Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 3 A representative western blot of the siRNA transfection experiment. A The blot of siRNA BAG2 and MDK, C the blot of siRNA MAD2L1. β-Tubulin was used as a loading control and GAPDH was the positive control of the siRNA transfection. As shown in the blots and also in graphs (B) and (D) there was a significant reduction of the protein level in the silenced cells, as compared to the negative CTRL. E Graph showing the comparison of protein expression in MeT-5A and MSTO cell lines of BAG2, MAD2L1, and MDK (Mann–Whitney test; p value < 0.05 were considered significant, p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****)).

Article Snippet: The membranes were blocked with 5% milk in TBST and probed overnight at 4 °C with the specific rabbit polyclonal antibodies (Abs) against BAG2 (cod A304-751A-T, 1:1000; Bethyl Laboratories Inc., Montgomery, TX, USA), MAD2L1 (cod 10337-1-AP, 1:800), Midkine (cod 28546-1- AP, 1:500), β-tubulin (cod 10068-1-AP, 1:8000), as well as with the mouse monoclonal Abs against epidermal growth factor receptor (EGFR) (cod 18986-1-A, P, 1:7000) and GAPDH (cod 60004-1-Ig, 1:10,000) (all from Proteintech, Rosemont, IL, USA). β-tubulin was used as the index of protein loading in the lanes.

Techniques: Western Blot, Transfection, Control, Positive Control, Comparison, Expressing, MANN-WHITNEY

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 4 Viability, proliferation and caspase assays after gene silencing. A AOC of MeT-5A and MSTO at 72 h after treatment with siRNA- negative-control (siRNA CTRL−), siRNA BAG2, siRNA MAD2L1, siRNA MDK. The AOC is normalized to the time of transfection (t0). B Overall, FI of MeT-5A and MSTO was measured at 72 h after silencing; the graphs show the comparison between MeT-5A and MSTO for the three-siRNA transfection. ANOVA test; p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). C–E End point of apoptosis analysis in MSTO and MeT-5A cells in the presence of 5 µM caspase 3/7 Green Dye and siRNA BAG2, siRNA MAD2L1, and siRNA MDK, respectively. Mann–Whitney test; p value 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).

Article Snippet: The membranes were blocked with 5% milk in TBST and probed overnight at 4 °C with the specific rabbit polyclonal antibodies (Abs) against BAG2 (cod A304-751A-T, 1:1000; Bethyl Laboratories Inc., Montgomery, TX, USA), MAD2L1 (cod 10337-1-AP, 1:800), Midkine (cod 28546-1- AP, 1:500), β-tubulin (cod 10068-1-AP, 1:8000), as well as with the mouse monoclonal Abs against epidermal growth factor receptor (EGFR) (cod 18986-1-A, P, 1:7000) and GAPDH (cod 60004-1-Ig, 1:10,000) (all from Proteintech, Rosemont, IL, USA). β-tubulin was used as the index of protein loading in the lanes.

Techniques: Negative Control, Transfection, Comparison, MANN-WHITNEY

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 7 Representative immunohistochemical staining of MPM samples showing intense expression of MDK, MAD2L1, and BAG2 proteins in tumor cells (brown staining). A–D Representative BAG2 immunostainings in RMP, EMM, BMM, and SMM, respectively. Moderate to strong expression in the three types of MPM, while no expression is visible in the hyperplastic mesothelium in RMP (red arrow). E–H Representative MAD2L1 immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is present in the three types of MPM, but there are areas of discernable expression in the mesothelial cells on the pleural surface in RMP as well (blue arrow positive mesothelial area, red arrow negative area). I–L Representative MDK immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is seen in the three types of MPM; however, some focal expression is also detectable in the mesothelial cells on the pleural surface in RMP (blue arrow focal staining, red arrow negative staining). Magnification: RMPs ×400, MPMs ×200.

Article Snippet: The membranes were blocked with 5% milk in TBST and probed overnight at 4 °C with the specific rabbit polyclonal antibodies (Abs) against BAG2 (cod A304-751A-T, 1:1000; Bethyl Laboratories Inc., Montgomery, TX, USA), MAD2L1 (cod 10337-1-AP, 1:800), Midkine (cod 28546-1- AP, 1:500), β-tubulin (cod 10068-1-AP, 1:8000), as well as with the mouse monoclonal Abs against epidermal growth factor receptor (EGFR) (cod 18986-1-A, P, 1:7000) and GAPDH (cod 60004-1-Ig, 1:10,000) (all from Proteintech, Rosemont, IL, USA). β-tubulin was used as the index of protein loading in the lanes.

