against γ-tubulin and EB1, a plus-end microtubule marker. Representative images of two independent experiments. Bars, 10 μm. B The concentration of acetylated tubulin at the spindle poles and the mitotic spindle length are reduced in ZO-2 KD MDCK cells. Sparse cultures of parental and ZO-2 KD MDCK cells that re-express or not ZO-2 were fixed and processed for immunofluorescence with DAPI to stain their DNA, with antibodies against ZO-1 or ZO-2 to stain the cell border, and with antibodies against acetylated tubulin to detect the mitotic spindle poles. (a,b and c) Representative images of two independent experiments. Bars, 5 μm. d) Quantitation of the area of acetylated tubulin present at the spindle poles. Statistical analysis was done using the Mann–Whitney test. Numbers in parentheses indicate the number of spindle poles analyzed per condition. *** p < 0.001. e) Quantification of the spindle length. Statistical analysis was done using the Mann–Whitney test. Numbers in parentheses indicate the number of spindle poles analyzed per condition. *** p < 0.001, **** p < 0.0001. C ZO-2 depletion reduces the expression of kinesin KIF14 at spindle poles. Subconfluent parental and ZO-2 KD cells were processed for immunofluorescence using DAPI to stain their DNA and rabbit antibodies against acetylated tubulin, and a mouse monoclonal anti-KIF14 antibody. A secondary antibody against rabbit IgG, coupled to Alexa594, was employed in conjunction with a mouse IgGκ light chain binding protein, coupled to FITC. a-d Representative images of two independent experiments. e Quantification of KIF14 fluorescent intensity. Statistical analysis was done using Student’s t test. *** p < 0.001. Bars, 5 μm. f KIF14 is detected by Western blot in a ZO-2 immunoprecipitate derived from suconfluent parental cells. D The absence of ZO-2 triggers the accumulation of NuMA condensates in the spindle poles. Subconfluent parental (a) and ZO-2 KD (b, c) MDCK cells were incubated or not for 60 s with 10% 1,6-hexanediol. They were then fixed and processed for immunofluorescence with DAPI to stain their DNA, along with antibodies against γ-tubulin and NuMA, a minus-end microtubule marker. Representative images of two independent experiments. Bars, 10 μm. Statistical analysis was done with the Mann–Whitney test (d). Numbers in parenthesis indicate the number of spindle poles analyzed in parental and ZO-2 KD cells ** p < 0.01, **** p < 0.0001. E In ZO-2 KD cells, active Aurora exhibits a more diffuse distribution around the spindle pole than in parental cells. Sparse parental and ZO-2 KD MDCK cells were fixed and processed for immunofluorescence with DAPI to stain their DNA, with antibodies against γ-tubulin to stain the centriole in the mitotic spindle poles, and with antibodies against phosphorylated Aurora (p-Aurora) to detect the active form of the kinase. Upper panel, representative images of three independent experiments. Bars, 10 μm. In the lower panel, linescan analysis with fluorescence intensities of the two channels is performed as a function of distance from the midline γ-tubulin labeling

Article Snippet: Mouse α-KIF14 (Dil. 1:20) Santa Cruz Biotechnology Cat. sc-365553 Dallas, TX. , Mouse α-ace-tubulin (Dil. 1:100) Santa Cruz Biotechnology Cat. sc-23950 Dallas, TX..

Techniques: Immunofluorescence, Staining, Marker, Concentration Assay, Quantitation Assay, MANN-WHITNEY, Expressing, Binding Assay, Western Blot, Derivative Assay, Incubation, Fluorescence, Labeling

