prism for macos version 6.0  (GraphPad Software Inc)

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Left: Western blot of endogenous GLI1 and cleaved PARP (c-PARP) in Colo205 cells treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox) for 48 h. Loading control: ACTIN. Images are representative of three independent experiments; right: bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. A. (B) Evaluation of cell viability, measured with Celltiter-GLO, of Colo205 cells treated with 5-FU + Ox in presence or absence of 10 μM GANT61 for 96 h. Data are representative of three independent experiments, *p < 0.05 versus untreated; § p < 0.05 5-FU + Ox versus GANT61 + 5-FU + Ox (two-way ANOVA test). (C) Immunofluorescence staining of GLI1 (green) in Colo205 cells treated with 10 μM GANT61 or 10 μM 5-FU + Ox for 48 h, as depicted in figure; Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of three independent experiments. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histogram showing the percentage of cells displaying nuclear GLI1. The experiment was performed in triplicate, *p < 0.05 (Mann–Whitney U test). (E) Left: representative western blot of endogenous GLI1, cleaved PARP (c-PARP) and PCNA in Colo205 cells treated with 10 μM 5-FU + Ox ,10 μM GANT61 or both (Combi) for 48 h. Loading control: ACTIN. Right: Bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Western blot of endogenous GLI1 in Colo205 at basal level, after short hairpin mediated GLI1 silencing using two different short hairpin clones (shGLI1a and shGLI1b) and non targeting control (PLKO) after 72 h from infection. Loading control: ACTIN. Images are representative of at least three independent experiments. Uncropped images are shown in Supplementary Fig. C. (B) Histograms showing mRNA levels expressed in arbitrary units of ABC transporters expressed by Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) after 72 h from infection. Dashed line: non targeting control (PLKO). *p < 0.05 versus PLKO; **p < 0.01 versus PLKO (one-way ANOVA test). Data are means ± SD from three independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques: Western Blot, Clone Assay, Infection

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Schematic representation of the promoter region of the indicated ABC transporters showing locations of putative GLI1 canonical and non canonical binding sites; see supplementary Fig. for detailed sequences. (B) qPCR-ChIP assay of endogenous GLI1 occupancy of the promoter region of the indicated ABC transporters in Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) versus non targeting control (PLKO) for 72 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus PLKO (Mann–Whitney U test). (C) qPCR-ChIP assay of overexpressed GLI1 occupancy of the promoter region of the indicated ABC transporters in HCT15 cells after GLI1 overexpression (oe GLI1) or control (oe empty) for 24 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus oe empty; **p < 0.01 versus oe empty (Mann–Whitney U test). (B, C) Data are means ± SD from at least 3 independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques: Binding Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY, Over Expression

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Histograms showing mRNA levels of ABC transporters in Colo205 treated with 10 μM GANT61, 10 μM 5-FU and Oxaliplatin (5-FU + Ox) or the combination (combi) for 48 h. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; § p < 0.05 GANT61 + 5-FU + Ox versus 5-FU + Ox (Two-way ANOVA test). (B) Western blot of endogenous ABC transporters in Colo205 cells treated with GANT61, 5-FU + Ox or the combination (combi). Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. A. (C) Immunofluorescence staining of MDR1/ABCB1 (red) and GLI1 (green) in Colo205 cells treated with 10 μM 5-FU + Ox or control (DMSO) for 48 h. Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of at least three independent experiments. Histograms of MDR1/ABCB1 number of positive cells and intensity are shown in supplementary Fig. A. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histograms showing mRNA levels of ABC transporters in HCT15 cells after GLI1 over expression (oe GLI1) for 24 h. Dashed line: Control transfected cells. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; **p < 0.01 versus Ctrl (One-way ANOVA test). (E) Western blot of endogenous GLI1 and ABC transporters in HCT15 after GLI1 over expression (oe GLI1), transfection with empty vector (oe empty) and in non-transfected cells (CTRL) for 24 h. Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. B. (F) Cell viability of HCT15 cells after overexpression of GLI1, after treatment with 10 μM 5-FU + Ox and after the combination of GLI1 overexpression and 5-FU + Ox. 5-FU + Ox treatment was added after 24 h from the transfection and it was carried out for 48 h. Data are representative of three independent experiments, *p < 0.05 versus control; § p < 0.05 GLI1 + 5-FU + Ox versus 5-FU + Ox (one-way ANOVA test). Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, Over Expression, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in Colo205 treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox), starting from 1 μM to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values (two-way ANOVA test). (B) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in HCT15 treated with increasing doses of 5-FU + Ox, starting from 1 to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values; § p < 0.05 5-FU + Ox versus 5-FU + Ox + GANT61 (two-way ANOVA test). Data are representative of three independent experiments, (A , B) . Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques:

