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scriptFig7A-7E-7H-7I-FigS8A-B.R
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## Plot qPCR results of marker genes in ST2 cells where Ahr was knocked down (ST2 siAHR) and Glis1 was knocked down (ST2 sGLIS1)##
library(readr)
library(dplyr)
library(tibble)
library(tidyr)
library(ggplot2)
# Ahr, Glis1 and marker gene expression in ST2 cells where GLIS1 is knocked-down
GLIS1.KD.ST2.qPCR = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/23022018-TF7_TF8_TF9_ST2_siGLIS1_qPCR.txt",
delim = "\t") %>%
filter(Samples == "ST2 siGLIS1 D1/ST2_NoDiff_siCTRL") %>%
filter(Gene != "Ahr") %>%
add_row(Samples = "ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", Gene = "empty1", Average = 0, SEM = 0, `Sig?` = "NS", .after = 1) %>% #Add a dummy sample to make a space between bars
add_row(Samples = "ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", Gene = "empty2", Average = 0, SEM = 0, `Sig?` = "NS", .after = 5) #Add a dummy sample to make a space between bars
GLIS1.KD.ST2.qPCR$Gene = factor(GLIS1.KD.ST2.qPCR$Gene, levels = c("Glis1", "empty1","Cebpa", "Pparg", "Lpl", "empty2", "Runx2", "Sp7", "Bglap"))
GLIS1.KD.ST2.qPCR.all.pl = ggplot(data = GLIS1.KD.ST2.qPCR, aes(x = Gene, y = Average, fill = Samples)) +
geom_bar(stat = "identity", size = 5, width = 0.9, position = position_dodge()) +
geom_errorbar(aes(ymin = Average - SEM, ymax = Average + SEM), width = 0.2, size = 1.0, position = position_dodge(width = 0.9)) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 1.5) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 30, colour = "black", angle = 45, hjust = 1),
axis.text.y = element_text(face = "bold", size = 30, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 30),
legend.position = "top", legend.text = element_text(size = 30, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6"), name = "Condition", breaks = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL"),
labels = c("ST2 siGlis1")) +
scale_x_discrete(breaks = c("Glis1", "empty1","Cebpa", "Pparg", "Lpl", "empty2", "Runx2", "Sp7", "Bglap"),
labels = c("Glis1", "","Cebpa", "Pparg", "Lpl", "", "Runx2", "Sp7", "Bglap")) +
geom_text(x = 1, y = 0.75, label = "*", color = "black", size = 20) +
geom_text(x = 3.15, y = 1.4, label = "**", color = "black", size = 20, angle = 90) +
geom_text(x = 5, y = 1.2, label = "*", color = "black", size = 20) +
geom_text(x = 9, y = 1.48, label = "*", color = "black", size = 20)
print(GLIS1.KD.ST2.qPCR.all.pl)
pdf("Y:/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/15032018-ST2-Glis1.KD.Fig7A.MarkerGenesExp.pdf", width = 10, height = 10)
GLIS1.KD.ST2.qPCR.all.pl
dev.off()
# Ahr, Glis1 and marker gene expression in ST2 cells where AHR is knocked-down
AHR.KD.ST2.qPCR = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/23022018-TF7_TF8_TF9_ST2_siGLIS1_qPCR.txt",
delim = "\t") %>%
filter(Samples == "ST2 siAHR D1/ST2_NoDiff_siCTRL") %>%
filter(Gene != "Glis1") %>%
add_row(Samples = "ST2 siAHR D1/ST2_NoDiff_siCTRL", Gene = "empty1", Average = 0, SEM = 0, `Sig?` = "NS", .after = 1) %>% #Add a dummy sample to make a space between bars
add_row(Samples = "ST2 siAHR D1/ST2_NoDiff_siCTRL", Gene = "empty2", Average = 0, SEM = 0, `Sig?` = "NS", .after = 5) #Add a dummy sample to make a space between bars
AHR.KD.ST2.qPCR$Gene = factor(AHR.KD.ST2.qPCR$Gene, levels = c("Ahr", "empty1","Cebpa", "Pparg", "Lpl", "empty2", "Runx2", "Sp7", "Bglap"))
AHR.KD.ST2.qPCR.all.pl = ggplot(data = AHR.KD.ST2.qPCR, aes(x = Gene, y = Average, fill = Samples)) +
geom_bar(stat = "identity", size = 5, width = 0.9, position = position_dodge()) +
geom_errorbar(aes(ymin = Average - SEM, ymax = Average + SEM), width = 0.2, size = 1.0, position = position_dodge(width = 0.9)) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 2.0) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 30, colour = "black", angle = 45, hjust = 1),
