Figure 1 A ) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous , CAF1 - knockdown - induced ( through p60 or p150 KD ) 2 - cell - like cells . Fold - changes relative to ESCs were calculated based on bulk RNA - seq data C ) Oxygen consumption rate of ES ( blue line ) and 2 - cell - like cells ( green line ) . Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose , L - glutamine and pyruvate . Basal , maximal ( FCCP - induced ) and non - mitochondrial ( Rotenone and Antimycin - A mediated ) respiratory rates are indicated . A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown . Due to the low number of 2 - cell - like cells available , compared to ESCs , one technical replicate of the former was analysed per biological replicate , while three or more technical replicates were performed for the latter . Accordingly , mean ± S . D . of technical replicates is shown for ESCs . D ) Basal oxygen consumption rate of ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser . Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose , L - glutamine and pyruvate . Boxes indicate the range between the first and third quantile , the band specifies the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each of the individual technical replicates . E ) Oxygen consumption rate of ES ( blue line ) , Zscan4 + ( red line ) and 2 - cell - like cells ( green line ) in glucose - free media and upon acute injection of sodium pyruvate or sodium L - lactate . Note that L - glutamine - but not glucose or pyruvate - was initially present in the assay medium . Maximal ( FCCP - induced ) and non - mitochondrial ( Rotenone and Antimycin - A mediated ) respiratory rates following pyruvate or lactate treatment are also are indicated . A graph including data from 3 independent biological replicates is presented , and the mean ± S . D . of technical replicates is shown . F ) Representative electron microscopy micrographs of mitochondria from ES ( n = 49 sections ) and 2 - cell - like cells ( n = 57 sections ) generated across 2 independent technical and biological replicates . Scale bar , 1 μm . Representative images from 2 independent biological and technical replicates are shown . 162 and 99 mitochondria were analysed for 2 - cell - like and ES cells , respectively . G ) Representative single section of CellROX - DeepRed fluorescence in ES and 2 - cell - like cells ( green arrow ) obtained using live cell microscopy . Scale bar , 10 μm . n , indicates the number of independent replicates . Representative images from 3 independent biological replicates are shown . H ) FACS - assisted quantification of CellROX - DeepRed fluorescence intensity in ES and 2 - cell - like cells . Measurements were obtained from 2 independent biological replicates . * * * P < 0 . 005 ; Mann - Whitney U test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous, CAF1-knockdown-induced (through p60 or p150 KD) 2-cell-like cells. Fold-changes relative to ESCs were calculated based on bulk RNA-seq data  C) Oxygen consumption rate of ES (blue line) and 2-cell-like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Basal, maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates are indicated. A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2-cell-like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± S.D. of technical replicates is shown for ESCs.  D) Basal oxygen consumption rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Boxes indicate the range between the first and third quantile, the band specifies the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates.  E) Oxygen consumption rate of ES (blue line), Zscan4+ (red line) and 2-cell-like cells (green line) in glucose-free media and upon acute injection of sodium pyruvate or sodium L-lactate. Note that L-glutamine - but not glucose or pyruvate - was initially present in the assay medium. Maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates following pyruvate or lactate treatment are also are indicated. A graph including data from 3 independent biological replicates is presented, and the mean ± S.D. of technical replicates is shown.  F) Representative electron microscopy micrographs of mitochondria from ES (n=49 sections) and 2-cell-like cells (n=57 sections) generated across 2 independent technical and biological replicates. Scale bar, 1 μm. Representative images from 2 independent biological and technical replicates are shown. 162 and 99 mitochondria were analysed for 2-cell-like and ES cells, respectively.  G) Representative single section of CellROX-DeepRed fluorescence in ES and 2-cell-like cells (green arrow) obtained using live cell microscopy. Scale bar, 10 μm. n, indicates the number of independent replicates. Representative images from 3 independent biological replicates are shown.  H) FACS-assisted quantification of CellROX-DeepRed fluorescence intensity in ES and 2-cell-like cells. Measurements were obtained from 2 independent biological replicates. *** P < 0.005; Mann-Whitney U test.


