kras g12v vector  (Addgene inc)

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Protein levels of HRAS, KRAS, V5-tag (BRAF), pERK1/2, ERK1/2 and α-TUBULIN (loading control) in BJ-hTERT cells after oncogene induction for the times indicated. (B) Densitometry quantification of pERK1/2 levels, normalised to loading and control, after oncogene induction for the times indicated. N=3. (C) Nascent RNA synthesis after oncogene induction, measured by nuclear incorporation of EU (red) for 1 h. (D) Nuclear EU intensity after oncogene induction, normalised to control. N=4. (E) Slot blots of genomic DNA stained with S9.6 antibody (RNA:DNA hybrids) and double-stranded DNA (dsDNA; loading control) 48 h after oncogene induction. RNase H treatment was used to validate S9.6 antibody specificity. (F) RNA:DNA hybrid quantification as in E. N=4. (G) Relative median replication fork speeds after oncogene induction, normalised to control. (N=4-10). (H) Replication fork speeds after 48 h KRAS G12V induction with DRB or DMSO treatment in the last 1 h. Data from 1 repeat. (I) Percentages of cells containing more than five 53BP1 foci after oncogene induction. N=3. (J) Representative images of cells with micronuclei and percentages of cells with micronuclei after RAS induction for 1-7 days. N=3-4. (K) Percentages of senescent cells, measured by β-galactosidase staining, after oncogene induction. N=4. (L) Relative levels of reactive oxygen species (ROS) after oncogene induction. N=3-6. Means +/-SEM (bars) are shown with 1-way or 2-way ANOVA or mixed effects analysis. Scatter graphs show median (line).

Article Snippet: KRAS G12V vector was purchased from (Addgene, #35635).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Experimental setup for steady-state RNA sequencing. (B) Numbers of up- or downregulated genes (log2-fold change >1) after 24 or 48 h HRAS G12V or KRAS G12V induction. (C) Numbers of unique and shared up-regulated genes after HRAS G12V or KRAS G12V induction. (D) Functional enrichment analysis (gene ontology, biological process) of genes downregulated 48 h after HRAS G12V or KRAS G12V induction. (E) Functional enrichment analysis (gene ontology, biological process) of genes upregulated 48 h after HRAS G12V or KRAS G12V induction. (G) Log2 fold-change in hallmark E2F target gene expression after HRAS G12V or KRAS G12V induction. (H) E2F1 expression (RNAseq, DEseq2) after HRAS G12V or KRAS G12V induction. N=3. (I) Log2 fold-change in hallmark MYC target gene expression after HRAS G12V or KRAS G12V induction. (J) MYC expression (RNAseq, DEseq2) after HRAS G12V or KRAS G12V induction. N=3. (K) Protein levels of pRB1 and α-TUBULIN after oncogene induction for the times indicated. (L) S phase percentage after HRAS G12V induction as determined by EdU labelling and flow cytometry. N=4. (M) S phase percentage after KRAS G12V induction. N=4. (N) S phase percentage after BRAF V600E induction. N=4. (O) Experimental setup for CDK4/6 inhibitor (CDK4/6i) treatment and release. (P) S phase percentage after HRAS G12V induction and treatment with CDK4/6i. N=2. (Q) Nuclear EU intensity after HRAS G12V induction and release from CDK4/6i. N=3. (R) Average replication fork speeds after HRAS G12V induction and release from CDK4/6i. N=3. Means +/-SEM (bars) are shown with 2-way ANOVA or mixed effects analysis.

Article Snippet: KRAS G12V vector was purchased from (Addgene, #35635).

