EMBO/soda-nel huggingface dataset:
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 1

Figure 1. LSD1 is secreted from gastric cancer cells through small extracellular vesicles (sEVs).

(A) Sphere formation in MGC-803 cells incubated with fresh medium or conditioned medium from MGC-803 cells for 7 days. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± standard error of mean (SEM); *P = 0.0146; two-tailed unpaired Student's t-test).

(B) Sphere formation in MGC-803 cells with indicated treatment. The number of spheres was quantified and indicated on the right. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; no significant differences; two-tailed unpaired Student's t-test).

(C) Sphere formation in MGC-803 cells incubated with an equal volume of phosphate-buffered saline, supernatant after differential centrifugation, or sEVs. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; ns = no significant difference; *P = 0.0390; two-tailed unpaired Student's t-test).

(D) Sphere formation in MGC-803 cells treated with sEVs (20 μg/mL) from five gastric cancer cell lines as indicated (n = 3 biological replicates; mean ± SEM; ***P = 0.0007 (MGC-803), **P = 0.0061 (HGC-27), and **P = 0.0018 (BGC-823); two-tailed unpaired Student's t-test).

(E) Expression levels of LSD1 in MGC-803, MKN-45, HGC-27, BGC-823, and NCI-N87 cell lines and their corresponding sEVs. The samples with equal amounts of proteins were loaded (n = 3 biological replicates; mean ± SEM; compared with the NCI-N87; *P = 0.0432 (cell/MGC-803), *P = 0.0300 (cell/HGC-27), **P = 0.0306 (cell/BGC-823), **P = 0.0018 (sEVs/MGC-803), **P = 0.0029 (sEVs/HGC-27), and ***P = 0.0003 (sEVs/BGC-823); two-tailed unpaired Student's t-test; GAPDH was used as a loading control for cell lysis; CD9 was used as a loading control for sEV lysis).

(F) Establishment of LSD1 knockout (KO) MGC-803 cell line. Con indicates MGC-803 cells, while KO indicates LSD1 KO MGC-803 cells.

(G and H) Transmission electron microscopy images (G) and the size distribution (H) of sEVs from MGC-803 and LSD1 KO MGC-803 cells. Scale bar = 100 nm.

(I) Expression levels of LSD1, CD63, CD9, TSG101, and calnexin in sEVs from MGC-803 and LSD1 KO MGC-803 cells. sEVs were extracted using two different extraction methods (ultracentrifugation (UC) and commercial kit). Calnexin is an sEV negative marker. UC, sEVs isolated using the ultracentrifugation method; Kit, sEVs isolated using the commercial kit.

(J) Expression levels of LSD1, CD63, and CD9 in sEVs with indicated treatment.
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 2

Figure 2. LSD1-containing small extracellular vesicles (sEVs) fuse to the recipient cell and deliver LSD1.

(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.

(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 3

Figure 3. LSD1 facilitates stemness and promotes the accumulation of SOX2.

(A) Expression level of LSD1 (KDM1A) in gastric cancer and non-cancerous tissues from UALCAN datasets (STAD, stomach adenocarcinoma; central band, boxes, and whiskers of the boxplot represent the median, first quartile, third quartile, minimum, and maximum values, respectively).

(B) Expression level of LSD1 (KDM1A) in gastric cancer or non-cancerous tissues from Gene Expression Profiling Interactive Analysis (GEPIA) datasets (central band, boxes, and whiskers of the boxplot represent the median, first quartile, third quartile, minimum, and maximum values, respectively).

(C) Overall survival analysis using GEPIA datasets (log-rank test; the solid line represents the survival curve, while the dashed line represents the 95% confidence interval).

(D) In vitro limiting dilution assay with MGC-803 and LSD1 knockout (KO) MGC-803 cells (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval).

(E) Sphere formation assay results of MGC-803 and LSD1 KO MGC-803 cells. Scale bar = 100 µm (n = 3 biological replicates; mean ± standard error mean (SEM); **P = 0.0058; two-tailed unpaired Student's t-test).

(F) Expression levels of LSD1, Nanog, OCT4, SOX2, and CD44 in LSD1 KO MGC-803 cells.

