length wild type rasal2  (Proteintech)

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 1 Residual tumours, but not adjacent normal tissues, are enriched with RASAL2. A UMAP plots of epithelial cells from primary TNBC tumours showing correlation between CytoTRACE score (1—least differentiation, 0 – most differentiation) and RASAL2 expression. B Violin plots of RASAL2 expression and residual tumour signature expression in the four clusters of epithelial cells identified in the TNBC tumours in (A). Cluster 1 expressions are significantly higher compared to all other clusters. Data are represented as mean ± SEM. P value by one-way ANOVA. C Heatmap of RASAL2 expression in pre- and post-treatment breast cancer patients [19]. Fold change (FC) was determined relative to pre-treatment expression level. P value by paired T-test. D Dot plots of RASAL2 expression in pre- versus post-treatment TNBC/BRCA-mutant breast cancer patients [20]. Probes that recognise RASAL2 variant 2 (ILMN_1813701 and ILMN_1673455) show significant increase following treatment. Data are represented as mean ± SEM. P value by two-tailed T-test. E Immunohistochemistry of fixed post-treatment TNBC patient breast specimens. RASAL2 (brown stain) was enriched in the tumour compartment versus adjacent normal epithelia. Data are represented as mean ± SEM. P value by two-tailed T-test. Scale bar, 20 µm. F Immunoblotting of fresh post-treatment TNBC patient specimens. RASAL2 was enriched in the tumour (T) versus adjacent normal (N) tissues in TNBC patients. LE long exposure.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Mutagenesis, Variant Assay, Two Tailed Test, Immunohistochemistry, Staining, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 2 RASAL2 promotes pan-resistance to cytotoxic agents beyond platinum. A Correlation between RASAL2 expression and sensitivity to indicated chemotherapy. The area under percent-viability curves (AUC) was computed as a metric of drug sensitivity, as derived from the Cancer Therapeutics Response Portal. Pearson r and P value are reported. B Cell viability assay. Vector control and RASAL2-overexpressing MDA-MB-468 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. C Spheroid assay. Viability of vector control and RASAL2-overexpressing TNBC spheroids was measured following treatment with vehicle DMSO, doxorubicin (DOXO) or gemcitabine (GEM). Representative images of HCC1806 spheroids are shown. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. Scale bar, 250 µm. Cell viability assay. Vector control and RASAL2- overexpressing (D) or -knockdown (E) HCC1937 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T-test. F Correlation between RASAL2 expression and in vivo tumour response to doxorubicin. Mice were treated with vehicle control or 2 mg/kg doxorubicin [22]. Each dot represents an independent TNBC PDX model. Tumour growth inhibition was defined as [1 −(mean volume of treated tumours)/(mean volume of control tumours)] × 100%. Pearson r and P value are reported. G Change in tumour volume following doxorubicin in two TNBC PDX models. Mice were treated as described in (F), n = 8–9 per group. TM00099 tumours had the lowest RASAL2 expression, whereas TM01278 had the highest RASAL2 expression. P value by two-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Derivative Assay, Viability Assay, Plasmid Preparation, Control, Knockdown, In Vivo, Inhibition

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 4 Transcription factor CREB1 drives RASAL2 and BCL2 expression. A Venn diagram showing the overlapped predicted transcription factors binding on the promoters of RASAL2 and BCL2 using computational tools, JASPAR and LASAGNA. B Analyses of candidate transcription factors in breast cancer patient cohorts. Heatmap shows the fold changes of RASAL2, BCL2 and candidate gene transcription factors in patient-matched breast tumour specimens post- versus pre-treatment (top, [19]). Correlation between RASAL2/BCL2 expression and candidate transcription factors in TNBC patients in the TCGA-BRCA cohort (bottom). P value by two-sided Pearson correlation analysis. C Consensus binding motifs of transcription factor CREB1. D Decrease in the relative mRNA expression of RASAL2 and BCL2 following siRNA- mediated knockdown of CREB1 in MDA-MB-468 cells. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T-test. E Immunoblotting of TNBC cells transfected with siCREB1 or control siRNA. CREB1, RASAL2 and BCL2 were decreased in expression in cells treated with siCREB1 compared to control. F ChIP-qPCR confirmation of CREB1 binding to predicted sites on RASAL2 and BCL2 promoters. TSS denotes transcription start site. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T-test. G Decrease in the relative luciferase units in siCREB1 MDA-MB-468 cells compared to siControl. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-way ANOVA. H Decrease in the relative luciferase units in MDA-MB-468 cells with truncated RASAL2 promoter without CREB1-binding sequence compared to those with wild-type (WT) RASAL2 promoter. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by one-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Binding Assay, Knockdown, Two Tailed Test, Western Blot, Transfection, Control, ChIP-qPCR, Luciferase, Plasmid Preparation, Sequencing

