Journal: Cancer medicine

Article Title: Cancer-associated fibroblasts secrete Wnt2 to promote cancer progression in colorectal cancer.

doi: 10.1002/cam4.2523

Figure Lengend Snippet: FIGURE 2 Analysis of gene expression profiling. The heat map demonstrated differentially expressed genes identified with RNA sequencing, including 283 upregulated genes and 301 downregulated genes in CAFs (A). Pathway analysis using Gene Set Enrichment Analysis revealed that the gene sets related to the Wnt signaling pathway were highly enriched in CAFs (B). CAFs had much higher WNT2 expression than NFs among the primary fibroblasts from each of the five cases (C). FPKM: fragments per kilobase of transcript per million mapped reads

Article Snippet: Then, samples were incubated with rabbit anti‐ Wnt2 polyclonal antibody (OriGene; dilution 1:300) as the primary antibodies, overnight at 4°C.

Techniques: Gene Expression, RNA Sequencing, Expressing

Journal: Cancer medicine

Article Title: Cancer-associated fibroblasts secrete Wnt2 to promote cancer progression in colorectal cancer.

doi: 10.1002/cam4.2523

Figure Lengend Snippet: FIGURE 3 Wnt2 expression in colon tissue by immunohistochemistry. The images show representative immunohistochemical staining in normal colon tissue (A), cancer tissue with a score of 0 (B), cancer tissue with a score of 1 (C), and cancer tissue with a score of 2 (D). Immunoreactivity was found in cancer stroma, particularly in the fibromatous area, rather than cancer calls, although staining for Wnt2 were rarely observed in both epithelial and stromal cells in normal tissues

Article Snippet: Then, samples were incubated with rabbit anti‐ Wnt2 polyclonal antibody (OriGene; dilution 1:300) as the primary antibodies, overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Cancer medicine

Article Title: Cancer-associated fibroblasts secrete Wnt2 to promote cancer progression in colorectal cancer.

doi: 10.1002/cam4.2523

Figure Lengend Snippet: FIGURE 4 Kaplan‐Meier analysis of disease‐free survival (A), overall survival (B), and disease‐specific survival (C) of patients with colorectal cancer according to the expression of Wnt2 protein. There was no significant correlation between the high‐expression group and the low‐expression group in these variables

Article Snippet: Then, samples were incubated with rabbit anti‐ Wnt2 polyclonal antibody (OriGene; dilution 1:300) as the primary antibodies, overnight at 4°C.

Techniques: Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 3. RNA sequencing reveals WNT2 up-regulation upon sotorasib treatment. Volcano plot representing gene expression changes between (A) H23 parental cells, sotorasib treated versus untreated, and (B) isogenic resistant H23 cells compared to parental cells. Statistical significance was calculated for control verse treatment. n = 3 per group. (C) Number of overlapping and unique genes that were up-regulated or down-regulated with respect to treatment represented as a Venn diagram. (D) Heatmap representing top 10 overlapping up-regulated and down-regulated genes based on average fold change. (E and F) Effect of 3.2 μM sotorasib on H23 parental or isogenic resistant (Iso R) cells having knockdown of ITGB4 or CTNNB1 or both represented as percent change in growth at 96 hours (bar graph), respectively. Two-way ANOVA was used to calculate the statistical significance for each time point and for each condition (si Control, si ITGB4, si CTNNB1, si CTNNB1 + si ITGB4; n = 3 per group; ****P < 0.0001. (G) Immunoblot confirmed knockdown of ITGB4 and β-catenin in H23 parental cells and H23 sotorasib (20 μM) resistant cells. These cells were also treated with 3.2 μM sotorasib for 72 hours to identify changes in protein expression and signaling. (H) Representing the effect of ITGB4 and CTNNB1 single knockdown or double knockdown together with 10 μM sotorasib as a percent change in growth. Two-way ANOVA was used to calculate the statistical significance for each time point and each condition (si Control, si PXN, si ITGB4, and si PXN + si ITGB4; n = 3 per group; ****P < 0.0001. (I) Immunoblot showing knockdown of ITGB4 and β-catenin and changes in expression of active β-catenin, γH2AX, and p27 in SW1573 cells. (J) Immunoblot showing the reduction in the expression of WNT2, ITGB4, phospho, and total β-catenin in the SW1573 treated with sotorasib and CFZ drug combination.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: RNA Sequencing, Gene Expression, Control, Knockdown, Western Blot, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 5. KRAS G12C inhibitors have a differential effect on cell growth and progression. (A) Effect of increasing concentrations (1 to 4 μM) of sotorasib and adagrasib exhibited different effects on cell proliferation over 24 hours in H23 cells. Two-way ANOVA was used for calculating statistical significance across various time points and for each drug concentration. n = 3. (B) Effect of KRAS inhibitors sotorasib or adagrasib increasing concentrations (1 to 8 μM) on SW1573 cells within 24 hours of drug treatment. Two-way ANOVA test was used to calculate the statistical significance. n = 3 sample per group. (C) Immunofluorescence image of SW1573 to support the dose dependent inhibitory effect of adagrasib on cell growth and expression of WNT2 (green) and phospsho-S675–β-catenin (magenta). The region of interest was zoomed 50% digitally to show membrane blebbing induced by adagrasib (white arrows). (D and E) Cell cycle dynamics were determined using IncuCyte Cell Cycle Lentivirus Reagent with fluorescence indicating cell cycle phase (brightfield image and schematic). Effect of increasing concentrations of sotorasib (1.25 to 20 μM) and adagrasib (0.6 to 1.25 μM) on cell cycle in SW1573 cells represented as pseudo color plots. The y axis of the plot represents events positive for GFP, and x axis represents the events positive for mKate2. mKate positive represents G1; GFP positive represents S, G2, and M; and double positive represents G1-S–transitioning cells.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: Concentration Assay, Immunofluorescence, Expressing, Membrane, Fluorescence