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A–C) Expression data of ABCG1 from datasets interrogated on R2 platform as indicated in main text. *p < 0.05; **p < 0.01, ****p < 0.0001 (Mann–Whitney U test). (D–F) Kaplan Meier curves of ABCG1 in the indicated datasets described in the main text. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Techniques: Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Left: Western blot of endogenous GLI1 and cleaved PARP (c-PARP) in Colo205 cells treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox) for 48 h. Loading control: ACTIN. Images are representative of three independent experiments; right: bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. A. (B) Evaluation of cell viability, measured with Celltiter-GLO, of Colo205 cells treated with 5-FU + Ox in presence or absence of 10 μM GANT61 for 96 h. Data are representative of three independent experiments, *p < 0.05 versus untreated; § p < 0.05 5-FU + Ox versus GANT61 + 5-FU + Ox (two-way ANOVA test). (C) Immunofluorescence staining of GLI1 (green) in Colo205 cells treated with 10 μM GANT61 or 10 μM 5-FU + Ox for 48 h, as depicted in figure; Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of three independent experiments. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histogram showing the percentage of cells displaying nuclear GLI1. The experiment was performed in triplicate, *p < 0.05 (Mann–Whitney U test). (E) Left: representative western blot of endogenous GLI1, cleaved PARP (c-PARP) and PCNA in Colo205 cells treated with 10 μM 5-FU + Ox ,10 μM GANT61 or both (Combi) for 48 h. Loading control: ACTIN. Right: Bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Western blot of endogenous GLI1 in Colo205 at basal level, after short hairpin mediated GLI1 silencing using two different short hairpin clones (shGLI1a and shGLI1b) and non targeting control (PLKO) after 72 h from infection. Loading control: ACTIN. Images are representative of at least three independent experiments. Uncropped images are shown in Supplementary Fig. C. (B) Histograms showing mRNA levels expressed in arbitrary units of ABC transporters expressed by Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) after 72 h from infection. Dashed line: non targeting control (PLKO). *p < 0.05 versus PLKO; **p < 0.01 versus PLKO (one-way ANOVA test). Data are means ± SD from three independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques: Western Blot, Clone Assay, Infection

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Schematic representation of the promoter region of the indicated ABC transporters showing locations of putative GLI1 canonical and non canonical binding sites; see supplementary Fig. for detailed sequences. (B) qPCR-ChIP assay of endogenous GLI1 occupancy of the promoter region of the indicated ABC transporters in Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) versus non targeting control (PLKO) for 72 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus PLKO (Mann–Whitney U test). (C) qPCR-ChIP assay of overexpressed GLI1 occupancy of the promoter region of the indicated ABC transporters in HCT15 cells after GLI1 overexpression (oe GLI1) or control (oe empty) for 24 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus oe empty; **p < 0.01 versus oe empty (Mann–Whitney U test). (B, C) Data are means ± SD from at least 3 independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques: Binding Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY, Over Expression