axis.text.y = element_text(face = "bold", size = 30, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 30),
legend.position = "top", legend.text = element_text(size = 30, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#808080"), name = "Condition", breaks = c("ST2 siAHR D1/ST2_NoDiff_siCTRL"),
labels = c("ST2 siAhr")) +
scale_x_discrete(breaks = c("Ahr", "empty1","Cebpa", "Pparg", "Lpl", "empty2", "Runx2", "Sp7", "Bglap"),
labels = c("Ahr", "","Cebpa", "Pparg", "Lpl", "", "Runx2", "Sp7", "Bglap")) +
geom_text(x = 1, y = 0.6, label = "*", color = "black", size = 20)
print(AHR.KD.ST2.qPCR.all.pl)
pdf("Y:/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/15032018-ST2-Ahr.KD.Fig7A.MarkerGenesExp.pdf", width = 10, height = 10)
AHR.KD.ST2.qPCR.all.pl
dev.off()
## Make the plot of Glis1 expression in Ahr knockdown (St2, adipocyte and osteoblast) ##
Glis1InAHR.KD = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/28022018-TF1_TF2_TF3_ST2&Ad&Ob_siAHR_Glis1Exp_qPCR.txt",
delim = "\t", progress = TRUE)
Glis1InAHR.KD$Samples = factor(Glis1InAHR.KD$Samples, levels = c("ST2 siAHR D1/ST2_NoDiff_siCTRL", "Adipo siAHR D1/Adipo_NoDiff_siCTRL","Osteo siAHR D1/Osteo_NoDiff_siCTRL"))
Glis1InAHR.KD.pl = ggplot(data = Glis1InAHR.KD, aes(x = Samples, y = Average, fill = Samples)) +
geom_bar(stat = "identity", size = 5, width = 0.6, position = position_dodge(width = 0.9)) +
geom_errorbar(aes(ymin = Average - SEM, ymax = Average + SEM), width = 0.2, size = 1.0, position = position_dodge(width = 0.9)) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 3.5) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 40, colour = "black", angle = 45, hjust = 1),
axis.text.y = element_text(face = "bold", size = 40, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 40),
legend.position = "none", legend.text = element_text(size = 20, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6", "#FF6666", "#0066CC"), breaks = c("ST2 siAHR D1/ST2_NoDiff_siCTRL", "Adipo siAHR D1/Adipo_NoDiff_siCTRL","Osteo siAHR D1/Osteo_NoDiff_siCTRL"),
labels = c("ST2 + siAhr", "Ad + siAhr", "Ob + siAhr")) +
scale_x_discrete(breaks = c("ST2 siAHR D1/ST2_NoDiff_siCTRL", "Adipo siAHR D1/Adipo_NoDiff_siCTRL","Osteo siAHR D1/Osteo_NoDiff_siCTRL"),
labels = c("ST2 + siAhr", "Ad + siAhr", "Ob + siAhr")) +
geom_text(x = 2, y = 3.3, label = "*", color = "black", size = 20) +
geom_text(x = 3, y = 1.8, label = "*", color = "black", size = 20)
print(Glis1InAHR.KD.pl)
pdf("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/02032018-TF1_TF2_TF3_ST2&Ad&Ob_siAHR_Glis1Exp_qPCR.pdf", width = 10, height = 10)
Glis1InAHR.KD.pl
dev.off()
## For Figure 7, plot the level of Notch1, Notch2 and Nocth3 from the knock-down of Ahr in ST2 cells, adipocyte and osteoblasts ##
Notch1 = "ENSMUSG00000026923"
Notch2 = "ENSMUSG00000027878"
Notch3 = "ENSMUSG00000038146"
Notches = c(Notch1, Notch2, Notch3)
# Load all genes from the knock-down of Ahr in ST2 cells, adipocyte and osteoblasts
ST2.AhrKD.AllGenes = read_delim("Y:/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/08062017-ST2-AHR-KD-AllGenes.txt",
delim = "\t", progress = TRUE, col_names = TRUE)
ST2.AhrKD.Notches = ST2.AhrKD.AllGenes %>%
filter(ensembl_gene_id %in% Notches) %>%
mutate(linearFoldChange = 2^log2FoldChange,
Condition = "ST2 siAhr",
GeneName = c("Notch1", "Notch2", "Notch3"))
Ad.AhrKD.AllGenes = read_delim("Y:/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/08062017-A-AHR-KD-AllGenes.txt",
delim = "\t", progress = TRUE, col_names = TRUE)
Ad.AhrKD.Notches = Ad.AhrKD.AllGenes %>%
filter(ensembl_gene_id %in% Notches) %>%
mutate(linearFoldChange = 2^log2FoldChange,
Condition = "Ad siAhr",
