plko 1 puro constructs  (Addgene inc)

Journal: Clinical and Translational Medicine

Article Title: Targeting KIF23 inhibits cell proliferation and primary chemoresistance in cervical cancer by inactivating the MYH9/MCM2/PCNA pathway

doi: 10.1002/ctm2.70652

Figure Lengend Snippet: KIF23 and myosin heavy chain 9 (MYH9) interact with ubiquitin‐specific protease 7 (USP7). (A) Molecular docking results for KIF23 and MYH9. (B) Co‐immunoprecipitation (Co‐IP) analysis to detect the interaction between KIF23 and MYH9 in CC cells. (C) Immunofluorescence (IF) analysis showing the co‐localisation of KIF23 and MYH9 in CC cells (scale bar: 10 µm). (D) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis of MYH9 mRNA levels in CC cells with or without KIF23 KO. (E) Western blotting (WB) analysis of MYH9 protein levels in CC cells with or without KIF23 KO. (F) Nuclear and cytoplasmic fractionation assay to detect MYH9 protein levels in overexpression KIF23 (oe‐ KIF23 ) CC cells. (G) Schematic diagram of the functional domains of KIF23. (H) Co‐IP assay demonstrating the interaction between different KIF23 domains and MYH9 in HEK‐293T cells transfected with the corresponding constructs. (I) Schematic diagram of the functional domains of MYH9. (J) Co‐IP assay demonstrating the interaction between different MYH9 domains and KIF23 in HEK‐293T cells. (K and L) Molecular docking diagrams of MYH9 with USP7 and KIF23 with USP7. (M) Co‐IP analysis in CC cells demonstrating the interactions of MYH9 and KIF23 with USP7. (N and O) Representative confocal images showing the co‐localisation of MYH9 with USP7 and KIF23 with USP7 (scale bar: 10 µm).

Article Snippet: CC cells were transfected with plasmid constructs encoding USP7 (Miaoling Biology), MYH9 (Vigene Biosciences), MCM2 (Miaoling Biology) and USP15 (Miaoling Biology) using Lipofectamine 3000 (model L2000‐015; Invitrogen) as per the manufacturer's protocols.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Fractionation, Over Expression, Functional Assay, Transfection, Construct

Journal: Clinical and Translational Medicine

Article Title: Targeting KIF23 inhibits cell proliferation and primary chemoresistance in cervical cancer by inactivating the MYH9/MCM2/PCNA pathway

doi: 10.1002/ctm2.70652

Figure Lengend Snippet: KIF23 induces K48‐linked deubiquitination of MYH9 through recruitment of USP7. (A) Western blotting (WB) analysis of MYH9 protein stability at different time points following cycloheximide (CHX) treatment in control and KIF23 KO groups. (B) CHX chase assay examining the regulation of MYH9 protein stability by USP7 overexpression (oe‐ USP7 ). (C) Effect of MG132 treatment (12 h) on MYH9 protein stability in KIF23 KO CC cells and their controls. (D) Co‐IP and WB analyses of the effect of KIF23 KO on MYH9 ubiquitination levels after 12 h of MG132 treatment. (E) Co‐IP and WB assays assessing the effects of control, KIF23 KO and KIF23 KO combined with USP7 overexpression (KO‐ KIF23 + oe‐ USP7 ) on MYH9 ubiquitination after 12 h of MG132 treatment in SIHA (left) and C33A (right) cells.

Article Snippet: CC cells were transfected with plasmid constructs encoding USP7 (Miaoling Biology), MYH9 (Vigene Biosciences), MCM2 (Miaoling Biology) and USP15 (Miaoling Biology) using Lipofectamine 3000 (model L2000‐015; Invitrogen) as per the manufacturer's protocols.

Techniques: Western Blot, Control, Over Expression, Co-Immunoprecipitation Assay, Ubiquitin Proteomics