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Histograms showing mRNA levels of ABC transporters in Colo205 treated with 10 μM GANT61, 10 μM 5-FU and Oxaliplatin (5-FU + Ox) or the combination (combi) for 48 h. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; § p < 0.05 GANT61 + 5-FU + Ox versus 5-FU + Ox (Two-way ANOVA test). (B) Western blot of endogenous ABC transporters in Colo205 cells treated with GANT61, 5-FU + Ox or the combination (combi). Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. A. (C) Immunofluorescence staining of MDR1/ABCB1 (red) and GLI1 (green) in Colo205 cells treated with 10 μM 5-FU + Ox or control (DMSO) for 48 h. Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of at least three independent experiments. Histograms of MDR1/ABCB1 number of positive cells and intensity are shown in supplementary Fig. A. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histograms showing mRNA levels of ABC transporters in HCT15 cells after GLI1 over expression (oe GLI1) for 24 h. Dashed line: Control transfected cells. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; **p < 0.01 versus Ctrl (One-way ANOVA test). (E) Western blot of endogenous GLI1 and ABC transporters in HCT15 after GLI1 over expression (oe GLI1), transfection with empty vector (oe empty) and in non-transfected cells (CTRL) for 24 h. Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. B. (F) Cell viability of HCT15 cells after overexpression of GLI1, after treatment with 10 μM 5-FU + Ox and after the combination of GLI1 overexpression and 5-FU + Ox. 5-FU + Ox treatment was added after 24 h from the transfection and it was carried out for 48 h. Data are representative of three independent experiments, *p < 0.05 versus control; § p < 0.05 GLI1 + 5-FU + Ox versus 5-FU + Ox (one-way ANOVA test). Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, Over Expression, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in Colo205 treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox), starting from 1 μM to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values (two-way ANOVA test). (B) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in HCT15 treated with increasing doses of 5-FU + Ox, starting from 1 to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values; § p < 0.05 5-FU + Ox versus 5-FU + Ox + GANT61 (two-way ANOVA test). Data are representative of three independent experiments, (A , B) . Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques:

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A–C) Expression data of ABCG1 from datasets interrogated on R2 platform as indicated in main text. *p < 0.05; **p < 0.01, ****p < 0.0001 (Mann–Whitney U test). (D–F) Kaplan Meier curves of ABCG1 in the indicated datasets described in the main text. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Techniques: Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Left: Western blot of endogenous GLI1 and cleaved PARP (c-PARP) in Colo205 cells treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox) for 48 h. Loading control: ACTIN. Images are representative of three independent experiments; right: bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. A. (B) Evaluation of cell viability, measured with Celltiter-GLO, of Colo205 cells treated with 5-FU + Ox in presence or absence of 10 μM GANT61 for 96 h. Data are representative of three independent experiments, *p < 0.05 versus untreated; § p < 0.05 5-FU + Ox versus GANT61 + 5-FU + Ox (two-way ANOVA test). (C) Immunofluorescence staining of GLI1 (green) in Colo205 cells treated with 10 μM GANT61 or 10 μM 5-FU + Ox for 48 h, as depicted in figure; Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of three independent experiments. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histogram showing the percentage of cells displaying nuclear GLI1. The experiment was performed in triplicate, *p < 0.05 (Mann–Whitney U test). (E) Left: representative western blot of endogenous GLI1, cleaved PARP (c-PARP) and PCNA in Colo205 cells treated with 10 μM 5-FU + Ox ,10 μM GANT61 or both (Combi) for 48 h. Loading control: ACTIN. Right: Bar graphs show densitometrically quantified band intensity values normalized to the loading control. Data are representative of three independent experiments, *p < 0.05 versus control (two-way ANOVA test). Uncropped images are shown in Supplementary Fig. B. Histograms were created using GraphPad Prism version 6.0 for macOS, https://ww.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Western blot of endogenous GLI1 in Colo205 at basal level, after short hairpin mediated GLI1 silencing using two different short hairpin clones (shGLI1a and shGLI1b) and non targeting control (PLKO) after 72 h from infection. Loading control: ACTIN. Images are representative of at least three independent experiments. Uncropped images are shown in Supplementary Fig. C. (B) Histograms showing mRNA levels expressed in arbitrary units of ABC transporters expressed by Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) after 72 h from infection. Dashed line: non targeting control (PLKO). *p < 0.05 versus PLKO; **p < 0.01 versus PLKO (one-way ANOVA test). Data are means ± SD from three independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques: Western Blot, Clone Assay, Infection

