Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DNA content of normal and tumor tissue from Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); p53(lf); alb(lf) zebrafish. B) Scales from wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. Melanin pigment is dispersed throughout the cytoplasm of zebrafish melanocytes, revealing markedly different cell sizes. Scale bar = 250μm, insets are at same scale as one another. C) Quantification of melanocyte densities of wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. One-way ANOVA with Tukey’s multiple comparisons test, ***p<0.001, **p<0.01, ns = not significant. Error bars represent mean ± SEM. D) Images from brightfield (left), anti-Mitfa (middle) and DAPI (right) staining of a single wildtype (top) or Tg(mitfa:BRAF V600E ) (bottom) epidermal melanocyte. Only the melanocyte nuclei stain positively for Mitfa. White arrowheads indicate nuclei within a single melanocyte. Scale bar = 5μm. E) Percent binucleate cells as determined by anti-Mitfa staining of pigmented melanocytes. One-way ANOVA with Tukey’s multiple comparisons test, ****p<0.0001, ns = not significant. Error bars represent mean ± SEM. F) Flow cytometry and DNA content analysis of control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) melanocytes with brightfield, EGFP and DAPI images of single melanocytes. G) Quantification of percent mononucleate and binucleate melanocytes from Tg(mitfa:EGFP);alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) strains. Chi Square test, p=0.000009. H) DNA content analysis of wild-type and Tg(mitfa:BRAF V600E ) melanocyte nuclei by confocal densitometry.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Generation of BRAF V600E -expressing RPE-1 FUCCI cell lines with a lentiviral-based doxycycline-inducible vector. B) Western blot showing inducible expression of BRAF V600E (+Dox) using a BRAF and BRAF V600E -specific antibody. Expression of the Tet repressor protein is shown. Tubulin is used as the loading control. C) Flow cytometry plots of control (-Dox) and BRAF V600E -expressing (+Dox) cells. Tetraploid cells accumulating in G1 were quantified based on Cdt1-mCherry positivity and Hoechst incorporation. Percentages of G1 tetraploid cells in control and BRAF V600E -expressing cultures are indicated. D) Fold change in G1 tetraploids relative to the control (-Dox) are shown for BRAF V600E , BRAF WT and BRAF K483W (kinase-dead) -expressing cell lines. Fold change from 3 independent experiments is shown; unpaired Student’s t test, ** p < 0.01, ns = not significant. Error bars represent mean ± SEM. E) Merged phase contrast and GFP photomicrographs of H2B-GFP expressing control (-Dox) and BRAF V600E -expressing (+Dox) cells that have recently undergone mitosis. White dotted lines indicate 2 cells with 1 nucleus each that have separated following a successful cytokinesis (-Dox) and 1 cell with 2 nuclei that has failed cytokinesis (+Dox). F) Quantification of cytokinesis failure in H2B-GFP RPE-1 control (-Dox) and BRAF V600E -expressing (+Dox) cells. Percent cells with cytokinesis failure from 3 independent experiments is shown (total cells are n= 934 for -Dox and n=568 for +Dox). Unpaired Student’s t test, **** p< 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI and anti-Anillin staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Mean Anillin fluorescence intensity at the equator of control (n=80) and BRAF V600E -expressing (n=61) anaphase cells. Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. Unpaired Student’s t test, ****p < 0.0001. Error bars represent mean ± SEM. C) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with MEKi or ERKi. Drugs were added coincident with Dox administration. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. D) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=41), BRAF V600E -expressing (+Dox) cells (n=68), and BRAF V600E -expressing (+Dox) cells treated with MEKi or ERKi (n=21, n=21, respectively). