Journal: Cell Communication and Signaling : CCS

Article Title: Constant light exposure impairs embryonic development via disrupting oocyte cytoskeleton integrity and redox homeostasis

doi: 10.1186/s12964-026-02879-z

Figure Lengend Snippet: Melatonin restores microtubule stability for spindle assembly in oocytes under constant light exposure. A Representative images and abnormal percentage of spindle in oocyte of the control ( n = 45), light ( n = 37), and light + MT ( n = 45) groups. Green, α-tubulin. Blue, DNA. Bar = 10 μm. B Measurement of spindle morphology in the control ( n = 53), light ( n = 54), and light + MT ( n = 63) group oocytes. We defined the distance of spindle pole to pole as L and the width across the middle of the spindle as D. The ratio of L/D was considerably reduced in the light exposure oocytes compared with control and light + MT groups. Cyan, α-tubulin. Bar = 10 μm. C Band intensity analysis of PLK1 in MⅠ-stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. D Representative images of chromosome numbers and percentage of aneuploidy in control ( n = 38), light ( n = 33), light + MT ( n = 31) oocytes of MⅡ stage. Blue, DNA. Bar = 5 μm. E Representative images and abnormal of attachments between kinetochore and microtubule/K-fibers (K-MT attachment) in oocyte from control ( n = 43), light ( n = 53), light + MT ( n = 44) groups. Red, ACA. Green, α-tubulin. Blue, DNA. Bar = 5 μm. F Representative images of microtubule in oocyte from control, light, and light + MT groups after cold treatment. Black, α-tubulin. Blue, DNA. Bar = 10 μm. G Relative intensity of microtubule in oocyte from control ( n = 45), light ( n = 36), and light + MT ( n = 37) groups after cold treatment. H Band intensity analysis of ac-tubulin in MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. I Band intensity analysis of HDAC6 in MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. Data were presented as mean ± SEM. For panels A, B, D, and E, the data were analyzed using one-way ANOVA test. For panels C, G, H, and I, the intensity of control group was normalized to 1, and these data were analyzed using t -test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. *, P < 0.05. **, P < 0.01. #, P < 0.05. ##, P < 0.01

Article Snippet: Rabbit anti-H3K27me3 antibody (A2363), rabbit anti-H3K27ac3 antibody (A7253), rabbit anti-PLK1 antibody (A2548), and rabbit anti-HDAC6 antibody (A11259) were from Abclonal.

Techniques: Control, Western Blot

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Schematic of AURKA activation mechanisms during mitotic entry (left panel) and spindle assembly (right panel) by its allosteric activators phospho-Bora and Tpx2, respectively. In the G2 phase of the cell cycle, AURKA is maintained in a dephosphorylated state on the T-loop (T288) by counteracting phosphatases. During mitotic entry, Cyclin A-Cdk1 phosphorylates Bora, which then binds and activates AURKA. Phospho-Bora preferentially binds unphosphorylated AURKA. Activated by pBora, AURKA phosphorylates the Plk1 T-loop (T210) to activate the kinase and trigger mitotic entry. Then, during mitosis, Tpx2, through its first 43 amino acids, recruits and activates phosphorylated AURKA (pT288) at the microtubules to promote mitotic spindle assembly. ( B ) Domain architecture of Homo sapiens Bora (orange, left panel) and Tpx2 (blue, right panel) with the minimal fragments required for AURKA activation highlighted in dark orange (Bora 18-120 ) and dark blue (Tpx2 1–43 ). Important sequence elements identified in these regions are indicated, including the aromatic anchors in the M1 and M2 motifs of Bora and Tpx2, as well as the phosphorylatable PSP motif (M3) (green) of Bora, located just downstream of the M2 motif. Based on these sequences, we engineered a phosphorylated Tpx2-Bora fusion (phosphopeptide MK51, right panel) that reconstitutes the regulatory properties of both proteins. We also engineered a minimal Bora construct (phosphopeptide GK51, left panel) by directly fusing the putative M1 motif to the M2 and M3 motifs via a Glycine linker. ( C ) Competitive binding assay where fluorescein-labeled Tpx2 1–43 polypeptide, in complex with AURKA T288V , is displaced by increasing amounts of competitor (cold Tpx2 1–43 , pBora 1–224 , pBora fusion, and pTxp2-Bora chimera) and monitored through fluorescence polarization signals. The displayed data points and the half-maximal inhibitory concentration (IC 50 ) value represent the average fluorescence polarization for each reaction condition, with standard deviations of the mean as error bars ( N = 3 independent experiments, each performed with n = 3 independent experimental samples). ND not determined. ( D ) Activation of AURKA T288V ATPase activity by different activators as assessed using the ADP Glo assay with Kemptide substrate. Displayed data points and EC 50 values represent the average luminescence for each reaction condition with standard deviations of the mean as error bars ( N = 3 independent experiments, each performed with n = 3 independent experiment samples). RLU relative light unit. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Activation Assay, Sequencing, Phospho-proteomics, Construct, Competitive Binding Assay, Labeling, Fluorescence, Concentration Assay, Activity Assay, Glo Assay