Figure 2 A ) ATP content in ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 4 independent biological replicates . B ) Extracellular acidification rate of ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser . C ) Glucose uptake rates in Zscan4 + ( red ) and 2 - cell - like cells ( green ) were measured using a luciferase - based assay across 4 independent biological replicates and are represented relative to those of control ES cells ( blue ) . F ) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES , Zscan4 + and 2 - cell - like cells . Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA . Shown are the mean ± s . d . of the indicated number of independent cell cultures , performed across 2 or more independent biological replicates each . G ) Glucose - 6 - phosphate dehydrogenase activity was measured in ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cell ( green ) lysates using a fluorometric assay . Measurements were obtained from 3 independent biological replicates , performed in 3 technical replicates each . n . s . - not significant ; one - way ANOVA . Data information : In panels A - C and G , boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each individual technical replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates.  B) Extracellular acidification rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser.  C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue).  F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each.  G) Glucose-6-phosphate dehydrogenase activity was measured in ES (blue), Zscan4+ (red) and 2-cell-like cell (green) lysates using a fluorometric assay. Measurements were obtained from 3 independent biological replicates, performed in 3 technical replicates each. n.s. - not significant; one-way ANOVA. Data information: In panels A-C and G, boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate.


Figure 3 B ) Heatmap showing the effect of the 20 metabolites tested on 2 - cell - like cell levels . Percentage of 2 - cell - like cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Percentage of Zscan4 + cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Percentage of Zscan4 + cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Percentage of 2 - cell - like cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 B) Heatmap showing the effect of the 20 metabolites tested on 2-cell-like cell levels.  Percentage of 2-cell-like cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.  Percentage of Zscan4+ cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.  Percentage of Zscan4+ cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.  Percentage of 2-cell-like cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


Figure 4 A ) Immunofluorescence staining for OCT4 , ZSCAN4 and 2C : : tbGFP in control and acetate treated ESC cultures . Green arrows indicate 2 - cell - like cells , and inlets highlight their DAPI structure . Scale bar , 20 μm . B ) RT - qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h . Shown are the mean ± s . d . of 3 independent cell cultures , performed in 2 technical replicates . C ) Percentage of 2 - cell - like cells obtained upon transfection of control or Dux - targeting siRNAs in control conditions or in combination with sodium acetate treatment . Measurements were obtained from 2 independent cell cultures , performed across 3 independent biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each technical replicate . D ) Percentage of 2 - cell - like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment . Shown are the mean ± s . d . of 4 independent cell cultures , performed across 2 independent biological replicates . F ) Proportion of Zscan4 + cells at various timepoints during the time - lapse experiment . Shown are the mean ± s . d . of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm.  B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates.  C) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate.  D) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates.  F) Proportion of Zscan4+ cells at various timepoints during the time-lapse experiment. Shown are the mean ± s.d. of 3 independent experiments.


Figure 1 Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( A ) Confocal micrographs of the representative field of the LN section . Scale bar : 10 μm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( B ) Histograms illustrated that the dynamic changes of GFP + 4T1 cells metastasized to the inguinal LN . Cells were counted in whole sections ( 2 non - consecutive sections per LN per mouse ) . Each group was composed of at least two mice . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( C ) Soft - agar colony forming activity was examined in 4T1PT and 4T1LN cells ( 5x102 cells / well , n = 4 wells per group ) . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( C ) , * * P = 0 . 000088 and * * P = 0 . 000004 for the 4T1LN cells at week 2 and week 3 , respectively . ( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( E ) , * * P = 0 . 000006 . In ( G ) * * P = 0 . 000734 . Level of significance was determined using two - tailed unpaired t - test . ( F and G ) BALB / c mice ( n = 3 mice per group ) were injected with 2x105 4T1PT or 4T1LN cells via tail - vein . Lung colonization was examined by lung morphology ( F ) and the numbers of tumor nodule ( G ) on day 21 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( F and G ) BALB / c mice ( n = 3 mice per group ) were injected with 2x105 4T1PT or 4T1LN cells via tail - vein . Lung colonization was examined by lung morphology ( F ) and the numbers of tumor nodule ( G ) on day 21 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . ( H and I ) NSG mice ( n = 5 mice per group ) were injected with 4T1PT ( 5x102 ) , 4T1LN ( 5x102 ) , EMT6PT ( 1x102 ) , or EMT6LN ( 1x102 ) cells . Distant organ metastasis was examined by bioluminscent images on day 28 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . ( H and I ) NSG mice ( n = 5 mice per group ) were injected with 4T1PT ( 5x102 ) , 4T1LN ( 5x102 ) , EMT6PT ( 1x102 ) , or EMT6LN ( 1x102 ) cells . Distant organ metastasis was examined by bioluminscent images on day 28 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (A) Confocal micrographs of the representative field of the LN section. Scale bar: 10 μm. Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (B) Histograms illustrated that the dynamic changes of GFP+ 4T1 cells metastasized to the inguinal LN. Cells were counted in whole sections (2 non-consecutive sections per LN per mouse). Each group was composed of at least two mice.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. (C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays: (C) Soft-agar colony forming activity was examined in 4T1PT and 4T1LN cells (5x102 cells/well, n = 4 wells per group). Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (C), **P = 0.000088 and **P = 0.000004 for the 4T1LN cells at week 2 and week 3, respectively.(C-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays:(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (E), **P = 0.000006. In (G) **P = 0.000734. Level of significance was determined using two-tailed unpaired t-test.(F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(F and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.(H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.(H and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