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM. E) Western blot analysis of immunoprecipitated RhoA-GTP from control (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Total RhoA protein and alpha tubulin were used as a controls. F) Western blot quantification of immunoprecipitated RhoA-GTP levels from (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Samples were normalized to the -Dox condition. Measurements from 3 independent experiments are shown. Unpaired Student’s t test, * p< 0.05, ** p < 0.01, *** p < 0.001. Error bars represent mean ± SEM. G) Fold change in G1 tetraploid cells following addition of RhoA activators. LPA (1μM) and S1P (1μM) were added coincident with DOX administration. Fold change in G1 tetraploids relative to the control (+Dox no drug) are shown. Measurements from 3 independent experiments are shown. Unpaired Student’s t test, *** p<0.001. Error bars represent mean ± SEM. H) G1 tetraploid generation following expression of HA-tagged-BRAF V600E in RHOA Q61L -inducible cells. Experimental design (top): RPE-1 FUCCI cells with Dox inducible RHOA Q61L were transiently transfected with an HA-tagged-BRAF V600E -expressing construct and selected accordingly. G1 tetraploids were quantified (left) by gating HA-positive, Cdt-1:mCherry-positive cells with increased Hoechst incorporation. Fold change in G1 tetraploids (right), normalized to control (-Dox) cells. Unpaired Student’s t test, *** p < 0.001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing, Fluorescence, Western Blot, Immunoprecipitation, Transfection, Construct

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Fold change in G1 tetraploids following inhibitor treatment. Fold changes are expressed relative to control (+ BRAF V600E , no drug) cells. Fold change from 3 independent experiments is shown; unpaired Student’s t test, * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars represent mean ± SEM. B) ELISA-based quantification of RAC1-GTP levels in control (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cells. Cells were measured at the indicated timepoints post thymidine release. RAC-1 GTP signal was measured using a colorimetric assay at 490nM absorbance. Unpaired Student’s t test, **** p < 0.0001. Error bars represent mean ± SEM. C) Fold change in G1 tetraploids following addition of RAC1 inhibitors. NSC2366 and EHT1864 were added coincident with BRAF V600E induction. Fold changes are expressed relative to control (+ BRAF V600E , no drug) cells. Fold change from 3 independent experiments is shown; unpaired Student’s t test, **** p<0.0001. Error bars represent mean ± SEM. D) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with NSC2366 or EHT1864. Drugs were added coincident with Dox administration. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. E) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=40), BRAF V600E -expressing (+Dox) cells (n=38), and BRAF V600E -expressing (+Dox) cells treated with NSC2366 or EHT1864 (n=38, n=36, respectively). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Colorimetric Assay, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI and anti-CENTRIN-2 staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Insets show centrioles at one pole. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Quantification of cells in mitosis with supernumerary (>4) centrioles. -Dox (n=131); +Dox (n=169); +Dox MEKi (n=85) and +Dox ERKi (n=91). Drugs were added coincident with Dox administration. Percent cells from 3 independent experiments is shown; one-way ANOVA with Tukey’s multiple comparisons test, ** p < 0.05. Error bars represent mean ± SEM. C) DAPI, anti-Mitfa and anti-CENTRIN-2 staining of control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) non-cycling zebrafish melanocytes. Scale bar = 7.5μM. Insets show centrioles. D) Percent cells with normal (2) and extra (>2) centrioles in control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) zebrafish melanocytes; chi squared test p = 0.000843.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI, anti-CENTRIN-2 and anti-RhoA staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Insets show centrioles at one pole. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Mean RhoA fluorescence intensity at the equator of control (n=30) and BRAF V600E -expressing anaphase cells with normal (n=54) and supernumerary (n=38) centrosomes. Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, ** p < 0.01, **** p < 0.0001. Error bars represent mean ± SEM. C) Quantification of cells in mitosis with supernumerary (>4) centrioles. -Dox (n=119); +Dox (n=124) and +Dox +Centrinone (n=122). Centrinone was added coincident with Dox administration. Percent cells from 3 independent experiments is shown; one-way ANOVA with Tukey’s multiple comparisons test, *** p < 0.001, ns = not significant. Error bars represent mean ± SEM. D) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with Centrinone. Centrinone was added coincident with Dox administration (G1/S/G2/M) or only during G1/S. