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Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, <t>ATACseq,</t> and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.
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Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, <t>ATACseq,</t> and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.
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Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, <t>ATACseq,</t> and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.
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Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, <t>ATACseq,</t> and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.
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Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, ATACseq, and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.

Journal: Cancers

Article Title: Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance

doi: 10.3390/cancers16030553

Figure Lengend Snippet: Study design and summary of multiomic measurements. ( a ) Study design—500,000 cells/well were plated in 6-well plates and cultured for 48 h in complete media with one media change after 24 h. Cells were serum starved using media containing 0.1% FBS for 14–16 h. After serum starvation, cells were treated with 50 ng/mL of epidermal growth factor (EGF), 10 ng/mL of heregulin (HRG), or vehicle (water) in freshly made serum starvation media for 30 min (for RPPA), 1 h, 12 h, or 24 h (for ATAC/RNAseq). Time-zero (T0) samples were collected after serum starvation. All treatments were performed in duplicate. ( b ) An infographic that highlights the types of comparisons performed within this manuscript. Differential expression (fold change) for each omics data modality (RNAseq, ATACseq, and RPPA) was computed as pairwise comparisons between treatment and their respective control for each time point. The results were then compared between the two cell lines to identify functional differences (functional comparisons). ( c ) Differentially expressed proteins (DEPs, adj p < 0.05) detected from RPPA measurements for 30 min, 1 h, 12 h, and 24 h for BT474R (R30, R1, R12, and R24, left panel) and sensitive cell line BT474 (S30, S1, S12, and S24, right panel) showed distinct and increased response in BT474 after treatment in contrast to BT474R. ( d ) A comparison of phosphorylated DEPs, which alludes to a highly active cell state, shows a more robust response in BT474 after both HRG and EGF. ( e ) A comparison of the differentially accessible regions (DARs) in BT474 and BT474R assessed from the ATACseq data reflected trends similar to DEPs, with BT474 overall being more accessible across time after treatments. ( f ) The proportion of DARs (as percentage of total DARs) that were observed in the promoter (upstream 1 kb downstream 1 kb of transcription start site (TSS)) and non-promoter regions of annotated genes (adj p < 0.05) for BT474R (left panel) and BT474 (right panel). BT474 clearly showed increased accessibility in its promoter region (compared to their respective control, “+”), especially after EGF. ( g ) Differentially expressed genes ( p adj < 0.05) identified from RNAseq data reflected trends observed in RPPA and ATACseq with both EGF and HRG treated BT474 (right panel) in a transcriptionally more active state compared to BT474R (left panel) across time. Of note, BT474 seems to be most sensitive to treatment at 12 h, as observed.

Article Snippet: Raw bulk RNAseq and ATACseq data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GEO: GSE237607.

Techniques: Cell Culture, Quantitative Proteomics, Control, Functional Assay, Comparison