EMBO/SourceData huggingface dataset:
Figure 3A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting.B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies.MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.
Figure 4(A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.MCF10A WT or two independent clones for MAVS, RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, (B cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.C, MCF10A WT or two independent clones for RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.D, MCF10A WT or two independent clones for MDA5, KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later E) cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.F) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.
Figure 5A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.B, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean + standard error of the mean of three biological replicates.C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.D, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO MC38 cells. Data are presented as mean + standard error of the mean of three biological replicates.
Figure 6A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.
f1(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(b) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulphate (SDS) and analysed by immunoblotting with indicated antibodies.(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(g,h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 h and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h; n=7). Scale bar, 5 μm. All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
f2HepG2 cells were treated with bovine serum albumin (Con or (−)), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 9 h. (a-d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a,b) or Fura-2-AM (c,d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b,d; n=8 and 30, respectively).(e-h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analysed by immunoblotting (e,f) and quantified (g,h; n=3).(i-l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i,k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j; n=10). Co-localization between LAMP1 and LC3 was quantified (l; n=4). Boxed areas in fluorescence images are magnified in right-most panels (i,k). Scale bar, 10 μm (a), 20 μm (c), 5 μm (i,k). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
f3Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet (LFD)-kept mice (n=5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period.(c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) were measured from indicated mice.(c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) and total epididymal white adipose tissue (eWAT) mass (e) were measured from indicated mice.(f-h) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription-PCR.(i,j) Liver sections were analysed by hematoxylin and eosin (HE, upper panels) and Oil Red O (ORO, lower panels) staining (i). ORO densities were quantified (j).(k,l) Calcium-induced CaMKII autophosphorylation in livers was analysed by immunoblotting (k) and quantified (l).(m) Primary hepatocytes from 2-month-old C57BL/6 mice kept on LFD were treated with bovine serum albumin (Con), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 12 h. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n=17). Scale bar, 1 cm (c), 200 μm (i). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
f4Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a-d) Livers were subjected to solubility fractionation. 1% Triton X-100-soluble (a) and -insoluble (b) fractions were analysed by immunoblotting (a,b) and quantified (c,d).(e,f) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining (e). p62-positive areas were quantified (f). Scale bar, 200 μm (e). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
f5Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a,b) Levels of LC3-II from 1% Triton X-100-insoluble fraction of livers were analysed by immunoblotting (a) and quantified (b).(c,d) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining (c). Co-localization between LC3 and LAMP1 was quantified (d). Boxed areas in fluorescence images are magnified in right-most panels (c). Scale bar, 5 μm (c). All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.
f6Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice of same age (n=5) were used as a negative control. (a,b) Liver sections were subjected to F4/80 immunostaining, which visualizes macrophage infiltration and hematoxylin counterstaining (a). F4/80-positive areas were quantified (b).(c-f) Glucose tolerance tests (GTT, c,d) and insulin tolerance tests (ITT, e,f) were conducted using indicated mice (c,e). Area under the curve (AUC) was quantified from GTT and ITT data (d,f).(g) Serum insulin levels were measured from indicated mice before (Basal) and 10 min after (Glucose stimulated) glucose injection (n=4).(h,i) Pancreas sections were analysed by hematoxylin and eosin (HE) staining (h). Islet areas were quantified (i; n=4). Scale bar, 200 μm (a), 100 μm (h). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test).
f7(a-g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=4) for 10 days. Systolic blood pressure (a), LV wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV) and cardiac output (g, CO) were analysed by tail-cuff method (a) or echocardiography (b-g). All data are shown as mean±s.e.m. *P0.05 (Student's t-test).
Figure 1A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 hours (Fasted group) and then refed for 4 hours (Fasted+Refed group) normalized to mice fed for 24 hours (Fed group). n=5, data represent mean ± SEM, unpaired t-test.C. Blood glucose levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.D. Plasma insulin levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, Welch's t-test.E. Blood glucose levels for insulin tolerance test of control (n=7) and NICDiOE-EC control (n=7) mice. Data represent mean ± SEM, unpaired t-test.F. Quantification of area under curve for insulin tolerance test in E. Data represent mean ± SEM, Welch's t-test.G. Blood glucose levels for glucose tolerance test of control (n=6) or NICDiOE-EC (n=7) mice. Data represent mean ± SEM, unpaired t-test. H. Quantification of area under the curve for glucose tolerance test in G. Data represent mean ± SEM, unpaired t-test.
Figure 2A. Blood glucose levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.B. Plasma insulin levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.C. Blood glucose levels for insulin tolerance test of control (n=10) and Rbpji∆EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.D. Quantification of area under curve for insulin tolerance test in C. Data represent mean ± SEM, Welch's t-test.E. Blood glucose levels for glucose tolerance test of control (n=6) or RbpjiΔEC (n=5) mice. Data represent mean ± SEM, unpaired t-test. F. Quantification of area under the curve for glucose tolerance test in E. Data represent mean ± SEM, Welch's t-test.
Figure 3A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm.B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm.C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test. E. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test.
Figure 4B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test.C. Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells (MCECs). D. Densitometric analysis of western blot in C. Data represented as fold change over control. n=3, data represent mean ± SEM, unpaired t-test. E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.
Figure 5A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation. C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test. D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction. E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test. F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test.G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test.H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test.I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05.Representative immune-histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.
Figure 6B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test. G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.
Figure 7B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM. C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test. D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test. F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test. K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.
Figure 1(C-D) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.(E-F) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.(G) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.(H-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.
Figure 2(A-B) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.(C-D) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.