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. E) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=32), BRAF V600E -expressing (+Dox) cells (n=40), and BRAF V600E -expressing (+Dox) cells treated with Centrinone coincident with Dox administration (G1/S/G2/M; n=44) or only during G1/S (n=41). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM. F) Fold change in G1 tetraploids in control cells (-Dox), BRAF V600E -expressing cells (+Dox) and BRAF V600E -expressing cells treated with Centrinone (+Dox +cent.). Fold changes are expressed relative to the control cells. Fold change from 3 independent experiments is shown; One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Anti-Mitfa and DAPI staining of nuclei in Tg(mitfa:BRAF V600E ) and Tg(mitfa:BRAF V600E ); p53(lf) melanocytes. Melanin pigment was bleached to more clearly visualize nuclear size. White arrowheads indicate 2 nuclei within a single melanocyte. Scale bar = 10μm. B) DNA content per nucleus as measured by confocal densitometry. Each data point represents a single nucleus. C) Flow cytometry plots of BRAF V600E -expressing (+Dox) p53 wild-type and p53 mutant tetraploid cells. Prior to analysis, G1 tetraploid cells were isolated and plated for 24 hours in the presence of nocodazole. Percentages of Geminin-GFP-positive cells in S/G2/M are indicated. D) Quantification of S/G2/M cells from flow cytometry analysis in (C). Percent cells from 3 independent experiments is shown; unpaired Student’s t test, ** p < 0.01. Error bars represent mean ± SEM. E) Flow cytometry and DNA content analysis of normal tissue (left, top) and nascent melanomas (left, bottom) from Tg(crestin:EGFP); Tg(mitfa:BRAF V600E ); p53(lf) zebrafish. Brightfield, EGFP and DAPI images of single melanocytes are shown. Quantification of percent mononucleate and binucleate cells in the 4N and 8N peaks of nascent melanomas (right).

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Flow Cytometry, Expressing, Mutagenesis, Isolation

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of PFS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for PFS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. (F, G) Kaplan-Meier plots of PFS for CRC patients undergoing exclusive first-line chemotherapy (F) and chemotherapy combined with bevacizumab (G) , stratified by BRAF mutation status. PFS, progression-free survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of OS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for OS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. OS, overall survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Construction of a four-gene mutation signature prediction disease progression and prognosis in FPHYP cohort. (A, B) Univariate and multivariate analyses were performed to assess the impact of clinicopathological features, individual somatic gene mutations, and the four-gene mutation signature on PFS (A) and OS (B) in CRC. (C) The Kaplan-Meier survival analysis for PFS in CRC patients between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (D) The Kaplan-Meier survival analysis for OS in CRC cases between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (E, F) ROC curves for PFS (E) and OS (F) that dependent on time were generated to evaluate the prognostic model’s performance, which is based on the gene mutation status within the FPHYP cohort. PFS, progression-free survival; OS, overall survival; MT, mutation type; WT, wiled type; ROC, receiver operating characteristic.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis, Biomarker Discovery, Generated

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Immunohistochemical analysis of BRAF and ARID2 in CRC. (A, B) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of the BRAF -MT (A) and BRAF -WT (B) groups. (C) Comparison of the IOD/Area value between BRAF -MT and BRAF -WT groups. (D, E) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of stage I (D) and stage IV (E) groups. (F) Comparison of the IOD/Area value between stage I and IV groups. (G, H) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of the ARID2 -MT (G) and ARID2 -WT (H) groups. (I) Comparison of the IOD/Area value between ARID2 -MT and ARID2 -WT groups. (J, K) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of stage I (J) and stage IV (K) groups. (L) Comparison of the IOD/Area value between stage I and IV groups. MT, mutation type; WT, wiled type; IOD, cumulative optical density.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Immunohistochemical staining, Expressing, Comparison, Mutagenesis