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Schematic representation of the promoter region of the indicated ABC transporters showing locations of putative GLI1 canonical and non canonical binding sites; see supplementary Fig. for detailed sequences. (B) qPCR-ChIP assay of endogenous GLI1 occupancy of the promoter region of the indicated ABC transporters in Colo205 cells after short hairpin mediated GLI1 silencing (shGLI1) versus non targeting control (PLKO) for 72 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus PLKO (Mann–Whitney U test). (C) qPCR-ChIP assay of overexpressed GLI1 occupancy of the promoter region of the indicated ABC transporters in HCT15 cells after GLI1 overexpression (oe GLI1) or control (oe empty) for 24 h. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 (a-acH3) antibody was used to detect ABC transporters transcriptional activation. *p < 0.05 versus oe empty; **p < 0.01 versus oe empty (Mann–Whitney U test). (B, C) Data are means ± SD from at least 3 independent experiments. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques: Binding Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY, Over Expression

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) Histograms showing mRNA levels of ABC transporters in Colo205 treated with 10 μM GANT61, 10 μM 5-FU and Oxaliplatin (5-FU + Ox) or the combination (combi) for 48 h. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; § p < 0.05 GANT61 + 5-FU + Ox versus 5-FU + Ox (Two-way ANOVA test). (B) Western blot of endogenous ABC transporters in Colo205 cells treated with GANT61, 5-FU + Ox or the combination (combi). Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. A. (C) Immunofluorescence staining of MDR1/ABCB1 (red) and GLI1 (green) in Colo205 cells treated with 10 μM 5-FU + Ox or control (DMSO) for 48 h. Nuclei are counterstained with Hoechst. Bars, 20 μm. Images are representative of at least three independent experiments. Histograms of MDR1/ABCB1 number of positive cells and intensity are shown in supplementary Fig. A. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing. (D) Histograms showing mRNA levels of ABC transporters in HCT15 cells after GLI1 over expression (oe GLI1) for 24 h. Dashed line: Control transfected cells. Data are representative of three independent experiments, *p < 0.05 versus Ctrl; **p < 0.01 versus Ctrl (One-way ANOVA test). (E) Western blot of endogenous GLI1 and ABC transporters in HCT15 after GLI1 over expression (oe GLI1), transfection with empty vector (oe empty) and in non-transfected cells (CTRL) for 24 h. Loading control: ACTIN. Data are representative images from 3 independent experiments. Uncropped images are shown in Supplementary Fig. B. (F) Cell viability of HCT15 cells after overexpression of GLI1, after treatment with 10 μM 5-FU + Ox and after the combination of GLI1 overexpression and 5-FU + Ox. 5-FU + Ox treatment was added after 24 h from the transfection and it was carried out for 48 h. Data are representative of three independent experiments, *p < 0.05 versus control; § p < 0.05 GLI1 + 5-FU + Ox versus 5-FU + Ox (one-way ANOVA test). Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques: Western Blot, Immunofluorescence, Staining, Software, Over Expression, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in Colo205 treated with increasing doses of 5-FU and Oxaliplatin (5-FU + Ox), starting from 1 μM to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values (two-way ANOVA test). (B) mRNA levels of GLI1 and indicated genes, expressed in arbitrary units, in HCT15 treated with increasing doses of 5-FU + Ox, starting from 1 to 10 μM and 10 μM GANT61 during 5 weeks. *p < 0.05 versus starting values; § p < 0.05 5-FU + Ox versus 5-FU + Ox + GANT61 (two-way ANOVA test). Data are representative of three independent experiments, (A , B) . Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques:

Journal: Scientific Reports

Article Title: Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

doi: 10.1038/s41598-020-70871-9

Figure Lengend Snippet: (A–C) Expression data of ABCG1 from datasets interrogated on R2 platform as indicated in main text. *p < 0.05; **p < 0.01, ****p < 0.0001 (Mann–Whitney U test). (D–F) Kaplan Meier curves of ABCG1 in the indicated datasets described in the main text. Histograms were created using GraphPad Prism version 6.0 for macOS, https://www.graphpad.com .

Article Snippet: Differences were analysed using the Mann–Whitney U-test for non-parametric values, One-way ANOVA and Two-way ANOVA test where appropriate, using the GraphPad Prism software Version 6.0 for macOS, GraphPad Software, San Diego, California USA, https://www.graphpad.com .

Techniques: Expressing, MANN-WHITNEY