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Schematic of the two-step in vitro reconstitution of Histone H3 phosphorylation on S10 by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera. In step 1, Bora 1–224 phosphorylated by ERK (noted pBora) or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 min. In step 2, the reaction mix from step 1 is incubated in the presence of Mg/ATP with Histone H3 for 5, 10, 20, 30, and 60 min. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-S10 Histone H3, or pan Histone H3, and AURKA (from top to bottom). Note that during step 2, pBora 1–224 itself is phosphorylated by activated AURKA T288V during the reaction, which is manifested by a mobility shift in SDS-PAGE, as reported previously (Tavernier et al, ). ppBora thus denotes Bora phosphorylated by ERK during step 1 and by activated AURKA during step 2 in this and other Figures. The graph presents the normalized quantification of pS10 Histone H3 signal over Histone H3 from n = 3 independent experiments. Error bars display the standard deviation. ( B ) Side-by-side comparison of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of Plk1 (pT210) by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera when the Plk1 kinase domain or only the isolated T-loop is used as a substrate. In step 1, pBora 1–224 or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 min. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with Plk1 kinase domain catalytically dead mutant (Plk1 K82R ) or the isolated Plk1 T-loop for 30 and 60 min. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-T210 Plk1, pan Plk1, or GST, and AURKA (from top to bottom). The graph presents the normalized quantification of pT210 Plk1 signal over either Plk1 KDom or GST-Plk1 T-loop from n = 3 independent experiments. Error bars display the standard deviation. ( C ) Schematics summarizing the main results. The pTpx2-Bora chimera and pBora 1–224 can similarly activate AURKA T288V toward Histone H3 (red arrows). However, in sharp contrast to pBora 1–224 , the pTpx2-Bora chimera is unable to activate AURKA T288V toward the Plk1 T-loop embedded in the kinase domain (crossed arrow). Both the pTpx2-Bora chimera and pBora 1–224 can stimulate AURKA T288V activity towards the isolated T-loop, but with poor efficacy (thin red arrows). .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: In Vitro, Phospho-proteomics, Incubation, Western Blot, Mobility Shift, SDS Page, Standard Deviation, Comparison, Isolation, Mutagenesis, Activity Assay

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of Plk1 (pT210) by AURKA T288V and pBora 1–224 or the pTpx2-Bora chimera. In step 1, pBora 1–224 or the pTpx2-Bora chimera is incubated with AURKA T288V for 15 min. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with Plk1 kinase domain catalytically dead mutant (Plk1 K82R ) for 5, 10, 20, 30, and 60 min. Samples were then analyzed by Western blot. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. ( B ) Same experiment as in panel A, except that AURKA T288V activated by pBora 1–224 or the pTpx2-Bora chimera is incubated with the isolated Plk1 T-loop fused to GST as substrate. Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). The graph presents the normalized quantification of pT210 Plk1 signal over GST-Plk1 T-loop from n = 3 independent experiments. ( C ) Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of the isolated Plk1 T-loop fused to GST by AURKA T288V and pBora 18–120 . In step 1, pBora 18–120 is incubated 1 h at 30 °C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and the isolated Plk1 T-loop fused to GST for 30 min at 30 °C. Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of the T-loop of Plk1 (aa 190-225) fused to GST (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, GST, and AURKA, as indicated (from top to bottom). The graph presents the normalized quantification of pT210 Plk1 signal over GST-Plk1 T-loop from n = 3 independent experiments. Error bars display the standard deviation.