Figure 2 ( C and D ) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor - bearing mice after tumor resection with ( n = 13 mice ) or without ( n = 10 mice ) tumor - draining lymph node dissection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section . ( E and I ) Summary table of distant organ metastasis derived from each surgery group . ( E and I ) Summary table of distant organ metastasis derived from each surgery group . ( G and H ) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre - removed tumor - bearing mice ( n = 9 mice ) or sham surgery tumor - bearing mice ( n = 5 mice ) after tumor resection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (C and D) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor-bearing mice after tumor resection with (n=13 mice) or without (n=10 mice) tumor-draining lymph node dissection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section. (E and I) Summary table of distant organ metastasis derived from each surgery group. (E and I) Summary table of distant organ metastasis derived from each surgery group.(G and H) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre-removed tumor-bearing mice (n=9 mice) or sham surgery tumor-bearing mice (n=5 mice) after tumor resection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section.


Figure 3 Figure 3 . Up - regulation of Il - 17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor - draining lymph node ( A ) Gene expression profiles were shown at 4T1PT to 4T1LN cells . Five genes encoding cell surface proteins were identified among up - regulated genes . ( B ) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT - qPCR . Gapdh was used as an internal control . ( C and D ) Il - 17rb , Gpr56 , and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis . ( C and D ) Il - 17rb , Gpr56 , and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis . ( E ) Western blotting and RT - qPCR analysis of Il - 17rb , Gpr56 , and Scara5 expression in 4T1 cells transduced with Il - 17rb , Gpr56 , Scara5 , or control LacZ shRNA lentivirus respectively . ( F ) Soft - agar colony forming activity was examined in lentivirus transduced shIl - 17rb , shGpr56 , shScara5 , or shLacZ 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) . ( G ) Western blotting analysis was used to detect ectopic expression of Il - 17rb in 4T1 cells infected with either lentiviruses carrying Il - 17rb cDNA or empty vector . ( H ) Soft - agar colony forming activity was examined using control or Il - 17rb over - expressing 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) . ( I ) Western blotting analysis was used to examine Il - 17rb in a representative Il - 17rbDel 4T1 clone derived from Il - 17rb knocked - out 4T1 cells using a CRISPR / Cas9 system . ( J and K ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1 cells from WT or Il - 17rbDel clone . Tumor mass ( J ) and weight ( K ) were measured on day 28 . Scale bar : 1 cm . ( L and M ) BALB / c mice ( n = 3 mice per group ) were injected with 5x105 4T1 cells from WT or Il - 17rbDel clone via tail - vein . Lung colonization was examined by lung morphology ( L ) and the numbers of tumor nodule ( M ) on day 21 . Scale bar : 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Figure 3. Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node (A) Gene expression profiles were shown at 4T1PT to 4T1LN cells. Five genes encoding cell surface proteins were identified among up-regulated genes. (B) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT-qPCR. Gapdh was used as an internal control.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.(E) Western blotting and RT-qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus respectively.(F) Soft-agar colony forming activity was examined in lentivirus transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5x102 cells/well, n = 6 wells per group).(G) Western blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.(H) Soft-agar colony forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5x102 cells/well, n = 6 wells per group).(I) Western blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb knocked-out 4T1 cells using a CRISPR/Cas9 system.(J and K) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.(L and M) BALB/c mice (n = 3 mice per group) were injected with 5x105 4T1 cells from WT or Il-17rbDel clone via tail-vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm.


