Fig 2: L3MBTL3-mediated promotion of BC cell migration and invasion is dependent on STAT3.The migration assays in (a) L3MBTL3 overexpression MCF7 cells and (b) L3MBTL3-rescued SUM159PT cells transfected with STAT3 siRNA (siSTAT3 1 and siSTAT3 2). c The invasion assays in L3MBTL3-rescued SUM159PT cells transfected with STAT3 siRNA (siSTAT3 1 and siSTAT3 2). The migration assays in (d) L3MBTL3-overexpressing MCF7 cells and (e) L3MBTL3-rescued SUM159PT cells treated or untreated with NIF. f The invasion assay in L3MBTL3-rescued SUM159PT cells treated or untreated with NIF. For (a–f), n = 3 biological replicates, mean ± SD, one-way ANOVA test. Source data are provided as a Source Data file.

Fig 3: L3MBTL3 upregulated the expression of SNAIL in BC.a ChIP-seq profiles (top) and heatmaps (bottom) showing the occupancy of genome-wide L3MBTL3 peaks in SUM159PT cells and STAT3 in MDA-MB-231 cells. The occupancy sites ranged from −2.5 kb before the transcription start site (TSS) to 2.5 kb after the transcription end site (TES). Technical replicates of L3MBTL3 rep-1 and L3MBTL3 rep-2 were used. b Venn diagram of the common L3MBTL3- and STAT3-occupied genes detected by ChIP-seq in SUM159PT cells and DEGs detected by RNA-seq in L3MBTL3 rescued SUM159PT cells. Technical replicates of L3MBTL3 rep-1 and L3MBTL3 rep-2 were used. Q-PCR analysis showing the transcript levels of SNAIL, E-cad, OCLN, and DSP in (c) L3MBTL3-overexpression MCF7 cells and (f) L3MBTL3 knockout and (h) rescued MDA-MB-231 and SUM159PT cells. n = 3 biological replicates, mean ± SD. Statistical analysis according to two-tailed unpaired t test (c, f) and one-way ANOVA test (h). d WB analyses showing the protein levels of SNAIL and E-cad in L3MBTL3-overexpression MCF7 cells. The antibody β-TUBULIN was used as a loading control. e Immunofluorescence showing the E-cad expression in L3MBTL3-overexpression MCF7 cells. Nuclei were stained with DAPI (scale bar, 20 μm). WB analyses showing the protein levels of SNAIL in L3MBTL3 (g) knockout and (i) rescued MDA-MB-231 and SUM159PT cells. For (d, e, g, i), each experiment included three biological replicates with similar results. j Scatter plots showing the positive correlation between L3MBTL3 and SNAIL transcript levels in TCGA BC dataset (TCGA BRCA 2017, n = 1218) and GEO dataset (GSE202203, n = 3207, two-tailed Spearman correlation test). k Distant metastasis free survival in breast cancer dataset (GSE12276, n = 204) was analyzed according to L3MBLT3 and SNAIL transcription level (L3MBTL3low SNAILlow, n = 37; L3MBTL3low SNAILhigh, n = 19; L3MBTL3high SNAILlow, n = 91; L3MBTL3high SNAILhigh, n = 57). The Distant metastasis free survival of L3MBTL3high SNAILhigh group (n = 57) was much lower than L3MBTL3high SNAILlow group (n = 91). The Distant metastasis free survival of L3MBTL3 high SNAILhigh group (n = 57) was much lower than L3MBTL3low SNAILlow group (n = 37). Statistical analysis according to log-rank test. Source data are provided as a Source Data file.