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: In Vitro, Phospho-proteomics, Incubation, Mutagenesis, Western Blot, Isolation, Standard Deviation

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) (i) View of the M1 and M2 motifs of Bora bound to AURKA predicted by AlphaFold 3. The Bora residues F25, V44, and F45 of the M1 motif binding to the F (green) and Y (yellow) pockets, as well as the residues F103 and F104 of the M2 motif binding to the W pocket (magenta), are shown. (ii) View of the Bora-specific motif bound to AURKA. I60 and I71 (blue arrows) anchor the motif to AURKA, whereas other hydrophobic and aromatic residues, such as F56, W58, and I66 (red arrows), are solvent-exposed. (iii) View of the Bora segment comprising amino acids 60 to 79 bound to AURKA. ( B ) Western blot analysis of total bacterial extracts reconstituting pBora and AURKA T288V -dependent T-loop (T210) phosphorylation of the isolated Plk1 T-loop fused to GST (GST T-loop ). Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). ERK K-dead kinase dead.

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Binding Assay, Solvent, Western Blot, Phospho-proteomics, Isolation

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Structural alignment of the five pBora•AURKA•Plk1 KDom complex predictions generated by Alphafold 3 in a 1:1:1 binding stoichiometry. AURKA is in gray, Plk1 is in blue, and Bora is in orange. The predicted template modeling (pTM) score and the interface predicted template modeling (ipTM) scores are indicated. ( B ) Predicted Alignment Error (PAE) map calculated by AlphaFold3 showing the confidence of the distances between the residues in contact in the H. sapiens AURKA (126–403) (gray) • Bora (18–116) (orange) • Plk1 (1–332) (blue) complex generated by AlphaFold 3 in a 1:1:1 binding stoichiometry. ( C ) Per-residue confidence (pLDDT) of the pBora•AURKA•Plk1 KDom complex predicted by AlphaFold 3.

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Generated, Binding Assay, Residue

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Schematic of MITOKINAC: E. coli BL21 are transformed with three plasmids containing different replication origins, antibiotic resistance, and expressing (i) the active ERK kinase (MAPKK constitutively activated S118E, S222D, which phosphorylates and activates the MAPK ERK), (ii) Bora 1–224 and AURKA T288V expressed from the same plasmid, and (iii) the Plk1 kinase domain (kinase-dead K82R but phosphorylable on the T-loop (T210)) or the isolated T-loop fused to GST as substrates. After co-transformation, culture, and protein induction with IPTG, Plk1 T-loop phosphorylation (T210) is monitored by Western blot directly on total bacterial extracts. ( B ) Schematic of the mitotic kinase cascade reconstituted in E. coli . The activated ERK kinase phosphorylates Bora at multiple sites, including S112 (M3 motif), which binds and activates non-phosphorylated AURKA (AURKA T288V ) towards the Plk1 kinase domain. ( C , D ) Western blot analysis of total bacterial extracts reconstituting pBora 1–224 and AURKA T288V -dependent T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) ( C ) or the isolated T-loop fused to GST (GST T-loop ) ( D ). Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1 ( C ) or GST ( D ), and AURKA (from top to bottom). ERK K-dead: kinase dead. Asterisk denotes a nonspecific band. ( E ) Schematic of MITOKINAC using Genetic Code Expansion to produce Bora 1–224, uniquely phosphorylated at serine 112, thus bypassing the need for a priming phosphorylation by ERK. The schematic illustrates the translation of Bora 1–224 (orange) by ribosomes (gray) in E. coli , which inserts a phosphoserine (orange) at the amber codon UAG. When produced and phosphorylated at S112, pBora 1–224 binds to and activates AURKA, which subsequently phosphorylates the Plk1 T-loop (T210). ( F ) Western blot analysis of total bacterial extracts reconstituting pBora and AURKA T288V -dependent T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using Genetic code expansion. Bacteria were transformed with a plasmid expressing wild-type Bora 1–224 (lane 1) or Bora 1–224 harboring the amber codon TAG at position S112 (lane 2). Blots were probed with antibodies to Bora, phospho-T210 Plk1, GST, and AURKA (from top to bottom). ERK K-dead: kinase dead. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Transformation Assay, Expressing, Plasmid Preparation, Isolation, Phospho-proteomics, Western Blot, Produced, Bacteria