Techniques: RNA Sequencing, Functional Assay, Targeted Gene Expression, Expressing, RNA sequencing, Flow Cytometry

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Principle of chromatin RNAseq approach. (B) Numbers of nascent transcripts up-or downregulated (log 2 -fold change) 48 h after HRAS G12V or KRAS G12V induction. HRAS G12V : N=3, KRAS G12V : N=2. (C) Numbers of nascent transcripts upregulated after HRAS G12V or KRAS G12V induction as in (B) that have been reported to be preferentially transcribed in either early G1 (EG1), G1 or late S phase, G2 and mitosis (G2) . (D) Numbers of protein coding gene transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (E) Numbers of long noncoding (lncRNA) transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (F) Numbers of enhancer RNA (eRNA) transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (G) Numbers of histone gene transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (H) Functional enrichment analysis (REACTOME) of transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. Numbers of significant REACTOME terms were counted. (I) Numbers of small nucleolar RNA (snoRNA) transcripts up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (J) RNA polymerase I (RNAPI) is activated by oncogene signalling to generate 45S pre-ribosomal RNA for ribosome biogenesis. (K) RT-qPCR analysis of chromatin-associated 45S pre-RNA transcripts after 48 h oncogene induction. N=3-4. (L) RNA polymerase III (RNAPIII) is activated by oncogene signalling to generate non-coding RNAs for ribosome biogenesis and translation. (M) Numbers of transcripts in tRNA regions up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (N) Components of RNAPIII type 2 and type 3 promoters. (O) Numbers of RNA polymerase III transcripts with type 2 promoters up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. (P) Numbers of RNAPIII transcripts with type 3 promoters up- or downregulated 48 h after HRAS G12V or KRAS G12V induction. Means +/-SEM (bars) are shown with 2-way ANOVA.

Article Snippet: KRAS G12V vector was purchased from (Addgene, #35635).

Techniques: RNA sequencing, Functional Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Experimental setup for PI3K inhibitor (PI3Ki) and MEK inhibitor (MEKi) treatment and release. (B) Protein levels of pERK1/2, ERK1/2, pAKT, and AKT after HRAS G12V induction and treatment with MEKi or PI3Ki. (C) S phase percentage after HRAS G12V induction with release from PI3Ki or MEKi as determined by EdU labelling and flow cytometry. N=4. (D) Nuclear EU intensity after HRAS G12V induction and release from MEKi or PI3Ki. N=5 (MEKi N=4). (E) Average replication fork speeds after HRAS G12V induction and release from MEKi or PI3Ki. N=4. (F) Protein levels of pAKT (S473), AKT, pERK1/2 and ERK1/2 after UCL-TRO-1938 treatment of uninduced BJ-hTERT-BRAF V600E cells for 48 h. (G) Nuclear EU intensity after KRAS G12V induction +/- UCL-TRO-1938 (5 μM) for 48 h. N=4. (H) Median replication fork speeds after KRAS G12V induction +/- UCL-TRO-1938 (5 μM) for 48 h. N=3. (I) Inducible expression of oncogenic PI3K E545K , alone or with KRAS G12V or BRAF V600E , was used to increase PI3K signalling, on its own or in combination with MAPK activation. (J) Protein levels of pAKT (S473), AKT, pERK1/2 and ERK1/2 after oncogene induction or doxycycline treatment (72 h) of parental cells (BJ-hTERT). (K) Nuclear EU intensity after oncogene induction. N=4. (EL) Median replication fork speeds after oncogene induction. N=4-6. Means +/-SEM (bars) are shown with 2-way ANOVA or mixed effects analysis.

Article Snippet: KRAS G12V vector was purchased from (Addgene, #35635).