(G) Correlation between LSD1 and SOX2 mRNA levels analyzed using the GEPIA dataset (Pearson's test).

(H) Correlation between LSD1 and SOX2 in 172 gastric cancer tissues (Pearson's test).

(I) The mRNA levels of SOX2 in different cells were detected using quantitative real-time polymerase chain reaction (n = 3 biological replicates; mean ± SEM).

(J) Stability of SOX2 in MGC-803 and LSD1 KO MGC-803 cells treated with cycloheximide (20 μM) at the indicated times.

(K) Relative intensity of SOX2 in (J) (n = 3 biological replicates, mean ± SEM).

(L) Immunoprecipitation of Kme1/2 and ubiquitin (Ub) with SOX2 in the presence or absence of LSD1. The cells were treated with MG132 (10 μM) for 8 h before analysis.

(M) Reverse immunoprecipitation of Kme1/2 on SOX2.

(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).

(O) Stability of SOX2 in HEK293T cells co-transfected with different plasmids (WT, wild type; SOX2-Mut, K42R and K117R mutations; LSD1-Mut: K661A mutation).

(P) Relative intensity of SOX2 in (O) (n = 3 biological replicates; mean ± SEM; **P = 0.0055; two-tailed unpaired Student's t-test).
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 4

Figure 4. LSD1-containing small extracellular vesicles (sEVs) promote cancer cell stemness in vitro.

(A) Sphere formation assay of MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 knockout (KO) MGC-803 cells for 7 days (n = 3 biological replicates; mean ± standard error of mean (SEM); **P = 0.0090 (MGC-803) and **P = 0.0012 (MKN-45); two-tailed unpaired Student's t-test; scale bar = 100 µm).

(B) In vitro limiting dilution assays performed using MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 14 days (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval. The circles and triangles represent data from different groups).

(C) Expression levels of LSD1, OCT4, and SOX2 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h.

(D) Quantification of the results of (C) (n = 3 biological replicates; mean ± SEM; *P = 0.0385 (LSD1), *P = 0.0160 (OCT4), and ***P = 0.0006 (SOX2) for MGC-803; *P = 0.0299 (LSD1), *P = 0.0147 (OCT4), and *P = 0.0258 (SOX2) for MKN-45; two-tailed unpaired Student's t-test).

(E) Expression level of CD44 in MGC-803 and MKN-45 cells incubated with 20 μg/mL sEVs from MGC-803 or LSD1 KO MGC-803 cells for 48 h.

(F) Sphere formation assay results of MGC-803 (upper panel) and MKN-45 (bottom panel) cells incubated with 20 μg/mL sEVs from HEK293T cells (left panel), WT-LSD1 sEVs (middle panel), and LSD1 K661A sEVs (right panel) for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0021 (MGC-803) and **P = 0.0020 (MKN-45); two-tailed unpaired Student's t-test).
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 5

Figure 5. LSD1-containing small extracellular vesicles (sEVs) promote gastric cancer cell stemness in vivo and clinical samples.

(A and B) In vivo limiting dilution assay results of MGC-803 cells treated with phosphate-buffered saline, BBI608, sEVs from control cells (Con sEVs), and sEVs from LSD1 knockout (KO) cells (KO sEVs) as indicated for 28 days in BALB/c-nu mice. Representative images of tumors excised from the mice (A) and the frequency of tumor formation (B) are shown (the solid line represents the sphere formation ability curve, while the dashed line represents the 95% confidence interval. The circles and triangles represent data from different groups).

(C) Tumor volume of each group subjected to in vivo limiting dilution assay with 5×106 cells with indicated treatment (n = 6 biological replicates; mean ± standard error of mean(SEM), *P = 0.0441 and ***P = 0.0002; two-tailed unpaired Student's t-test).

(D) Tumor weight of each group subjected to in vivo limiting dilution assay (n = 6 biological replicates; mean ± SEM; ***P < 0.0001, *P = 0.0285, *P = 0.0162, *P = 0.0498, and *P = 0.0049; two-tailed unpaired Student's t-test).

(E) Expression levels of LSD1, CD44, SOX2, and OCT4 in tumor tissues subjected to in vivo limiting dilution assay performed with 5×106 cells.