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Multiple protein sequence alignments of the M1 motif of Bora. Identical residues are in dark blue. The mutated residues and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutation on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles mean no effect, while red rectangles indicate a loss of function. ( B ) Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants of the M1 motif. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. ( C ) View of the extended M1 motif of Bora bound to AURKA predicted by AlphaFold3. The M1 motif of Bora is anchored at one end by F25 binding to the F pocket of and V44, and F45 binding to the Y pocket of AURKA, respectively. ( D ) Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. Error bars display the standard deviation. ( E ) Multiple protein sequence alignments of the M2 and M3 motifs of Bora. Identical residues are shown in dark blue. The mutated residues and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutation on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles indicate no effect, while red rectangles indicate a loss of function. ( F ) Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants of the M2 and M3 motifs. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. Note that Bora F103D and F104D mutants are expressed in E. coli ( H ) but not detected by our anti-Bora antibody. ( G ) View of the M2 and M3 motifs of Bora bound to AURKA predicted by AF3. AURKA is shown as a surface colored according to electrostatic potential, with blue indicating positive and red indicating negative. Bora is shown as an orange ribbon. Bora residues F103 and F104 bind to the W pocket of AURKA, while pS112 engages an electropositive pocket. ( H ) Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to MBP, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). ERK K-dead kinase dead. As membranes were sequentially revealed with different antibodies, the orange asterisk indicates the signal revealed by the anti-Bora antibody. The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. Error bars display the standard deviation. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Sequencing, Mutagenesis, Western Blot, Phospho-proteomics, Binding Assay, Standard Deviation

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) Multiple protein sequence alignments of the Bora region located between the M1 and M2/M3 motifs. Identical residues are shown in dark blue. The residues mutated, and the nature of the substitutions are indicated at the bottom of the alignment. The impact of the mutations on Bora’s function is indicated by the rectangle’s color around the amino acids. Green rectangles indicate no effect, while red rectangles indicate a loss of function. ( B ) Western blot analysis of bacterial extracts reconstituting the mitotic kinase cascade in E. coli BL21, resulting in T-loop (T210) phosphorylation of the Plk1 kinase domain (Plk1 Kdom ) using wild-type and Bora mutants. Blots were probed with antibodies to Bora, phospho-T210 Plk1, or pan Plk1, and AURKA (from top to bottom). ERK K-dead: kinase dead. ( C ) Schematic of the two-step in vitro reconstitution of T-loop phosphorylation on T210 of the Plk1 kinase domain (pT210) by AURKA T288V and pBora 18–120 . In step 1, pBora 18–120 is incubated 1 h at 30 °C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and Plk1 KDom for 30 min at 30 °C. ( D ) Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Plk1 KDom (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoT210 Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). As membranes were sequentially revealed with different antibodies, the orange asterisk indicates the signal revealed by the anti-Bora antibody. The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 KDom from n = 3 independent experiments. ( E ) Schematic of the two-step in vitro reconstitution of Histone 3 phosphorylation on serine 10 (pS10) by AURKA T288V and pBora 18–120 . In step 1, pBora 18–120 is incubated 1 h at 30 °C with the ERK kinase and Mg/ATP. In step 2, the reaction mix in step 1 is incubated in the presence of Mg/ATP with AURKA T288V and Histone H3 for 30 min at 30 °C. ( F ) Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of Histone H3 (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phosphoSer10 Histone H3, or pan Histone H3, and AURKA, as indicated (from top to bottom). The graph presents the normalized quantification of pS10 Histone H3 signal over Histone H3 from n = 3 independent experiments. Error bars display the standard deviation. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Sequencing, Western Blot, Phospho-proteomics, In Vitro, Incubation, Mutagenesis, Standard Deviation