Figure 4 ( B and C ) Inguinal lymph node - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the RT - qPCR ( B ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control . ( B and C ) 4T1 - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the Western blotting ( C ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control . ( D ) Soft - agar colony forming activity was examined using co - cultured 4T1 cells at lower well ( 5x102 cells / well , n = 4 wells per group ) . ( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the RT - qPCR ( E ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control . ( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the Western blotting ( F ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control . ( G ) Percentage of CD4 + Foxp3 + Tregs of total CD4 + cells in inguinal LN , spleen and peripheral blood of BALB / c mice ( n = 3 mice per group ) injected with 4T1 ( Blue ) or γ - irradiated 4T1 ( Red ) cancer cells was analyzed by FACS . ( H ) 4T1 cells co - cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor - bearing mice by FACS sorting . After 5 - day co - culture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B and C) Inguinal lymph node-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the RT-qPCR (B)analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(B and C) 4T1-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the Western blotting (C) analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.(D) Soft-agar colony forming activity was examined using co-cultured 4T1 cells at lower well (5x102 cells/well, n = 4 wells per group).(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the RT-qPCR (E)analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the Western blotting (F) analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.(G) Percentage of CD4+Foxp3+ Tregs of total CD4+ cells in inguinal LN, spleen and peripheral blood of BALB/c mice (n = 3 mice per group) injected with 4T1 (Blue) or γ-irradiated 4T1 (Red) cancer cells was analyzed by FACS.(H) 4T1 cells co-cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor-bearing mice by FACS sorting. After 5-day co-culture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.


Figure 5 Figure 5 . Depletion of Tregs in the tumor - draining lymph node microenvironment abolishes Il - 17rb induction and enhanced malignancy in breast cancer cells . ( A ) 4T1 - injected BALB / c mice were treated with control IgG , anti - CD4 or anti - CD25 neutralizing antibody at the day 15 and day 18 after initial injection . Mice were sacrificed at three weeks after initial injection . Depletion of CD4 + Foxp3 + Tregs in inguinal LNs was confirmed by FACS analysis . ( B ) Western blotting analysis of Il - 17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( C and D ) BALB / c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( n = 3 mice per group ) . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . ( C and D ) BALB / c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( n = 3 mice per group ) . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . ( E ) NSG mice ( n = 5 mice per group ) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . Distant organ metastasis was examined by bioluminscent images on day 21 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 Figure 5. Depletion of Tregs in the tumor-draining lymph node microenvironment abolishes Il-17rb induction and enhanced malignancy in breast cancer cells. (A) 4T1-injected BALB/c mice were treated with control IgG, anti-CD4 or anti-CD25 neutralizing antibody at the day 15 and day 18 after initial injection. Mice were sacrificed at three weeks after initial injection. Depletion of CD4+Foxp3+ Tregs in inguinal LNs was confirmed by FACS analysis. (B) Western blotting analysis of Il-17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(C and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.(E) NSG mice (n = 5 mice per group) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. Distant organ metastasis was examined by bioluminscent images on day 21. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