GeneName = c("Notch1", "Notch2", "Notch3"))
Ob.AhrKD.AllGenes = read_delim("Y:/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/08062017-O-AHR-KD-AllGenes.txt",
delim = "\t", progress = TRUE, col_names = TRUE)
Ob.AhrKD.Notches = Ob.AhrKD.AllGenes %>%
filter(ensembl_gene_id %in% Notches) %>%
mutate(linearFoldChange = 2^log2FoldChange,
Condition = "Ob siAhr",
GeneName = c("Notch1", "Notch2", "Notch3"))
Notches.all = bind_rows(ST2.AhrKD.Notches, Ad.AhrKD.Notches, Ob.AhrKD.Notches)
Notches.all$Condition = factor(Notches.all$Condition, levels = c("ST2 siAhr", "Ad siAhr", "Ob siAhr"))
Notches.pl = ggplot(data = Notches.all, aes(x = GeneName, y = linearFoldChange, fill = Condition)) +
geom_bar(stat = "identity", size = 5, width = 0.6, position = position_dodge()) +
geom_errorbar(aes(ymin = 2^(log2FoldChange - lfcSE), ymax = 2^(log2FoldChange + lfcSE)), width = 0.2, size = 1.0, position = position_dodge(width = 0.6)) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 2.0) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 40, colour = "black", angle = 45, hjust = 1),
axis.text.y = element_text(face = "bold", size = 40, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 40),
legend.position = "none", legend.text = element_text(size = 40, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6", "#FF6666", "#0066CC")) +
geom_text(x = 2.8, y = 1.4, label = "*", color = "black", size = 20) +
geom_text(x = 1, y = 1.4, label = "*", color = "black", size = 20) +
geom_text(x = 2, y = 1.5, label = "0.07", color = "black", size = 10) +
geom_text(x = 3.1, y = 1.8, label = "***", color = "black", size = 20, angle = 90) +
geom_text(x = 3.3, y = 1.8, label = "***", color = "black", size = 20, angle = 90)
print(Notches.pl)
pdf("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/02032018-RNASeqST2&Ad&Ob_siAHR_Notches_Exp.pdf", width = 10, height = 10)
Notches.pl
dev.off()
## For Figure 7, plot the level of Notch1, Notch2 and Nocth3 from the knock-down of Glis1 in ST2 cells, adipocyte and osteoblasts ##
# Load all genes from the knock-down of Glis1 in ST2 cells, adipocyte and osteoblasts
Notches.siGlis1 = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/01032018-TF7_TF8_TF9_ST2&Ad&Ob_siGLIS1_Notches_qPCR.txt",
delim = "\t", progress = TRUE, col_names = TRUE)
Notches.siGlis1$Samples = factor(Notches.siGlis1$Samples, levels = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", "Ad siGLIS1 D1/Ad_siCTRL",
"Ob siGLIS1 D1/Ob_siCTRL"))
Notches.siGLIS1.pl = ggplot(data = Notches.siGlis1, aes(x = Gene, y = Average, fill = Samples)) +
geom_bar(stat = "identity", size = 5, width = 0.6, position = position_dodge()) +
geom_errorbar(aes(ymin = Average - SEM, ymax = Average + SEM), width = 0.2, size = 1.0, position = position_dodge(width = 0.6)) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 2.0) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 40, colour = "black", angle = 45, hjust = 1),
axis.text.y = element_text(face = "bold", size = 40, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 40),
legend.position = "none", legend.text = element_text(size = 20, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6", "#FF6666", "#0066CC"), breaks = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", "Ad siGLIS1 D1/Ad_siCTRL","Ob siGLIS1 D1/Ob_siCTRL"),
labels = c("ST2 + siGlis1", "Ad + siGlis1", "Ob + siGlis1")) +
geom_text(x = 0.8, y = 1.2, label = "*", color = "black", size = 20) +
geom_text(x = 1, y = 1.6, label = "*", color = "black", size = 20) +
geom_text(x = 1.9, y = 1.2, label = "**", color = "black", size = 20, angle = 90) +
geom_text(x = 2.0, y = 1.8, label = "*", color = "black", size = 20)
print(Notches.siGLIS1.pl)
pdf("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/02032018-RNASeqST2&Ad&Ob_siGLIS1_Notches_Exp.pdf", width = 10, height = 10)