Journal: The EMBO Journal

Article Title: Molecular basis for the activation of Aurora A and Plk1 kinases during mitotic entry

doi: 10.1038/s44318-025-00679-8

Figure Lengend Snippet: ( A ) AlphaFold 3 model of the trimeric complex composed of pBora 18–120 (orange), AURKA (gray), and the Plk1 kinase domain (blue). The residue T210 of the Plk1 T-loop, T288 of the AURKA T-loop are highlighted in red, and the phosphorylated residue S112 of Bora is highlighted in green. ( B , C ) Zoom in view of the AURKA/Bora/Plk1 interface, highlighting contacting residues. Color scheme as in ( A ). ( D ) Western blot analysis of 2-step kinase reactions carried out with MBP-Bora 18–120 wild-type or mutant phosphorylated (+) or not (−) by the ERK kinase (step 1) in the presence of the Plk1 kinase domain (substrate) and AURKA T288V (step 2). Blots were probed with antibodies to Bora, phospho-T210 of Plk1, or pan Plk1, and AURKA, as indicated (from top to bottom). ( E ) The graph presents the normalized quantification of pT210 Plk1 signal over Plk1 from n = 3 independent experiments. .

Article Snippet: Rabbit anti-human Plk1 , Bethyl , Cat#A300-250A.

Techniques: Residue, Western Blot, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: TRIM47-facilitated PLK1 stabilization promotes the proliferation of liver cancer cells

doi: 10.1007/s13402-025-01130-0

Figure Lengend Snippet: TRIM47 interacted with PLK1. A. Correlation between TRIM47 and PLK1 mRNA levels in clinical HCC samples was analyzed via the TIMER database. B. The interaction between TRIM47 and PLK1 was verified via a yeast two-hybrid system. C. Co-immunoprecipitation analysis of the interaction between GFP-TRIM47 and Flag-PLK1 in HEK293T cells. D. Co-immunoprecipitation analysis and protein blotting were conducted in primitive Hep3B cells. E. The purified GST or GST-TRIM47 protein was incubated with the lysates of Flag-PLK1-transfected HEK293T cells. After the GST pull-down experiment, the eluted protein and cell lysates were analyzed via western blotting. The expression of the GST and GST-TRIM47 proteins was confirmed by Coomassie brilliant blue staining and an anti-GST antibody. F. Co-localization of TRIM47 and PLK1 in Huh7 cells. The TRIM47 proteins were stained with a green fluorescent antibody (Alexa Fluor 488), with red fluorescence (Alexa Fluor 594) representing PLK1 and blue representing the nucleus. G. Representative images of the cytosolic and nuclear location of TRIM47 and PLK1 measured by western blot in cytosolic and nuclear fractions, using TBP (TATA-binding protein) as the nuclear control and GAPDH as the cytoplasmic control. H. Schematic diagram of PLK1 deletion mutants generated in the pEGFP-C vector. I. The interaction of TRIM47 with PLK1 and its deletion mutants was determined by GST pull-down using lysates of HEK293T cells transfected with PLK1 and its deletion mutants as indicated. J. The interaction of GFP-PLK1 with Flag-TRIM47 and its deletion mutants was determined by co-IP in HEK293T cells co-transfected with GFP-PLK1 and Flag-TRIM47 deletion mutants. K. Schematic diagram of TRIM47 deletion mutants generated in pcDNA3.1-Flag vector. L. Co-IP and immunoblotting were performed in HEK293T cells co-transfected with Flag-PLK1 and either GFP-empty vector (EV), full-length GFP-TRIM47 (WT), or the N-terminal GFP-TRIM47 truncation mutant (1–80 aa) to characterize their interaction