(F) Immunofluorescence image and quantification of the expression of SOX2 in tumor tissues subjected to in vivo limiting dilution assay. Scale bar = 50 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0024, **P = 0.0088, and *P = 0.0342; two-tailed unpaired Student's t-test).

(G) Representative images of second-generation tumors (left) and the tumor formation rate (right) in each group.

(H) Second-generation tumor weight in each group (n = 6 biological replicates; mean ± SEM; *P = 0.0091; two-tailed unpaired Student's t-test).

(I and J) Expression levels of LSD1 in sEVs isolated from the plasma. CD9 and CD63 were used as markers of sEVs. CD9 was used as a loading control for sEV lysis (n = 3 biological replicates; mean ± SEM; *P = 0.0256 (patient 1), **P = 0.0068 (patient 2), **P = 0.0013 (patient 3), **P = 0.0031 (patient 4), ***P = 0.0009 (patient 5), **P = 0.0012 (patient 6), ***P = 0.0007 (patient 7), ***P = 0.0010 (patient 8), ***P = 0.0001 (patient 9), and *P = 0.0461 (patient 10); two-tailed unpaired Student's t-test).

(K) Sphere formation assay results of MGC-803 cells treated with sEVs as indicated for 7 days. Scale bar = 100 µm (n = 3 biological replicates; mean ± SEM; **P = 0.0019 and **P = 0.0050; two-tailed unpaired Student's t-test).
Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness (2021)
Figure 6

Figure 6. LSD1-containing small extracellular vesicles (sEVs) induce oxaliplatin resistance in gastric cancer cells.

(A-D) Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C), and LSD1 knockout (KO) MGC-803 (D) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).

Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).

(G) Tumor growth rate in mice treated with oxaliplatin in the presence or absence of sEVs. Tumor growth rate was measured according to tumor volume (n = 3 biological replicates; mean ± SEM; *P = 0.0439; two-tailed unpaired Student's t-test).

(H) Proliferation assay results of MGC-803 cells treated with oxaliplatin in the presence or absence of sEVs from the plasma of patients with gastric cancer or healthy individuals (n = 3 biological replicates; mean ± SEM; *P = 0.0487; two-tailed unpaired Student's t-test).

(I) Schematic model for the shuttling of LSD1 from parent cells to recipient cells using sEVs as vehicles. The sEV-delivered LSD1 promotes recipient gastric cancer stemness and chemoresistance.
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 2

Figure 2. DVL3 genomic binding in MDA-MB-468 and MCF7 cells

(A) An assembly of IgG (first row) and DVL3 (second row) ChIP-Seq data in MDA-MB-468 for the ARAP1, RFX5-GBAT2, WDR74, POU2F3, SNORD3D and HLA-A genes, visualized by IGV.

(B) An assembly of IgG (first row) and DVL3 (second row) ChIP-Seq data in MCF7 for the ARAP1, RFX5-GBAT2, ZNF672, SSC5D, HLA-A and HLA-E genes, visualized by IGV.

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).

(D) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).

(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.

(F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells.

Data information: For (A) and (B) each column is 4000 bp wide. The third rows show the gene nearest to the ChIP-Seq alignment including its location, and orientation. The medium thick dark lines are the UTRs of the gene and the thicker dark regions are exons followed by thin lines with arrows which are the introns. C and D panels show a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For C and D panels an independent t test was used for statistical comparison. * p < 0.05; n=3, technical replicates; data show means ± SD. For E and F panels transcript levels were normalized to actin transcript levels, an independent t test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3, biological replicates.
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 4

Figure 4. Localization of H3K4me3 mark respect to DVL3 genomic binding in MDA-MB-468 and MCF7 cells

(A) An assembly of IgG (first row), DVL3 (second row) and H3K4me3 peak (third row) ChIP-Seq data in MDA-MB-468 for the ARAP1, RFX5-GBAT2, APOC1P1, PSMB8, TAP1 and HLA-A genes, visualized by IGV. The green rectangular block is the location of the statistically-qualified peak of H3K4me3 in MDA-MB-468.

(B) An assembly of IgG (first row), DVL3 (second row) and H3K4me3 (third row) ChIP-Seq data in MCF7 for the HAUS5, APOC1P1, ZNF672, HLA-A and HLA-E genes, visualized by IGV.