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 3. RNA sequencing reveals WNT2 up-regulation upon sotorasib treatment. Volcano plot representing gene expression changes between (A) H23 parental cells, sotorasib treated versus untreated, and (B) isogenic resistant H23 cells compared to parental cells. Statistical significance was calculated for control verse treatment. n = 3 per group. (C) Number of overlapping and unique genes that were up-regulated or down-regulated with respect to treatment represented as a Venn diagram. (D) Heatmap representing top 10 overlapping up-regulated and down-regulated genes based on average fold change. (E and F) Effect of 3.2 μM sotorasib on H23 parental or isogenic resistant (Iso R) cells having knockdown of ITGB4 or CTNNB1 or both represented as percent change in growth at 96 hours (bar graph), respectively. Two-way ANOVA was used to calculate the statistical significance for each time point and for each condition (si Control, si ITGB4, si CTNNB1, si CTNNB1 + si ITGB4; n = 3 per group; ****P < 0.0001. (G) Immunoblot confirmed knockdown of ITGB4 and β-catenin in H23 parental cells and H23 sotorasib (20 μM) resistant cells. These cells were also treated with 3.2 μM sotorasib for 72 hours to identify changes in protein expression and signaling. (H) Representing the effect of ITGB4 and CTNNB1 single knockdown or double knockdown together with 10 μM sotorasib as a percent change in growth. Two-way ANOVA was used to calculate the statistical significance for each time point and each condition (si Control, si PXN, si ITGB4, and si PXN + si ITGB4; n = 3 per group; ****P < 0.0001. (I) Immunoblot showing knockdown of ITGB4 and β-catenin and changes in expression of active β-catenin, γH2AX, and p27 in SW1573 cells. (J) Immunoblot showing the reduction in the expression of WNT2, ITGB4, phospho, and total β-catenin in the SW1573 treated with sotorasib and CFZ drug combination.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: RNA Sequencing, Gene Expression, Control, Knockdown, Western Blot, Expressing

Journal: Science advances

Article Title: Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

doi: 10.1126/sciadv.ade3816

Figure Lengend Snippet: Fig. 5. KRAS G12C inhibitors have a differential effect on cell growth and progression. (A) Effect of increasing concentrations (1 to 4 μM) of sotorasib and adagrasib exhibited different effects on cell proliferation over 24 hours in H23 cells. Two-way ANOVA was used for calculating statistical significance across various time points and for each drug concentration. n = 3. (B) Effect of KRAS inhibitors sotorasib or adagrasib increasing concentrations (1 to 8 μM) on SW1573 cells within 24 hours of drug treatment. Two-way ANOVA test was used to calculate the statistical significance. n = 3 sample per group. (C) Immunofluorescence image of SW1573 to support the dose dependent inhibitory effect of adagrasib on cell growth and expression of WNT2 (green) and phospsho-S675–β-catenin (magenta). The region of interest was zoomed 50% digitally to show membrane blebbing induced by adagrasib (white arrows). (D and E) Cell cycle dynamics were determined using IncuCyte Cell Cycle Lentivirus Reagent with fluorescence indicating cell cycle phase (brightfield image and schematic). Effect of increasing concentrations of sotorasib (1.25 to 20 μM) and adagrasib (0.6 to 1.25 μM) on cell cycle in SW1573 cells represented as pseudo color plots. The y axis of the plot represents events positive for GFP, and x axis represents the events positive for mKate2. mKate positive represents G1; GFP positive represents S, G2, and M; and double positive represents G1-S–transitioning cells.

Article Snippet: We first generated a WNT2 knockout (KO) H23 cell line using the combination of WNT2 CRISPR-Cas9 KO and WNT2 homology directed repair (HDR) plasmids from Santa Cruz Biotechnologies.

Techniques: Concentration Assay, Immunofluorescence, Expressing, Membrane, Fluorescence