atcc 23943  (ATCC)

Journal: BMC Cancer

Article Title: Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells

doi: 10.1186/1471-2407-14-52

Figure Lengend Snippet: Regulation of AR protein level and cell growth via the MID1-α4/PP2A transcriptional regulator complex. MID1, α4 and PP2A form the core of a ribonuclear protein complex that enhances translation of associated mRNAs such as AR mRNA. Physical interaction of the complex components was confirmed in LNCaP cells overexpressing a flag-tagged MID1. MID1 and PP2A were co-precipitated in an α4 pull-down (α4); normal rabbit IgG was used as negative control (IgG). A representative western blot of two independent experiments is shown in (A) . Disruption of the complex by MID1 or α4 protein knockdown mimics the effect of metformin. MID1 knockdown in LNCaP and LNCaP-abl cells or α4 in LNCaP cells resulted in a reduction of AR protein levels (B) and the inhibition of cell growth (C) . In the AR-negative PC3 cells, MID1 overexpression enhanced, whereas MID1 knockdown inhibited cell growth (D) . Successful knockdown or overexpression, respectively, was verified by western blot; inserts show representative fluoroscans (B, D) . Luciferase (Luci) siRNA was used as negative control for siRNA transfections, empty vector for overexpression control. Cell numbers were determined after 10 days. Statistical significant differences are indicated as *, p < 0.05; **, p = <0.01 and ***, p < 0.001.

Article Snippet: After incubation with α4 antibody or rabbit control IgG (Santa Cruz, Dallas, TX, USA) overnight, the antigen-antibody complexes were immunoprecipitated with pansorbin cells.

Techniques: Negative Control, Western Blot, Inhibition, Over Expression, Luciferase, Transfection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells

doi: 10.1186/1471-2407-14-52

Figure Lengend Snippet: Metformin disrupts the MID1-α4/PP2A complex and releases associated AR mRNA. DuCaP or VCaP prostate cancer cells were treated with 5 mM of metformin or vehicle control for 24 h. Afterwards the MID1-α4/PP2A complex was immunoprecipitated using an α4 specific antibody. Normal rabbit IgG was used as negative control. Complex-associated RNA was isolated and transcribed to cDNA. Using PCR and real-time PCR amplification of an AR cDNA fragment containing the CAG-repeat region (A) or a fragment of the AR hormone-binding region (B) , respectively, were amplified. The agarose gel image (A) shows a representative PCR result of 3 independent experiments with DuCaP cells, the histogram (B) shows the relative real-time PCR AR fragment levels standardized to the input amount and normalized to the control for 3 independent experiments for DuCaP and VCaP cells. NeCo, negative control; Inpt, input. PCR primer and fragment information is provided in the supplementary data. Statistical significant differences are indicated as *, p < 0.05; **, p <0.01 and ***, p < 0.001.

Article Snippet: After incubation with α4 antibody or rabbit control IgG (Santa Cruz, Dallas, TX, USA) overnight, the antigen-antibody complexes were immunoprecipitated with pansorbin cells.

Techniques: Immunoprecipitation, Negative Control, Isolation, Real-time Polymerase Chain Reaction, Amplification, Binding Assay, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.3390/ijms23020584

Figure Lengend Snippet: DNAJC3 sensitizes targets to CADA. ( A ) Western blot images of cell lysates from non-transfected (NT) HEK293T cells, and huCD4-V5, ERLEC1-V5 or PTK7-V5, co-transfected with WT DNAJC3. Cells were treated for 24 h with different CADA concentrations and subjected to immunoblotting. Protein bands were visualized with an antibody against the V5 tag, and an antibody against clathrin (huCD4 and ERLEC1) or β-actin (PTK7) was used for the cell loading controls. An antibody against the FLAG tag was used to detect the co-transfected WT DNAJC3 in the samples. One representative experiment out of three to six is shown. The clathrin loading control shown is that of the ERLEC1 sample. The respective loading control for huCD4 and WT DNAJC3 is presented in . ( B ) Same as in ( A ) but for co-transfection with pPL-DNAJC3. One representative experiment out of three to six is shown. The clathrin loading control shown is that of the huCD4 sample. The respective loading control for ERLEC1 and pPL-DNAJC3 is presented in . ( C ) Concentration–response curves of CADA for huCD4, ERLEC1 and PTK7 in transfected HEK293T cells. Samples from ( A , B ) were quantified and normalised to the clathrin (huCD4 and ERLEC1) or β-actin (PTK7) internal control. A four-parameter concentration–response curve was fitted to data from at least three replicate experiments. Values are mean ± SD; n ≥ 3. Statistical analysis (multiple unpaired t -tests) showed significantly decreased expression of huCD4, ERLEC1 and PTK7 when co-expressed with pPL-DNAJC3 as compared to WT DNAJC3 (* = p < 0.05). HEK293T: human embryonic kidney 293T cells; huCD4: human CD4; ERLEC1: endoplasmic reticulum lectin 1; PTK7: inactive tyrosine-protein kinase 7; pPL: pre-prolactin; DNAJC3: DnaJ homolog subfamily C member 3; CADA: cyclotriazadisulfonamide.

Article Snippet: As described in a previous report [ ], the ERLEC1 expression vector (pGEM-T backbone) was purchased from Sino biological (Beijing, China), the PTK7 expression vector (pDONR223 backbone) from Addgene (Watertown, MA, USA) and the DNAJC3 expression vector (pDONR223 backbone) from the DNASU plasmid repository (Tempe, AZ, USA).