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 5 Mitochondria is a common homing site for BCL2 and RASAL2. A Immunofluorescence of BCL2 and RASAL2 in primary TNBC patient tumour. Scale bar, 20 µm. B Immunofluorescence of BCL2 and RASAL2 in TNBC cells. Bottom graph shows the line scan quantification of BCL2 (red) and RASAL2 (green). Scale bar, 30 µm. C Confocal imaging of BCL2 and RASAL2 in TNBC cells. Panels on the left show exemplary co-localisation of signals within the boxed region of the cell. Scale bar, 10 µm. D AlphaFold prediction of the interaction between BCL2 and the N-terminus of RASAL2. pLDDT score (0–100) is a confidence score, and pTM score (0–1) is a metric for the structural congruency between two folded protein structures, with higher scores corresponding to higher confidence. PAE plot of the top ranked model is shown on the right [33, 34]. E Co-immunoprecipitation of BCL2 and RASAL2 in MDA-MB-468 cells. F Immunoblotting of cytoplasmic versus mitochondrial fractions of mammary cell lines. BCL2 was not detected in 4T1 murine cells as the antibody used was reactive only to human. AKT and TOM20 serve as cytoplasmic and mitochondrial markers, respectively. G Confocal imaging of RASAL2 and MitoTracker in TNBC cells. Scale bar, 5 µm.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Imaging, Immunoprecipitation, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 6 BCL2 upregulation attenuate mitochondrial depolarisation by attenuating BAX oligomerisation. A Live-cell imaging. RASAL2 depletion increases the rate of GFP-BAX accumulation (green) in HCC1937 cells following exposure to 20 µM staurosporine. Number denotes time in seconds. Scale bar, 10 µm. B Quantification of change in BAX intensity. Fluorescence intensity of individual BAX foci was tracked over time and quantified, n = 4 foci per condition. Data are represented as mean ± SEM. P value by two-tailed T-test. C Schematic for live mitochondrial outer membrane permeabilisation (MOMP) assay. D MOMP assays revealing attenuated cytochrome c release in RASAL2-overexpressing TNBC cells. TOM20 serves as mitochondrial marker. E JC-1 mitochondrial membrane potential assay. TNBC cells were treated with vehicle DMSO or 5 µM doxorubicin (DOXO), and subsequently stained with JC-1 reagent. JC-1 aggregates (indicating high mitochondrial membrane potential) were observed as red, while JC-1 monomers (indicating low mitochondrial membrane potential) were green. Representative images of vector control and RASAL2-overexpressing TNBC cells are shown. Scale bar, 100 µm. F Quantification of the ratio of integrated intensity of red to green fluorescence in (E). Data are represented as mean ± SEM, n = 5 random fields of view per condition. P value by two-tailed T-test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Live Cell Imaging, Fluorescence, Two Tailed Test, Membrane, Marker, Staining, Plasmid Preparation, Control

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance.

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: Fig. 7 Mechanism of apoptotic regulation by the CREB1-RASAL2-BCL2 axis. RASAL2 and BCL2 share common transcription factor motifs in their promoter regions. Transcription factor CREB1 binds to these promoter regions, and drives the expression of RASAL2 and BCL2. This upregulation is supported by CREB1-interactor YAP, a transcription co-factor that is regulated by RASAL2, thus forming a positive loop in the CREB1-RASAL2-BCL2 axis. Both RASAL2 and BCL2 colocalise at the mitochondria. Their presence confers mitochondrial resilience by mitigating mitochondrial outer membrane depolarisation, which occurs, for example, during BAX/tBID-triggered apoptosis. Consequently, in high RASAL2/BCL2 chemoresistant tumour cells, there is reduced cytochrome c release upon apoptosis induction and thereby attenuation of cell death.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Membrane