Figure 6 Figure 6 . TGF - β1 secreted from Tregs induces Il - 17rb expression of breast cancer cells via Smad2 / 3 / 4 signaling . ( A ) CD4 + CD25 + Tregs were isolated from inguinal LN of BALB / c mice injected with 4T1 cells for three weeks , and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL - 10 or TGF - β1 . After 5 - day coculture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control . ( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant TGF - β1 ( B ) proteins for 5 days . ( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant IL - 10 ( C ) proteins for 5 days . ( D ) Western blotting analyzed Il - 17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF - β1 for 5 days . ( E ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . After 5 - day coculture , soft - agar colony forming activity was determined in co - cultured shLacZ or shTgfbr1 4T1 cells at lower well . ( 5x102 cells / well , n = 6 wells per group ) . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad2 ( F ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad3 ( G ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad4 ( H ) 4T1 cells treated with recombinant TGF - β1 for 5 days . ( I and J ) WT 4T1 ( I ) or Il - 17rbDel 4T1 ( J ) cells were treated with or without recombinant TGF - β1 for 5 days . After treatment , nuclear translocation of NF - κB p65 was assayed by Western blotting in cells treated with recombinant Il - 17b for 2 hours . Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts . f triplicate measurement . In ( B , C , D , F , G , H , I , and J ) , the intensity of each band was quantified using the Image J software . Gapdh , α - tubulin , or p84 was used as a loading control . Relative expression ( RE ) of protein levels in each sample to control 4T1 cells or Il - 17rbDel 4T1 clone is indicated .

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Figure 6 Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling. (A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control. (B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B)proteins for 5 days.(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant IL-10 (C) proteins for 5 days.(D) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days.(E) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group).(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ,shSmad3 (G)4T1 cells treated with recombinant TGF-β1 for 5 days.(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days. (I and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.f triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated.


Figure 7Figure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10 μmFigure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10 μm .

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Figure 7Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μmFigure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm.


Figure 8 Figure 8 . Elevated IL - 17RB expression in cancer cells derived from tumor - draining lymph nodes in breast cancer patients correlates with aggressive growth nature . ( A ) Freshly collected humanbreast cancer ( hBC ) specimens ( PT , primary tumors ; LN , paired LN metastasis from the same breast cancer patient ) were digested with collagenase - containing buffer overnight . Depletion of hematopoietic cells by CD45 Dyna beads ( Invitrogen ) enriched the EpCAM + cells for following xenografts . Percentages of EpCAM + cells in human primary breast cancer cells were confirmed by FACS analysis ( Left panel : before enrichment ; Right panel : after enrichment ) . All of the histograms were shown that they were EpCAM expressing ( black line ) , compared to isotype control ( gray filled ) . ( B ) Tumorigenesis assay were determined in NOD / SCID / IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells . Representative data on day 180 were shown . ( D and E ) H & E stainings and IHC stainings of pan - keratin were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 1 mm ( left panel ) , 100 μm ( right panel ) . ( D and E ) H & E stainings and IHC stainings of pan - keratin were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 1 mm ( left panel ) , 100 μm ( right panel ) . ( F ) IHC stainings of IL - 17RB were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 10 μm . ( G ) The correlation of tumor volumes and IL - 17RB expression ( composite IHC score ) in hBCLN cells - injected NOD / SCID / IL2Rγnull mice .

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Figure 8 Figure 8. Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature. (A) Freshly collected humanbreast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dyna beads (Invitrogen) enriched the EpCAM+ cells for following xenografts. Percentages of EpCAM+ cells in human primary breast cancer cells were confirmed by FACS analysis (Left panel: before enrichment; Right panel: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled). (B) Tumorigenesis assay were determined in NOD/SCID/IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells. Representative data on day 180 were shown.(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(D and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).(F) IHC stainings of IL-17RB were shown in the representative cases of fat-pad sections from (B). Scale bar: 10 μm.(G) The correlation of tumor volumes and IL-17RB expression (composite IHC score) in hBCLN cells-injected NOD/SCID/IL2Rγnull mice.