Techniques: Western Blot, Transfection, FLAG-tag, Cotransfection, Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.3390/ijms23020584

Figure Lengend Snippet: Inhibition of the proteasome in the presence of CADA rescues a preprotein fraction which is DNAJC3-dependent for PTK7. ( A ) Western blot images of cell lysates from non-transfected (NT) HEK293T cells, and huCD4-V5, ERLEC1-V5 or PTK7-V5, co-transfected with WT DNAJC3. Cells were treated for 24 h with different CADA concentrations and a constant dose of MG132 (200 nM). Protein bands were visualized with an antibody against the V5 tag, and an antibody against clathrin (huCD4 and ERLEC1) or β-actin (PTK7) was used for the cell loading controls. One representative experiment out of two to four is shown. The clathrin loading control shown is that of the ERLEC1 sample. The respective loading control for huCD4 is presented in . ( B ) Same as in ( A ) but for co-transfection with pPL-DNAJC3. One representative experiment out of two to four is shown. ( C ) Preprotein fraction of MG132-treated samples quantified from ( A , B ) and normalised to the internal loading control of the respective sample. Bars are mean ± SE; n = 2 for huCD4 and PTK7; n = 4 for ERLEC1. Statistical analysis (multiple unpaired t -tests) showed increased detection of the PTK7 preprotein when co-expressed with pPL-DNAJC3 as compared to WT DNAJC3 (* = p < 0.05). HEK293T: human embryonic kidney 293T cells; huCD4: human CD4; ERLEC1: endoplasmic reticulum lectin 1; PTK7: inactive tyrosine-protein kinase 7; pPL: pre-prolactin; DNAJC3: DnaJ homolog subfamily C member 3; CADA: cyclotriazadisulfonamide; DMSO: dimethyl sulfoxide.

Article Snippet: As described in a previous report [ ], the ERLEC1 expression vector (pGEM-T backbone) was purchased from Sino biological (Beijing, China), the PTK7 expression vector (pDONR223 backbone) from Addgene (Watertown, MA, USA) and the DNAJC3 expression vector (pDONR223 backbone) from the DNASU plasmid repository (Tempe, AZ, USA).

Techniques: Inhibition, Western Blot, Transfection, Cotransfection

Journal: International Journal of Molecular Sciences

Article Title: Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

doi: 10.3390/ijms23020584

Figure Lengend Snippet: DNAJC3 differentially affected BFP expression in the presence of CADA-stalled proteins. ( A ) Representation of the tGFP-P2A-BFP construct. The construct expressed huCD4 that was anchored in the plasma membrane via its transmembrane domain (TMD) and with tGFP at the cytosolic tail. As the SP was cleaved by the ER lumenal signal peptidase during protein biogenesis, the mature huCD4 variants differed in only 62 amino acids at their N-terminus. ( B ) Four-parameter concentration–response curves for the CADA of huCD4, ERLEC1, ERLEC1 with no SP, and PTK7 cloned in the same tGFP-P2A-BFP plasmid backbone as shown in ( A ). HEK293T cells were transiently transfected with the tGFP-P2A-BFP constructs and incubated with different CADA concentrations for 24 h. Transfected tGFP-P2A-BFP plasmid DNA was equal to the transfected tGFP-P2A-BFP plasmid DNA in the co-transfected conditions of ( C , D ). Protein levels of tGFP (representing the level of substrate) in CADA-treated samples were normalised to the DMSO control (set at 1.00). Curves were fitted to data from three to five replicate experiments. Values are mean ± SD; n ≥ 3. ( C ) Four-parameter concentration–response curves for CADA of the BFP signal of huCD4, ERLEC1 and PTK7 cloned in the same tGFP-P2A-BFP plasmid backbone as shown in ( A ). HEK293T cells were transiently transfected with the tGFP-P2A-BFP construct, or co-transfected with the tGFP-P2A-BFP construct and WT DNAJC3 or pPL-DNAJC3 and incubated with different CADA concentrations for 24 h. Transfected tGFP-P2A-BFP plasmid DNA was equal to the transfected tGFP-P2A-BFP plasmid DNA in the co-transfected conditions. BFP levels in CADA-treated samples were normalised to the DMSO control (set at 1.0). Curves were fitted to data from three to five replicate experiments. Values are mean ± SD; n ≥ 3. Statistical analysis (multiple unpaired t -tests) showed significantly increased expression of BFP for huCD4 and ERLEC1 when co-expressed with pPL-DNAJC3 and for ERLEC1 when co-expressed with WT DNAJC3 as compared to the control (* = p < 0.05). ( D ) Same as in ( C ). Four-parameter concentration–response curve for the CADA of the BFP signal of ERLEC1 with no SP cloned in the tGFP-P2A-BFP plasmid backbone. Curves were fitted to data from three replicate experiments. Values are mean ± SD; n = 3. tGFP: turbo green fluorescent protein; BFP: blue fluorescent protein; huCD4: human CD4; SP: signal peptide; CADA: cyclotriazadisulfonamide; ERLEC1: endoplasmic reticulum lectin 1; PTK7: inactive tyrosine-protein kinase 7; HEK293T: human embryonic kidney 293T cells; DMSO: dimethyl sulfoxide; pPL: pre-prolactin; DNAJC3: DnaJ homolog subfamily C member 3; TMD, transmembrane domain.

Article Snippet: As described in a previous report [ ], the ERLEC1 expression vector (pGEM-T backbone) was purchased from Sino biological (Beijing, China), the PTK7 expression vector (pDONR223 backbone) from Addgene (Watertown, MA, USA) and the DNAJC3 expression vector (pDONR223 backbone) from the DNASU plasmid repository (Tempe, AZ, USA).

Techniques: Expressing, Construct, Concentration Assay, Clone Assay, Plasmid Preparation, Transfection, Incubation