Notches.siGLIS1.pl
dev.off()
## Plot Glis1 levels in ST2, adipocytes and osteoblasts samples where Glis1 is knocked down ##
Glis1.siGLIS1 = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/07032018-TF7_TF8_TF9_ST2&Ad&Ob_siGLIS1_Glis1_qPCR.txt",
delim = "\t", progress = TRUE)
Glis1.siGLIS1$Samples = factor(Glis1.siGLIS1$Samples, levels = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL",
"Adipo siGLIS1/Adipo siCTRL",
"Osteo siGLIS1/Osteo siCTRL"))
Glis1.siGLIS1.pl = ggplot(data = Glis1.siGLIS1, aes(x = Samples, y = Average, fill = Samples)) +
geom_bar(stat = "identity", size = 5, width = 0.3) +
geom_errorbar(aes(ymin = Average - SEM, ymax = Average + SEM), width = 0.2, size = 1.0) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 1.0) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 30, colour = "black"),
axis.text.y = element_text(face = "bold", size = 30, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 30),
legend.position = "none", legend.text = element_text(size = 20, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6", "#FF6666", "#0066CC"), breaks = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", "Adipo siGLIS1/Adipo siCTRL","Osteo siGLIS1/Osteo siCTRL")) +
scale_x_discrete(breaks = c("ST2 siGLIS1 D1/ST2_NoDiff_siCTRL", "Adipo siGLIS1/Adipo siCTRL","Osteo siGLIS1/Osteo siCTRL"),
labels = c("ST2 + siGlis1", "Ad + siGlis1", "Ob + siGlis1")) +
geom_text(x = 1.0, y = 0.75, label = "*", color = "black", size = 10) +
geom_text(x = 3.0, y = 0.80, label = "*", color = "black", size = 10)
print(Glis1.siGLIS1.pl)
pdf("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/Figure7/07032018-ST2&Ad&Ob_siGLIS1_Glis1_qPCRExp.pdf", width = 10, height = 10)
Glis1.siGLIS1.pl
dev.off()
## Plot Ahr levels in ST2, adipocytes and osteoblasts samples where Ahr is knocked down ##
Ahr.siAHR = read_delim("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/qPCR/14042017-ST2.AhrKD-Ad.AhrKD-Ob.AhrKD_qPCR.txt",
delim = "\t", progress = TRUE)
View(Ahr.siAHR)
Ahr.siAHR$`Ahr NO DIFF` = factor(Ahr.siAHR$`Ahr NO DIFF`, levels = c("ST2siAhr/ST2siCTRL",
"AdsiAhr/AdsiCTRL",
"ObsiAhr/ObsiCTRL"))
Ahr.siAHR.pl = ggplot(data = Ahr.siAHR, aes(x = `Ahr NO DIFF`, y = Average_1, fill = `Ahr NO DIFF`)) +
geom_bar(stat = "identity", size = 5, width = 0.3) +
geom_errorbar(aes(ymin = Average_1 - SEM_1, ymax = Average_1 + SEM_1), width = 0.2, size = 1.0) +
geom_hline(yintercept = 1.0, linetype = "dashed", size = 2.0) +
theme_classic() +
ylim(0, 1.0) +
labs(y = "Relative expression") +
theme(axis.line = element_line(colour = "black", size = 1, linetype = "solid"),
axis.text.x = element_text(face = "bold.italic", size = 30, colour = "black"),
axis.text.y = element_text(face = "bold", size = 30, colour = "black"),
axis.title.x = element_blank(),
axis.title.y = element_text(face = "bold", size = 30),
legend.position = "none", legend.text = element_text(size = 20, face = "bold.italic"),
legend.title = element_blank(),
plot.title = element_text(face = "bold", size = 30, hjust = 0.5)) +
scale_fill_manual(values = c("#A6A6A6", "#FF6666", "#0066CC"), breaks = c("ST2siAhr/ST2siCTRL", "AdsiAhr/AdsiCTRL","ObsiAhr/ObsiCTRL")) +
scale_x_discrete(breaks = c("ST2siAhr/ST2siCTRL", "AdsiAhr/AdsiCTRL","ObsiAhr/ObsiCTRL"),
labels = c("ST2 + siAhr", "Ad + siAhr", "Ob + siAhr")) +
geom_text(x = 1.0, y = 0.60, label = "*", color = "black", size = 10) +
geom_text(x = 2.0, y = 0.62, label = "*", color = "black", size = 10) +
geom_text(x = 3.0, y = 0.75, label = "0.055", color = "black", size = 10)
print(Ahr.siAHR.pl)
pdf("//atlas/FSTC_SYSBIO/Deborah.GERARD/Gerard et al. - Manuscript 1/Figures/SUPPLEMENT/Supplementary Figure S6/08032018-ST2&Ad&Ob_siAHR_Ahr_qPCRExp.pdf", width = 10, height = 10)
Ahr.siAHR.pl
dev.off()