Figure 1A Mass spectrometric analysis of LSD1 methylation . Immunoprecipitation ( IP ) was performed using anti - Flag antibody in HEK - 293T cells overexpressing Flag - tagged LSD1 , followed by liquid chromatography coupled tandem mass spectrometry ( LC - MS / MS ) analysis . The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838 . B Interactions between LSD1 and CARM1 were detected by co - immunoprecipitation ( Co - IP ) assay with anti - Flag in HEK - 293T cells transfected with Flag - LSD1 or Flag - CARM1 , respectively . C Interaction of endogenous LSD1 with CARM1 was analyzed by Co - IP assay using anti - CARM1 antibody in MDA - MB - 231 cells . Purified GST - tagged CARM1 was pulled down with the Flag - LSD1 purified from HEK - 293T cells . The amounts of GST and GST - tagged CARM1 were visualized by Coomassie Blue staining ( asterisk : GST - CARM1 ) . E In vitro methylation assays . Purified fusion protein of GST - tagged LSD1 WT or the four methylated arginine mutants ( R - A ) , or four methylated arginine mutants in combination ( 4RA ) , were incubated with GST - CARM1 in the presence of 3H - SAM . Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining ( arrows : the position of LSD1 ; arrowheads : the position of CARM1 ) . F Increasing amounts of GST - CARM1 incubated with GST - LSD1 in presence of SAM at 30 ° C for 1 h . And methylation of LSD1 was analyzed by immunoblotting with anti - LSD1 R838me2a antibody . G GST - CARM1 incubated with GST - LSD1 WT or GST - LSD1 R838A / R838K in presence of SAM at 30 ° C for 1 h , followed by immunoblotting with anti - LSD1 R838me2a to detect LSD1 methylation .

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Figure 1A Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838.B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively.C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells.Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.


Figure 2A Methylation of LSD1 was assessed in MCF10A , MCF7 and MDA - MB - 231 cells by immunoblotting using anti - LSD1 R838me2a . Protein expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by western blotting ( B ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) . relative mRNA expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by qPCR analysis ( C ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) . D MDA - MB - 231 cells were treated with the indicated amounts of MS049 for 48 h , LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies . E MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 ( 10 μM ) or DMSO , before LSD1 level was analyzed by immunoblotting . F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h , and the cell lysates were analyzed by western blotting . G MDA - MB - 231 cells were transfected with CARM1 shRNA # 2 or control vector , and then treated with CHX ( 100 μg / ml ) for the indicated time ; LSD1 protein level was examined by western blotting . H MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated by anti - LSD1 antibody and then analysed by immunoblotting . I MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h . The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti - LSD1 antibody . J HEK - 293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time . LSD1 were examined by western blotting analysis . K HEK - 293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - Flag antibody and then subjected to immunoblotting . L HEK - 293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h . Co - IP assay was performed using anti - Flag and then subjected to western blotting .

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Figure 2A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.Protein expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.


Figure 3A Immunoprecipitation assay was performed using anti - Flag antibody in MM - 231 - shLSD1 - WT / R838A cells . The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies . B MDA - MB - 231 cells were treated with the indicated amounts of P5091 for 48 h , LSD1 protein levels were assessed by immunoblotting . C MDA - MB - 231 cells were treated with P5091 ( 10 μM ) for 48 h and MG132 for 10 h . The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti - LSD1 antibody . D HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - LSD1 antibody followed by immunoblotting analysis . E MDA - MB - 231 cells overexpressing Flag - tagged LSD1 WT / R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated by anti - Flag antibody and then analysed by immunoblotting . F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h . And cell lysates were immunoprecipitated with anti - LSD1 then subjected to immunoblotting . G MDA - MB - 231 cells were treated with or without MS049 for 48 h and MG132 for 10 h , Cell lysates were immunoprecipitated using anti - LSD1 antibody and then analysed by immunoblotting . H HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with or without MS049 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated with anti - LSD1 antibody followed by immunoblotting analysis .

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Figure 3A Immunoprecipitation assay was performed using anti-Flag antibody in MM-231-shLSD1-WT/R838A cells. The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies.B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.


Figure 4Migration assays ( 24 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( A ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . invasion assays ( 48 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( B ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . Migration assays ( 24 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( C ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . D invasion assays ( 48 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( D ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . E MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( 1 × 106 ) were injected into the tail veins of 5 - week - old female nude mice , and 7 weeks later , mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions . The number of visible surface metastatic lesions in lungs was counted ( mean of 7 mice ) ( Data are mean ± SD , n = 7 mice for each group , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . F Lung metastatic nodules confirmed by haematoxylin and eosin staining . Scale bars , 100 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O

Figure 4Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.


