pcdna3 ha gsk3β  (Addgene inc)

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of STARD3. The MENTAL domain in blue presents four transmembrane helices (dark blue). The sequence of the phospho-FFAT motif is highlighted in a red box. The four serine residues phosphorylated within and around the FFAT motif are numbered. The consensus sequence for the GSK3 recognition site is also shown. B-D: HA-tagged GSK3P (green) was expressed alone (B) or together with WT (C) and S209A mutant (D) STARD3 (magenta) in MCF7 cells. GSK3P and STARD3 were labeled with anti-HA and anti-STARD3 antibodies, respectively. The subpanels are higher magnification images of the area outlined in white. The overlay panel shows merged images of green, magenta and blue (nuclei labelled with Hoechst-33258) images. Scale bars: 1O µm. Inset scale bars: 2 µm. E: Pearson’s correlation coefficients between GSK3P-HA and STARD3 (WT or S209A) staining. Data are displayed as Superplots with Pearson’s correlation coefficient for individual cells (small dots) and the mean per independent experiment (large dots). Number of cells: GSK3P-HA-STARD3: 29; GSK3P-HA-STARD3 S209A: 33, from three independent experiments. Independent experiments are color-coded. Mean values with error bars (SD) are shown. Unpaired t-test (**P < 0.01). F, H: Western blot analysis of HCC1954 (G) and MCF7/STARD3 (H) cells treated or not with the GSK3 inhibitor CHIR99021 (5 µM; overnight). STARD3 protein level (Total) and S209 phosphorylation (pS209) were analyzed. Right: quantification of relative S209 phosphorylation levels. Means± SD. Student’s t-test (***, P < 0.001; n = 3 independent experiments). G: Western blot analysis of control MCF7 cells (left) and MCF7 cells stably overexpressing STARD3 (right). 1-J: Western blot analysis of HCC1954 (I) and MCF7/STARD3 (J) cells transfected with control siRNAs (siCtrl) or siRNAs targeting GSK3a (siGSK3a), GSK3p (siGSK3p), or both (siGSK3a + siGSK3a). Bottom: quantification of relative S209 phosphorylation levels. Means± SD. One-way ANOVA with Dunnett’s multiple comparison test(*, P < 0.05; **, P < 0.01; ***, P < 0,001, n = 3 independent experiments).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Sequencing, Mutagenesis, Labeling, Staining, Western Blot, Phospho-proteomics, Control, Stable Transfection, Transfection, Comparison

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Western blot analysis of MCF? cells overexpressing TIP60 treated or not with the GSK3 inhibitor CHIR99021 (5 µM; overnight). TIP60 protein levels (Total) and S586 phosphorylation (pS586) were analyzed. B: Western blot analysis of Hela/STARD3 cells treated overnight with different concentration of CHIR99021 (1, 2 and 5 µM) or left untreated. STARD3 protein level (Total) and S209 phosphorylation (pS209) were analyzed. C: (a) Western blot analysis of HCC1954 cells treated with 5 µM of CHIR99021 for 2, 4, 6, 8 and 16 h, or left untreated. STARD3 protein levels (Total) and S209 phosphorylation (pS209) were analyzed. (b) Quantification of relative S209 phosphorylation levels. Means± SD. One-way ANOVA with Dunnett’s multiple comparison test(**, P < 0.01; n = 3 independent experiments). (c) Representative images of HCC1954 cells expressing WT STARD3 and treated with the GSK3 inhibitor CHIR99021 for 2, 4, 6, 8 and 16 h, or left untreated. Cells were labelled with an anti-LAMP1 antibody (magenta), an anti-STARD3 antibody (green) and Hoescht-33258 for nuclei (blue).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Western Blot, Phospho-proteomics, Concentration Assay, Comparison, Expressing

Journal: Cells

Article Title: Interleukin 24 Promotes Mitochondrial Dysfunction, Glucose Regulation, and Apoptosis by Inactivating Glycogen Synthase Kinase 3 Beta in Human Prostate Cancer Cells.