Article Snippet: PLK1 (A2548) antibodies were purchased from ABclonal (Wuhan, China). p65 (8242S), p-p65 (3033S), Erk1/2 (4695S), p-Erk1/2 (4370S), p21 (2947S), p27 (3686S), p38 (9212S), p-p38 (4511S), Akt (9272S), p-Akt (4060S), K63-linked specific Ub (5621S), K48-linked specific Ub (8081S), and Ub (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Purification, Incubation, Transfection, Western Blot, Expressing, Staining, Fluorescence, Binding Assay, Control, Generated, Plasmid Preparation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: TRIM47-facilitated PLK1 stabilization promotes the proliferation of liver cancer cells

doi: 10.1007/s13402-025-01130-0

Figure Lengend Snippet: TRIM47 can stabilize PLK1 by mediating its K63-linked ubiquitination. A. Western blotting was used to detect the protein expression of PLK1 in stably transfected LC cells. B. The protein expression of TRIM47 and PLK1 in tumors from Hep3B xenografts was detected by western blot. C. Representative IHC staining of TRIM47 and PLK1 in Hep3B xenograft tumors. D/E. TRIM47-knockdown stable Huh7 cells/control cells D. or TRIM47-overexpressed stable Hep3B cells/control cells E. were treated with CHX (100 µg/mL) for 0, 2, 4, 6, 8, or 10 h. Western blotting was used to detect the protein expression of TRIM47 and PLK1 in the above cells, and ImageJ software was used for gray quantification analysis. * P < 0.05, ** P < 0.01. F. The ubiquitination level of the PLK1 protein was detected after immunoprecipitation with a PLK1 antibody in both TRIM47-overexpressed and control Huh7 cells. G. GFP-PLK1 was co-expressed with EV, Flag-TRIM47 (FL) or Flag-TRIM47 ΔRING (Mutant) in HEK293T cells, followed by anti-GFP IP and HA immunoblotting to examine PLK1 ubiquitination. H. HEK293T cells were co-transfected with GFP-PLK1 and seven different HA-ubiquitin (HA-Ub) mutants (K6O, indicating K6 only, which means that Ub ligation only occurs via the K6-linked pathway) with or without Flag-TRIM47 for 24 h. Then, the cell lysates were collected, immunoprecipitated with an anti-GFP antibody, and immunoblotted with an anti-HA antibody. I. HEK293T cells were co-transfected with GFP-PLK1 plus HA-Ub and/or Flag-TRIM47. After 24 h, the cell lysates were harvested, immunoprecipitated with anti-GFP antibody, and immunoblotted with the indicated antibodies. J. GFP-PLK1 and Flag-TRIM47 were co-transfected with HA-Ub, HA-Ub K48R or K63R into HEK293T cells. 24 h later, the cell lysates were immunoprecipitated with an anti-PLK1 antibody

Article Snippet: PLK1 (A2548) antibodies were purchased from ABclonal (Wuhan, China). p65 (8242S), p-p65 (3033S), Erk1/2 (4695S), p-Erk1/2 (4370S), p21 (2947S), p27 (3686S), p38 (9212S), p-p38 (4511S), Akt (9272S), p-Akt (4060S), K63-linked specific Ub (5621S), K48-linked specific Ub (8081S), and Ub (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Stable Transfection, Transfection, Immunohistochemistry, Knockdown, Control, Software, Immunoprecipitation, Mutagenesis, Ligation

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: TRIM47-facilitated PLK1 stabilization promotes the proliferation of liver cancer cells

doi: 10.1007/s13402-025-01130-0

Figure Lengend Snippet: The effect of TRIM47 on LC cell proliferation is dependent on PLK1. A. Western blot analysis of 6 groups of co-transfected Huh7 cells (Flag (empty vector) + shRNA control, Flag-PLK1 + shRNA control, Flag + TRIM47 shRNA-1, Flag-PLK1 + TRIM47 shRNA-1, Flag + TRIM47 shRNA-2, Flag-PLK1 + TRIM47 shRNA-2). B. CCK-8 assay. The above cells were co-transfected with the indicated vectors. After 48 h, the cells were plated into 96-well plates and then subjected to CCK-8 analysis ( n = 8). C. Colony formation assay. A total of 5 × 10 3 cells from each group were plated in 6-well plates containing 5% FBS medium. After 10–14 days, the cells were fixed and stained with 2% crystal violet. D. The number of cell colonies was calculated and statistically analyzed ( n = 3). E-F. Soft agar colony formation assay. 6 groups of Huh7 cells were seeded in 6-well plates in full growth medium (10% FBS) containing 0.35% agar (1 mL per well) on top of a layer of growth medium containing 0.6% agar (1 mL per well). After 20–25 days, the cells were imaged, and colonies larger than 50 μm were counted, after which the area was calculated ( n = 8). n.s. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA followed by Tukey’s test