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: The amount of CEP64, IFT-B protein IFT57, and kinesin KIF14 diminishes at the base of the procilia in ZO-2 KD cells. Parental and ZO-2 KD MDCK cells were plated to confluence and incubated in CDMEM for 24 h. The monolayers were then transferred to DMEM without serum to stimulate cilium development. After 48 h, the monolayers were fixed and processed for immunofluorescence using antibodies against acetylated tubulin and CEP164 ( A ), IFT57 ( B ), or KIF14 ( C ). A The concentration of CEP164 at the ciliary base diminishes in ZO-2-depleted cells in comparison to parental cells. Panels a and b, representative immunofluorescence images of CEP164 present in the base of cilia and procilia in parental and ZO-2 KD MDCK cells. Bars 1 μm. Panel c, quantification of CEP164 immunofluorescent intensity in the ciliary base. The statistical analysis was done using the Mann–Whitney test. *** p < 0.001. B In parental cells, IFT57 concentrates at the ciliary base and the ciliary tip. The accumulation of IFT57 at the ciliary base diminishes in ZO-2KD cells. Panels a,b,e, and f, representative immunofluorescence images of IFT57 present in the base of procilia in parental (a,b) and ZO-2 KD (e,f) MDCK cells. Bars, 1 μm. Panels c and d, representative immunofluorescence images showing the concentration of IFT57 in the base and tip of cilia present in parental MDCK cells. Bars, 5 μm. Panel g, quantification of IFT57 immunofluorescent intensity in the ciliary base of parental and ZO-2 KD cells. The statistical analysis was done using the Mann–Whitney test. *** p < 0.001. h) IFT57 is detected by Western blot in a ZO-2 immunoprecipitate derived from confluent parental MDCK cells. C In parental cells, KIF14 is present at the ciliary base and along the ciliary axoneme. The accumulation of KIF14 at the ciliary base diminishes in ZO-2KD cells. Panels a,b,e, and f, representative immunofluorescence images of KIF14 present in the base of procilia in parental (a,b) and ZO-2 KD (e,f) MDCK cells. Bars, one μμm. Panels c and d, representative immunofluorescence images of KIF14 present in the ciliary base and along the axoneme of elongated cilia in parental MDCK cells. Bars, 2 μm. Panel g, quantification of KIF14 immunofluorescent intensity in the ciliary base of parental and ZO-2 KD cells. The statistical analysis was done using the Mann–Whitney test. ** p < 0.01

Article Snippet: Mouse α-KIF14 (Dil. 1:20) Santa Cruz Biotechnology Cat. sc-365553 Dallas, TX. , Mouse α-ace-tubulin (Dil. 1:100) Santa Cruz Biotechnology Cat. sc-23950 Dallas, TX..

Techniques: Incubation, Immunofluorescence, Concentration Assay, Comparison, MANN-WHITNEY, Western Blot, Derivative Assay

Journal: Cell and Tissue Research

Article Title: ZO-2 is a scaffold at the centriole and mitotic spindle poles that enhances microtubule stability and supports the proper development of mitotic spindles and cilia

doi: 10.1007/s00441-025-03992-0

Figure Lengend Snippet: Schematic representation of the changes triggered in epithelial cells by ZO-2 depletion on the mitotic spindle and primary cilia. A ZO-2 is present at the distal appendages of the mother centriole. DA, distal appendage; SDA, subdistal appendage; MC, mother centriole; DC, daughter centriole. B ZO-2 depletion concentrates NuMA at the spindle pole and reduces the spindle pole accumulation of active Aurora (p-Aurora), TPX2, and KIF14. This suggests an increase in poleward spindle microtubule flux, which might explain the poor development of astral and mitotic spindle microtubules and the reduction in mitotic spindle length. The accumulation of NuMA at the spindle pole may also explain the previously reported spindle pole misorientation in ZO-2 KD cells (Raya-Sandino, et al. ), as illustrated by the dashed line. C In parental cells, ZO-2 functions as a scaffold at the ciliary basal body that promotes the concentration of CEP164, IFT57, and KIF14. Instead, ZO-2 depletion stabilizes the presence of p-Aurora and p-AKT at the basal body and reduces the accumulation of CEP164. Consequently, the development of primary cilia and Gli1 signaling through the SHH pathway is impaired

Article Snippet: Mouse α-KIF14 (Dil. 1:20) Santa Cruz Biotechnology Cat. sc-365553 Dallas, TX. , Mouse α-ace-tubulin (Dil. 1:100) Santa Cruz Biotechnology Cat. sc-23950 Dallas, TX..

Techniques: Concentration Assay