doi: 10.3390/cells14050357

Figure Lengend Snippet: Figure 2. IL-24 inactivates GSK3β kinase in prostate cancer cells in a dosage-dependent manner. (A) DU145 cells were treated with Ad.IL-24 (50, 100, or 200 pfu per cell) or Ad.vector (200 pfu per cell). Cells were collected, protein extracts were purified, and western blot analysis was performed to detect phospho-GSK3β (serine 9) and β actin proteins. (B) Cells were treated with Ad.IL-24 (200 pfu per cell) or Ad.vector (200 pfu per cell) or control, and the GSK3β activity was determined. An average of three independent experiments is shown ± SD; **** p < 0.0001. Numbers represent the ratio of specific treatments to values in control cells.

Article Snippet: After reaching 60–70% confluence, media was replaced with Opti-MEM (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and cells were transfected with either plasmids encoding wild-type GSK3β (Addgene, Watertown, MA, USA, #14753), or GSK3β S9A (Addgene, Watertown, MA, USA, #14754) using Lipofectamine 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Purification, Western Blot, Control, Activity Assay

Journal: Cells

Article Title: Interleukin 24 Promotes Mitochondrial Dysfunction, Glucose Regulation, and Apoptosis by Inactivating Glycogen Synthase Kinase 3 Beta in Human Prostate Cancer Cells.

doi: 10.3390/cells14050357

Figure Lengend Snippet: Figure 5. IL-24-dependent phosphorylation of GSK3β is necessary to mediate cell death in DU145 prostate cancer cells. (A) DU145 cells were treated with Ad.IL-24, or Ad.vector, in cells that expressed constitutively active GSK3β protein, and cell viability was determined by the colorimetric prolifera- tion assay five days after treatment. An average of three independent experiments is shown ± SD, *** p < 0.001 and **** p < 0.0001 compared to the control. (B) Cells were treated as described in A and assayed for cell death using Annexin V staining to measure apoptosis 48 h later by FACS analysis using the CellQuest software (Becton Dickinson). An average of three independent experiments is shown ± SD, * p < 0.05 and *** p < 0.001 compared to the control. (C) Cells were treated as described in B, and the western blot determined the cleaved caspase 3.

Article Snippet: After reaching 60–70% confluence, media was replaced with Opti-MEM (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and cells were transfected with either plasmids encoding wild-type GSK3β (Addgene, Watertown, MA, USA, #14753), or GSK3β S9A (Addgene, Watertown, MA, USA, #14754) using Lipofectamine 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Plasmid Preparation, Control, Staining, Software, Western Blot

Journal: Cells

Article Title: Interleukin 24 Promotes Mitochondrial Dysfunction, Glucose Regulation, and Apoptosis by Inactivating Glycogen Synthase Kinase 3 Beta in Human Prostate Cancer Cells.

doi: 10.3390/cells14050357

Figure Lengend Snippet: Figure 6. IL-24-dependent phosphorylation of GSK3β is necessary to mediate mitochondrial dys- function in DU145 prostate cancer cells. (A) DU145 cells were treated with Ad.IL-24, or Ad.vector, in cells that expressed constitutively active GSK3β protein, and mitochondrial membrane potential was analyzed by monitoring TMRE fluorescence intensity 72 h after treatment. An average of three independent experiments is shown ± SD, with significance levels indicated as **** p < 0.0001. (B) Cells were treated as described in (A), cells were collected, protein extracts were purified, and subjected to western blot analysis to detect Bax and β actin proteins. (C) Cells were treated as described in (A), and the glucose levels were determined. An average of three independent experiments is shown ± SD; Numbers represent the ratio of specific treatments to values in control cells; ns: not significant; ** p < 0.01.

Article Snippet: After reaching 60–70% confluence, media was replaced with Opti-MEM (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and cells were transfected with either plasmids encoding wild-type GSK3β (Addgene, Watertown, MA, USA, #14753), or GSK3β S9A (Addgene, Watertown, MA, USA, #14754) using Lipofectamine 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Plasmid Preparation, Membrane, Fluorescence, Purification, Western Blot, Control