plko.1-p53-shrna vector  (GenScript corporation)

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Knockdown of DNAJA1 specifically reduces protein levels of conformational mutp53, but not DNA contact mutp53 and wtp53. ( A , B ) Western blotting for p53, DNAJA1, and GAPDH ( A ) and immunofluorescence for p53, DNAJA1, and DAPI ( B ) using multiple cancer cells with different p53 status with or without knockdown of DNAJA1 (JA1) and p53 (p53). Scale bar: 50 µm.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Knockdown, Western Blot, Immunofluorescence

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Identification of a compound that binds DNAJA1 to specifically reduce conformational mutp53. ( A ) Chemical structure of PLIHZ compound, derived from plumbagin, which was identified through a molecular docking ( left ). Ribbon and crystal structures of DNAJA1 protein (PDB: 2LOI) showing binding of DNAJA1 to PLIHZ at tyrosine residue Y7 ( right ) with a 2.6 Å bond distance and binding free energy of −6.6 kcal/mol. ( B ) CETSA showing intracellular binding of PLIHZ to DNAJA1. A representative Western blotting for DNAJA1 and GAPDH using protein extracts from CAL33 (p53 R175H ) cells with treatment with DMSO and PLIHZ at 80 µM for 4 h, followed by incubation at different temperatures for 3 min ( left ). A representative blot using protein extracts from unheated cells are also shown. A summarized graph showing normalized DNAJA1 band densities at different temperatures of 40, 43, 46, 49, 52, 55, and 58 °C ( right ). Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-way ANOVA. ( C ) Western blotting for p53, DNAJA1, and GAPDH using indicated cells with different p53 status, treated with DMSO or PLIHZ at ~1/2 of 24h-IC50 for 24 h. ( D ) Immunofluorescence for p53, DNAJA1, and DAPI using indicated cells treated with DMSO or PLIHZ at ~1/2 of 24h-IC50 for 24 h.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Derivative Assay, Binding Assay, Residue, Western Blot, Incubation, Immunofluorescence

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: PLTFBH, an analog of PLIHZ, specifically reduces conformational mutp53 levels similar to PLIHZ. ( A ) Chemical structures of PLIHZ analogs including PLIHZ, PLFBH, PLTFBH, PLFUH, and PLOCT. ( B ) Western blotting for p53 and GAPDH using HN31 cells treated with different PLIHZ analogs (40 µM for 24 h). ( C ) Western blotting for p53, DNAJA1, and GAPDH using HN31, MDA-MB-231, and U2OS cells treated with PLTFBH at ~1/2 of 24h-IC 50 . ( D ) Immunofluorescence for p53, DNAJA1, and DAPI using HN31, MDA-MB-231, U2OS, and H1299 cells treated with PLTFBH at ~1/2 of 24h-IC 50 . Scale bar: 50 µm. ( E ) CETSA showing intracellular binding of PLTFBH to DNAJA1 in CAL33 cells: a representative Western blotting for DNAJA1 and GAPDH using protein extracts from HN31 cells ( left ); a summarized graph showing normalized DNAJA1 band densities at different temperatures ( right ). Mean ± SEM from three independent experiments ( n = 3). * p < 0.05 for two-way ANOVA. ( F ) Western blotting for p53, DNAJA1, and GAPDH using HN31 cells with or without knockout for DNAJA1 ( JA1 ) or p53 ( p53 ) using the CRISPR-Cas9 strategy. ( G ) Summary of MTT assays using control, DNAJA1 knockout, or p53 knockout HN31 treated with different concentrations of PLTFBH for 72 h. Mean ± SEM from three independent experiments ( n = 3). n.s.: not significant for one-way ANOVA. IC 50 values of PLTFBH for each sub-cell line are shown on the right.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Western Blot, Immunofluorescence, Binding Assay, Knock-Out, CRISPR, Control

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: PLTFBH inhibits migratory potential of cancer cells in a manner dependent on DNAJA1 and conformational mutp53. ( A ) Transwell migration assays using indicated cells with different p53 status treated with PLTFBH at ~1/2 IC 50 for 12 h. All cells were pre-treated with PLTFBH for 12 h, followed by trypan blue staining and transwell migration assays. Top : summarized graphs. Bottom : representative images. Mean ± SEM from three independent experiments ( n = 3). *** p < 0.001 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 100 µm. ( B ) F-actin staining showing inhibition of filopodia formation in HN31 cells, but not MDA-MB-231, U2OS, and H1299 cells, by PLTFBH (TF). Top: summarized graph. Bottom : representative images. Mean ± SEM from three independent experiments ( n = 3). **** p < 0.0001 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 10 µm. ( C ) Transwell migration assays using DNAJA1- or p53 -knockdown HN31 cells treated with PLTFBH at ~1/2 IC 50 for 12 h. Cells were pre-treated with PLTFBH for 12 h. Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 100 µm. ( D ) Rac1/Cdc42 activation assays following pulldown of active Rac1 and Cdc42 using protein extracts from HN31 cells treated with DMSO (D) or PLTFBH (TF) at ~1/2 of 24h-IC 50 . Left : representative immunoblots. Right : summarized graph. Mean ± SEM from three independent experiments ( n = 3). * p < 0.05, *** p < 0.001 for two-tailed Student’s t -test.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Migration, Staining, Two Tailed Test, Inhibition, Knockdown, Activation Assay, Western Blot

Journal: Cancers

Article Title: Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

doi: 10.3390/cancers14174187

Figure Lengend Snippet: Mutations at Y7 and Y8 residues in DNAJA1 abrogate the ability of PLTFBH to deplete DNAJA1 and conformational mutp53. ( A ) Western blotting to detect exogenous DNAJA1 and GAPDH, as well as endogenous p53 (p53 C176F ), using DNAJA1 -knockout HN31 cells (JA1KO) expressing exogenous wild-type (wt), Y7A mutant (Y7A), and Y8A mutant (Y8A) DNAJA1, treated with DMSO (D) or PLTFBH (TF) at ~1/2 IC 50 for 24 h. ( B ) Immunofluorescence for p53, DNAJA1, and DAPI using the same experimental set of HN31 sub-cell lines as in A. Scale bar: 50 µm. ( C ) F-actin staining using the HN31 sub-cell lines treated with DMSO or PLTFBH at ~1/2 IC 50 for 24 h. Mean ± SEM from three independent experiments ( n = 3). ** p < 0.01 for two-tailed Student’s t -test. n.s.: not significant. Scale bar: 10 µm. Left : representative images. Right : summarized graph.

Article Snippet: A lentiviral vector encoding a DNAJA1 shRNA (TRCN0000275847, Clone ID: NM_001539.2-1078s21c) was purchased from Sigma-Aldrich, Inc and a lentiviral vector encoding p53 shRNA (shp53 pLKO.1 puro, #19119) was purchased from Addgene (Watertown, MA, USA), while pGIPz control encoding-vectors were purchased from Open Biosystems, Inc. (Huntsville, AL, USA).

Techniques: Western Blot, Knock-Out, Expressing, Mutagenesis, Immunofluorescence, Staining, Two Tailed Test