Figure 5MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( A ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) . B MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( B ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) . MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the E - cadherin ( C ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . D MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the vimentin ( D ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( E ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . F MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( F ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


Figure 6A Typical pictures of the immunohistochemical staining of LSD1 R838me2a , LSD1 and CARM1 in breast cancer samples . Scale bars , 100 μm . The percentage of samples with high level of LSD1 R838me2a ( B ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) . C , D The percentage of samples with high level of , LSD1 ( C ) and CARM1 ( D ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) . The correlation of LSD1 R838me2a with LSD1 ( E ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al . F , G The correlation of , CARM1 with LSD1 R838me2a ( F ) and CARM1 with LSD1 ( G ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Typical pictures of the immunohistochemical staining of LSD1 R838me2a, LSD1 and CARM1 in breast cancer samples. Scale bars, 100 μm.The percentage of samples with high level of LSD1 R838me2a (B) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).C, D The percentage of samples with high level of , LSD1 (C) and CARM1 (D) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).The correlation of LSD1 R838me2a with LSD1 (E) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.F, G The correlation of , CARM1 with LSD1 R838me2a (F) and CARM1 with LSD1 (G) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


Figure 1SMC4 was depleted in G1 through the addition of 5 - Ph - IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins . NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment . B , Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated . C , Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated . D , Quantification of sister chromatid separation in one - sister labelled prometaphase cells for experimental conditions as indicated . Dots represent individual cells ; red bars indicate the mean . Wild type ( n = 83 cells ) , ΔSMC4 ( n = 75 cells ) and ΔNIPBL ( n = 48 cells ) were analysed . Significance was tested using a two - tailed Mann Whitney U test ; P = 2 . 33 x 10 - 27 . Two - sister labelled wild type prometaphase ( n = 4 ) , one - sister labelled wild type prometaphase ( n = 6 ) , ΔSMC4 prometaphase ( n = 4 ) , ΔNIPBL prometaphase ( n = 3 ) , wild type G2 ( n = 4 ) , ΔSMC4 G2 ( n = 3 ) , ΔNIPBL G2 ( n = 5 ) . Technical replicates Two - sister labelled wild type prometaphase ( n = 9 ) , one sister - labelled wild type prometaphase ( n = 14 ) , ΔSMC4 prometaphase ( n = 9 ) , ΔNIPBL prometaphase ( n = 6 ) , wild type G2 ( n = 8 ) , ΔSMC4 G2 ( n = 5 ) , ΔNIPBL G2 ( n = 8 ) . All images show single Z - slices from 3D - stacks . Yellow boxes indicate inset regions . Scale bars large panels : 5 μm , insets : 1 μSMC4 was depleted in G1 through the addition of 5 - Ph - IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins . NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment . F , Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid . G , Representative images from ΔSMC4 G2 cells labelled on one sister chromatid . H , Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid . I , Representative images from ΔNIPBL G2 cells labelled on one sister chromatid . Two - sister labelled , ΔSMC4 prometaphase ( n = 4 ) , ΔNIPBL prometaphase ( n = 3 ) , ΔSMC4 G2 ( n = 3 ) , ΔNIPBL G2 ( n = 5 ) . Technical replicates : Two - sister labelled , ΔSMC4 prometaphase ( n = 9 ) , ΔNIPBL prometaphase ( n = 6 ) , ΔSMC4 G2 ( n = 5 ) , ΔNIPBL G2 ( n = 8 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. B, Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated. C, Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated. D, Quantification of sister chromatid separation in one-sister labelled prometaphase cells for experimental conditions as indicated. Dots represent individual cells; red bars indicate the mean. Wild type (n = 83 cells), ΔSMC4 (n = 75 cells) and ΔNIPBL (n = 48 cells) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 2.33 x 10-27. Two-sister labelled wild type prometaphase (n = 4), one-sister labelled wild type prometaphase (n = 6), ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3), wild type G2 (n = 4), ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates Two-sister labelled wild type prometaphase (n = 9), one sister-labelled wild type prometaphase (n = 14), ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6), wild type G2 (n = 8), ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μSMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. F, Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid. G, Representative images from ΔSMC4 G2 cells labelled on one sister chromatid. H, Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid. I, Representative images from ΔNIPBL G2 cells labelled on one sister chromatid. Two-sister labelled , ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3) , ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates: Two-sister labelled , ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6) , ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8).