Article Snippet: PLK1 (A2548) antibodies were purchased from ABclonal (Wuhan, China). p65 (8242S), p-p65 (3033S), Erk1/2 (4695S), p-Erk1/2 (4370S), p21 (2947S), p27 (3686S), p38 (9212S), p-p38 (4511S), Akt (9272S), p-Akt (4060S), K63-linked specific Ub (5621S), K48-linked specific Ub (8081S), and Ub (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, shRNA, Control, CCK-8 Assay, Colony Assay, Staining, Soft Agar Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: TRIM47-facilitated PLK1 stabilization promotes the proliferation of liver cancer cells

doi: 10.1007/s13402-025-01130-0

Figure Lengend Snippet: PLK1 inhibition attenuated the oncogenic effects of TRIM47 in vivo. A. Scheme of the xenograft model experiment. First, Huh7 cells stably expressing the empty vector (EV) or TRIM47 (2 × 10 6 ) were subcutaneously injected into male BALB/c-nu mice. After the tumors reached 100 mm 3 , the mice were randomly divided into two groups ( n = 6 for each group) and treated i.p. with control vehicle (DMSO) or the PLK1 inhibitor GSK461364 (25 mg/kg) at the same dose. Treatments were administered every 3 days for 3 doses. B-E. Overexpression of TRIM47 promoted the growth of Huh7 cells in a xenograft model, whereas a PLK1 inhibitor attenuated the oncogenic effect of TRIM47. 14 days after injection, the tumor volume C . was measured every 3 days, and the animals were sacrificed on the 26th day. Images of the tumors are shown in B , the tumor volume and weight data are listed in D and E ( n = 6). F. The expression of proliferation-, apoptosis- and cyclin-related proteins in tumor tissues was detected via western blotting. G. Statistical analysis of the data in panel F ( n = 6). H. Tumors were isolated and fixed, and relevant indicators were detected via HE and IHC. n.s. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA followed by Tukey’s test

Article Snippet: PLK1 (A2548) antibodies were purchased from ABclonal (Wuhan, China). p65 (8242S), p-p65 (3033S), Erk1/2 (4695S), p-Erk1/2 (4370S), p21 (2947S), p27 (3686S), p38 (9212S), p-p38 (4511S), Akt (9272S), p-Akt (4060S), K63-linked specific Ub (5621S), K48-linked specific Ub (8081S), and Ub (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Control, Over Expression, Western Blot, Isolation

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: TRIM47-facilitated PLK1 stabilization promotes the proliferation of liver cancer cells

doi: 10.1007/s13402-025-01130-0

Figure Lengend Snippet: Working model. The expression of TRIM47 is up-regulated in LC cells, which increases the stability of PLK1 by ubiquitin modification and then phosphorylates p65 and p38 to promote their nuclear regulation, which leads to the reduction of cell apoptosis and shortening of cell cycle. Meanwhile, TRIM47 can promote the degradation of p53 and be involved in the regulation of cell apoptosis. Thus, the proliferation ability of LC cells was enhanced

Article Snippet: PLK1 (A2548) antibodies were purchased from ABclonal (Wuhan, China). p65 (8242S), p-p65 (3033S), Erk1/2 (4695S), p-Erk1/2 (4370S), p21 (2947S), p27 (3686S), p38 (9212S), p-p38 (4511S), Akt (9272S), p-Akt (4060S), K63-linked specific Ub (5621S), K48-linked specific Ub (8081S), and Ub (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Ubiquitin Proteomics, Modification