Fig 4: The interaction between L3MBTL3 and STAT3 is required for BC metastasis.a The migration assays in L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) expression MCF7 cells. The migration and invasion assays in L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) rescued SUM159PT cells. (n = 3 biological replicates, one-way ANOVA test, mean ± SD) b L3MBTL3 and Flag antibodies were used to detect the expression of L3MBTL3 in the control (NC + V), L3MBTL3 knockdown (L3-KD2 + V) and L3MBTL3 wildtype (L3-KD2 + L3) and mutant (L3-KD2 + L3-Δ176-193) rescued stable 4T1 cell lines. β-TUBULIN and β-ACTIN antibodies were used as loading controls. The double asterisks (**) indicated the mouse L3mbtl3 protein, and the single asterisks (*) indicated the human L3MBTL3 protein in left panel. Each blotting experiment include three biological replicates with similar results. c–f (c) The lung metastasis foci (scale bar, 5 mm) and (d) the HE staining of lung sections (scale bar, 4 mm) in the lung metastasis model induced by NC (NC + V) and L3MBTL3 knockdown, L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) rescued 4T1 cells. The metastasis foci and the metastatic areas in the lung sections were indicated by single asterisks (*), respectively. e The number of metastatic nodules or f the percentage of metastatic area in the lung sections were quantified and shown as scatter plots (n = 10 mice, Welch one-way ANOVA test, mean ± SD). g–j (g)The lung metastasis foci (scale bar, 5 mm) and (h) the HE staining of lung sections (scale bar, 4 mm) in the spontaneous lung metastasis model induced by NC (NC + V) and L3MBTL3 knockdown, L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) rescued 4T1 cells. The metastasis foci and the metastatic areas in the lung sections were indicated by single asterisks (*). i The number of metastatic nodules (n = 8 mice, Welch one-way ANOVA test, mean ± SD) or j the percentage of metastatic area in the lung sections (n = 8 mice, Kruskal-Wallis one-way ANOVA test, mean ± SD) were quantified and shown as scatter plots. k A schematic of how L3MBTL3 and STAT3 collaboratively upregulate SNAIL expression to promote metastasis in BC. Source data are provided as a Source Data file.

Fig 5: The interaction between L3MBTL3 and STAT3 is required for SNAIL expression.a Comparable enrichment analysis from ChIP sequence between L3-KO5 + L3 vs. L3-KO5 + V and STAT3 using MSigDB database. b Ratio plot depicting the differential binding signal at the differential binding genes in the L3-KO5 + L3 vs. L3-KO5 + L3-Δ176-193 group and the L3-KO5 + L3 vs. L3-KO5 + V group of SUM159PT cells. c Ratio plot depicting the differential binding signal at the differential binding genes in the L3-KO5 + L3 vs. L3-KO5 + L3-Δ176-193 group and differential expression level of the DEGs in C vs. L3-KO5 group of SUM159PT cells. d Volcano plot representing the transcript level of DEGs in common group in (c). The STAT3 target genes SNAIL, CEBPD, UGCG, SOD2, PIM1, NFKBIA, NFATC1, JUNB and FANCC were found in transcriptional upregulation group and labeled. e Venn diagram of the L3-KO5 + L3 vs. L3-KO5 + V different binding genes detected by ChIP-seq and DEGs detected by RNA-seq in L3MBTL3 knockout group and STAT3 knockdown group. f UCSC genome browser tracks of Flag ChIP-seq signals in the vicinity of the SNAIL gene in L3MBTL3 knockout (L3-KO5 + V), L3MBTL3 wildtype (L3-KO5 + L3) and mutant rescued (L3-KO5 + L3-Δ176-193) SUM159PT cells. Rep-1 and rep-2 were used for technical replicates. g ChIP assays were performed in (c) L3MBTL3 knockout (L3-KO5 + V), L3MBTL3 wildtype (L3-KO5 + L3) and mutant (L3-KO5 + L3-Δ176-193) rescued SUM159PT cells using L3MBTL3 and p-STAT3Y705 antibodies. Primer specific for the P3 region on the SNAIL gene promoter were used (n = 3 biological replicates, mean ± SD, one-way ANOVA test). h Q-PCR showing the transcription levels of L3MBTL3 and SNAIL in L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) overexpression MCF7 cells and in L3MBTL3-knockout, L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) rescued SUM159PT cells (n = 3 biological replicates, mean ± SD, one-way ANOVA test). i Western blot showing the protein levels of L3MBTL3 and SNAIL in L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) overexpression MCF7 cells and in L3MBTL3-knockout, L3MBTL3 wildtype (L3) and mutant (L3-Δ176-193) rescued SUM159PT cells. The β-TUBULIN antibody was used as a loading control. The blotting experiment include three biological replicates with similar results. Source data are provided as a Source Data file.