Figure S4 ,
Tables S3 and ). (C) Frequency of siRNA EGR2 spheroid formation in nonadherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; ∗p value < 0.05; ∗∗p value < 0.01 ( t -test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in nonadherent cell culture (scale bars = 100 μm). (E) Immunofluorescence staining of control (top panel) and siRNA EGR2 (lower panel) PDOs for EGR2 (yellow) and F-ACTIN (red) in Matrigel culture. Nuclei are stained blue with DAPI (scale bars, 20 μm). (F) Heatmap showing scRNA-seq generated log(nTPM) z-scores for EGR2 , stem cell genes ( ALDH1A1 , EPHB2 , OLFM2 ), Wnt signaling genes ( AXIN2 , LGR5 ), differentiation ( ATOH1 , KRT20 , MUC1 ), and proliferation genes ( MKI67 , MYC ) in neuropod cells, stem cells, mucus-secreting cells, enterocytes, granulocytes, and Paneth cells from healthy human colon tissue. Results based on Single Cell Type information from the Human Protein Atlas (
Karlsson et al., 2021 ) ( https://www.proteinatlas.org/ENSG00000122877-EGR2/single+cell+type/colon ). (G) Representative images of control virus (top panel) and shRNA EGR2 (lower panel) transduced 195-CB-P cells in non-adherent cell culture (scale bars, 100 μm). (H) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 two, and shRNA EGR2 3 195-CB-P cells are 6.9 × 10 −9 , 4.9 × 10 −6 , and 6.92 × 10 −8 , respectively. (I) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (See also
Table S4 ). " width="100%" height="100%">
Journal: iScience
Article Title: Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis
doi: 10.1016/j.isci.2022.104498
Figure Lengend Snippet: EGR2 is required for CSC survival and tumorigenicity (A) Proliferation of siRNA transfected patient-derived colon cancer cells in nonadherent cell culture compared to untreated control cells (mean ± SD; data from three independent experiments). ∗p value < 0.05; ∗∗p value < 0.01 ( t -test). (B) Fold expression of ALDH1A1 , EGR2 , EGR3 , HDGFRP3 , OLFML2 , OLFML3 , PCP4 , PEG10 , PLK1 , PRKACB , and THBS1 RT-PCR gene expression data (±95% confidence intervals) in siRNA transfected 278-ML-P cells (n = 3 independent cell preparations) compared to untreated control cells. Nontargeting siRNA and siRNA PLK1 were used as negative and positive controls, respectively (see also Figure S4 , Tables S3 and ). (C) Frequency of siRNA EGR2 spheroid formation in nonadherent cell culture compared to control transfected cells (mean ± SD; data from three independent experiments). ns = not significant; ∗p value < 0.05; ∗∗p value < 0.01 ( t -test). (D) Representative images of a 278-ML-P control spheroid (LHS) and a siRNA EGR2 spheroid (RHS) in nonadherent cell culture (scale bars = 100 μm). (E) Immunofluorescence staining of control (top panel) and siRNA EGR2 (lower panel) PDOs for EGR2 (yellow) and F-ACTIN (red) in Matrigel culture. Nuclei are stained blue with DAPI (scale bars, 20 μm). (F) Heatmap showing scRNA-seq generated log(nTPM) z-scores for EGR2 , stem cell genes ( ALDH1A1 , EPHB2 , OLFM2 ), Wnt signaling genes ( AXIN2 , LGR5 ), differentiation ( ATOH1 , KRT20 , MUC1 ), and proliferation genes ( MKI67 , MYC ) in neuropod cells, stem cells, mucus-secreting cells, enterocytes, granulocytes, and Paneth cells from healthy human colon tissue. Results based on Single Cell Type information from the Human Protein Atlas ( Karlsson et al., 2021 ) ( https://www.proteinatlas.org/ENSG00000122877-EGR2/single+cell+type/colon ). (G) Representative images of control virus (top panel) and shRNA EGR2 (lower panel) transduced 195-CB-P cells in non-adherent cell culture (scale bars, 100 μm). (H) Table shows results of limiting dilution transplantation of control virus transduced and shRNA EGR2 transduced 195-CB-P cells. The number of established tumors as a fraction of the number of animals transplanted is given. P values for pairwise tests of differences in CSC frequencies between control virus versus shRNA EGR2 1, shRNA EGR2 two, and shRNA EGR2 3 195-CB-P cells are 6.9 × 10 −9 , 4.9 × 10 −6 , and 6.92 × 10 −8 , respectively. (I) Growth curves for xenografts derived from control virus transduced cells and shRNA EGR2 transduced cells. (See also Table S4 ).
Article Snippet: PEG10 (Hs00248288_s1) , Thermo Fisher , 4331182.
Techniques: Transfection, Derivative Assay, Cell Culture, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Immunofluorescence, Staining, Generated, Virus, shRNA, Transplantation Assay
Journal: iScience
Article Title: Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis
doi: 10.1016/j.isci.2022.104498
Figure Lengend Snippet:
Article Snippet: PEG10 (Hs00248288_s1) , Thermo Fisher , 4331182.
Techniques: RNA Extraction, RNA Library Preparation, Viability Assay, shRNA, Transduction, Control, Software