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).

(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).

(E) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H3K4me3 ChIP-Seq

(F) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H3K4me3 ChIP-Seq

(G) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and FAIRE Seq.

Data information: For (A) and (B) each column is 4000 bp wide. The fourth rows show the gene nearest to the ChIP-Seq alignment, including its location and orientation. C and D panels show a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For C and D panels, an independent t-test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3, technical replicate.
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 5

Figure 5. Localization of KMT2D and epigenetic enhancer marks with respect to DVL3 genomic binding in MDA-MB-468 and MCF7 cells

(A) An assembly of IgG (first row), DVL3 (second row) and KMT2D (third row) ChIP-Seq data in MCF7 for the HAUS5, WDR74, YWHAQ, CDNF/HSPA14 and PRDM10 genes, visualized by IGV. The bar below the DVL3 peak is the location of the statistically-qualified peak of KMT2D in MCF7.

(B) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and KMT2D ChIP-Seq.

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).

(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.

(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.

(F) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq.

(G) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H3K27ac ChIP-Seq.

(H)Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq

(I) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and H4K8ac ChIP-Seq.

(J) Venn diagram showing the overlap between MDA-MB-468 DVL3 ChIP-seq peaks and super-enhancer targets in basal breast carcinoma (BRCA)

(K) Venn diagram showing the overlap between MCF7 DVL3 ChIP-seq peaks and super-enhancer targets in luminal breast carcinoma.

Data information: Panel C shows a representative ChIP-qPCR of three independent ChIP-qPCR experiments. For panel C, an independent t-test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3, technical replicate. For (A), (D) and (E) each column is 4000 bp wide. The fourth rows (panel A) and the fifth rows (panel D and E) show the gene nearest to the ChIP-Seq alignment, including its location and orientation.
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 6

Figure 6. The effects of silencing DVL3 on KMT2D expression and subcellular localization

(A) DVL3 co-immunoprecipitates KMT2D in MDA-MB-468 cells (upper panel) and MCF7 cells (lower panel). IgG heavy chain (Hc) was blotted for as a control for equal antibody capture for immunoprecipitation and whole cell extracts (WCE) as a positive control.

(B) MDA-MB-468 histone extracts from NTC, shA DVL3 and shB DVL3 subjected to immunoblot analysis for H3K4me3 and H3.

(C) An assembly of IgG (first row), DVL3 (second row) ChIP-Seq in MDA-MB-468 cells, IgG (third row), and, DVL3 (fourth row) ChIP-Seq data in MCF7 for the KMT2D gene, visualized by IGV. Each column is 20000 bp wide. The fifth row show the location and orientation of the KMT2D gene.

(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.

(E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells.

(F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells.

(G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells.

(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm

(I) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and KMT2D at RFX5, HAUS5, COX14, APOC1P1, WDR74, JUNB, and, HLA-A promoters analyzed by end-point PCR.

(J) Sequential ChIP (ChIP-reChIP) to examine occupancy of DVL3 and H3K4me3 at RFX5, APOC1P1, JUNB, and, HLA-A promoters analyzed by end-point PCR.

Data information: For D-G panels transcript levels were normalized to actin transcript levels and an independent t test was used for statistical comparison. * p < 0.05; data show means ± SD, n=3 biological replicates.
Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth (2021)
Figure 7

Figure 7. The effects of silencing DVL3 on the formation and progression of spheroid and orthotopic xenograft tumor models.

(A) Quantification of spheroids' area in MDA-MB-468 cell line (NTC vs. shDVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 32h *p < 0.05.

(B) Quantification of the spheroids' area in the MCF7 cell line (NTC, shA DVL3 and shB DVL3). The data are presented as the mean ± SD, n = 3 showing statistical difference after 100h *p < 0.05.

(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal.

(D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9.

(E) Tumor pictures of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Scale shown in cm.

(F) Final weight of tumors resulting from the injections of MDA-MB-468 (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, One-way ANOVA was used for statistical comparison, n ≥ 9.

(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal.

(H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.

Data information: For all panels: T-Test was used for statistical comparison, unless is specificated, data show means ± SD, * indicates significant difference from control p˂0.05.