Techniques: Flow Cytometry, Expressing, Activation Assay

Journal: bioRxiv

Article Title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

doi: 10.64898/2026.03.16.711577

Figure Lengend Snippet: (A) Illustration of the approach used to generate hypertranscription scores . (B) Hypertranscription scores (fold-change) in colorectal adenocarcinoma (COAD) samples with the indicated oncogene mutations. Lines denote mean. (D) Illustration of the approach to generate replication stress transcription signatures . (E) Replication stress transcription signatures as in C after 48 h induction of KRAS G12V or HRAS G12V in BJ-hTERT cells (weighted log2-fold change). (E) Replication stress transcription signatures in COAD samples with the indicated oncogene mutations (weighted z-score). (F) Percentages of samples with homozygous deletions in the common fragile sites WWOX , FHIT or MACROD2 across pan-cancer samples with the indicated oncogene mutations. (G) Percentages of samples with amplifications in the common fragile sites WWOX , FHIT or MACROD2 across pan-cancer samples with the indicated oncogene mutations. (H) Model of the signalling pathways that promote TRCs downstream of RAS. PI3K and AKT contribute to S phase entry for example through GSK3β and CYCLIN D1, and to hypertranscription through AKT, MYC and potentially MTORC1. MAPK and PI3K pathways, and S phase entry and hypertranscription, cooperate in promoting TRCs and replication stress.

Article Snippet: KRAS G12V vector was purchased from (Addgene, #35635).

Techniques:

Journal: Nature Communications

Article Title: Intrinsic resistance to RAS inhibitors is driven by dysregulation of KRAS degradation

doi: 10.1038/s41467-025-67109-5

Figure Lengend Snippet: A , B Hemalun eosin and Immunostaining analysis of KRAS-G12D and HA (corresponding to KRAS-WT overexpression) of lung tumor sections from the Kras-G12D; GFP and Kras-G12D; WT-KRAS-OE mice 10 weeks post-Cre injection. Scale bar, 50 µm. C Tumor nodule growth was assessed by tumor volumetric analysis of micro-CT scans in the indicated mice. Data are shown as mean ± SEM; n = 5 mice per group with p -value calculated by two-sided mixed model analysis with no adjustments. D Survival analysis in the indicated mice; n = 5 mice per group with p -value calculated using the Log-rank (Mantel-Cox) test. E Heatmaps illustrating the proximity-based interaction signals of KRAS proteins determined by BioID analysis. The data were extracted from , . F Relative GPS-based stability of KRAS proteins in HEK293T cells expressing sh GFP or sh LZTR 1. Data are shown as mean ± SEM, n = 4 technical replicates per group. G Immunoblot analysis of the indicated proteins in A427 and H727 cells expressing sh GFP or sh LZTR1 . The samples derive from the same experiment. LZTR1, KRAS-G12V, and Vinculin were detected on the same gel; total KRAS was run on a separate gel processed in parallel. Data are shown as mean ± SEM, n = 4 technical replicates per group. H KRAS mutations and LZTR1 alterations in the TCGA LUAD dataset obtained from cBioPortal. Homdel, homozygous deletion; hetloss, shallow deletion. n = 507 patients with co-occurence q -value calculated. I Immunoblot analysis of the indicated proteins in lung tumor cells isolated from individual mice at 14 weeks post-Cre injection. The samples derive from the same experiment. LZTR1, KRAS-G12D, and Vinculin were detected on the same gel; total KRAS was run on a separate gel processed in parallel. Data are shown as mean ± SEM, n = 4 mice per group with p -values calculated by two-sided Mann‒Whitney test. J Expression of RAS proteins in lung cancer cells isolated from the indicated mice by TMT-labeled MS proteomics. Data are shown as mean ± SEM, n = 4 mice per group with p -values calculated by two-sided Mann‒Whitney test. K Tumor nodule growth was assessed by tumor volumetric analysis of micro-CT scans. Data are shown as mean ± SEM; n = 12 mice per group with p -value calculated by two-sided mixed model analysis with no adjustments. L H&E staining of the lung tissues from the indicated mice at 6 and 14 weeks post-Cre injection. Scale bar, 100 µm.

Article Snippet: pDONR223-KRAS4B G12V , Addgene plasmid #81665.

Techniques: Immunostaining, Over Expression, Injection, Micro-CT, Expressing, Western Blot, Isolation, Labeling, Staining