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Staining

Journal: Cancer gene therapy

Article Title: BAG2, MAD2L1, and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma.

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Fig. 8 Quantification of protein expression in RMP vs MPM as assessed by IHC and H-score. Graphs for BAG2 immunostaining in RMP vs MPM regardless of subtype (A) and RMP vs the different subtypes of MPM (B), respectively. Graphs for MAD2L1 immunostaining in RMP vs MPM regardless of subtype (C) and RMP vs the different subtypes of MPM (D), respectively. Graphs for MDK immunostaining in RMP vs MPM regardless of subtype (E) and RMP vs the different subtypes of MPM (F), respectively. Kruskal–Wallis test followed by a post hoc Dunn’s test with Bonferroni correction was used to evaluate the statistical difference between the median of each group, p value <0.001(***), <0.01(**), <0.05(*).

Article Snippet: The membranes were blocked with 5% milk in TBST and probed overnight at 4 °C with the specific rabbit polyclonal antibodies (Abs) against BAG2 (cod A304-751A-T, 1:1000; Bethyl Laboratories Inc., Montgomery, TX, USA), MAD2L1 (cod 10337-1-AP, 1:800), Midkine (cod 28546-1- AP, 1:500), β-tubulin (cod 10068-1-AP, 1:8000), as well as with the mouse monoclonal Abs against epidermal growth factor receptor (EGFR) (cod 18986-1-A, P, 1:7000) and GAPDH (cod 60004-1-Ig, 1:10,000) (all from Proteintech, Rosemont, IL, USA). β-tubulin was used as the index of protein loading in the lanes.

Techniques: Expressing, Immunostaining

Journal: Cell reports

Article Title: The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization.

doi: 10.1016/j.celrep.2024.114689

Figure Lengend Snippet: Figure 3. BAG2 is a constitutive interaction partner of ULK1 modulating autophagy (A) BAG2 binds to ULK1 similarly to its complex members in AP-MS. Shown are normalized and log2-transformed SILAC ratios using N- and C-terminal-tagged ULK1 variants. GAPDH is depicted as negative control (n = 3 biological replicates each). Boxplots show spreads of ratios. Single values are highlighted as white dots. (B and C) (B) Co-immunoprecipitation (coIP) of SHA-tagged BAG2 and its interaction partners ULK1 and ATG13. Shown is one representative experiment of n = 3 biological replicates. Equal protein amounts per lane were loaded. Samples were run on one gel, and black lines indicate cropping of unrelated lanes; these data were also used to generate Figures 6B and 7D. (C) Quantification of n = 3 replicates. (D–F) BAG2 KO reduces autophagy flux. Shown is one representative blot of n = 3 biological replicates (D). Actin serves as loading control. Cells were starved in HBSS and/or treated with BafA1 for 3 h. Respective quantification of LC3-II and GABARAP-II bands relative to actin are shown in (E). Red dotted lines indicate protein levels in DMEM control conditions. Comparing BafA1-treated with non-treated lanes indicates reduced autophagy flux in KO compared to WT cells (F). (G–I) BAG2 KD increases autophagy flux. Shown is one representative blot of n = 3 biological replicates (G). Actin serves as loading control. Cells were starved in HBSS and/or treated with BafA1 for 3 h. Respective quantification of LC3-II and GABARAP-II bands are shown in (H). Red dotted lines indicate protein levels in DMEM control conditions. Comparing BafA1-treated with non-treated lanes indicates increased autophagy flux in KD compared to control cells (I). Student‘s t test, unpaired; *p < 0.05, **p < 0.01, ***p < 0.001. Single values are highlighted as white dots. Error bars indicate 95% confidence interval.

Article Snippet: In addition to those mentioned above, the following primary antibodies were used only in IF experiments: BAG2 (Novus Biologicals, NBP2-59476, mouse), ERGIC53 (Enzo, ENZ-ABS300-0100, mouse), SNX1 (BD Biosciences, 611482, mouse).

Techniques: Protein-Protein interactions, Transformation Assay, Multiplex sample analysis, Negative Control, Immunoprecipitation, Control