Figure 3.
Collectively invading cells overexpress and abundantly secrete laminin-332. (A) Integration of RNA sequencing log2 fold change values (x-axis) and mouse methylation array beta difference values (y-axis) for the leaders (L) versus singles (S) pair-wise comparison. Lama3, Lamb3, and Lamc2 are highlighted as red dots among gene transcripts with high mRNA transcription in leaders and significant hypomethylation at the promoter region compared to singles. (B) Raw mRNA counts for gene transcripts of the laminin-332 subunits from RNA sequencing analysis (n = 3, ****P < 0.0001). (C) Volcano plot of the leaders versus singles pair-wise comparison derived from RNA sequencing data highlighting Lama3, Lamb3, and Lamc2 (bordered red dots) as prominent DEGs. (D) Beta value comparison of distinct CpG loci within the promoter regions of Lama3, Lamb3, and Lamc2 between leaders and singles. (E) Volcano plot of differentially secreted peptides extracted via LC-MS/MS from CM from leaders and singles. Red, unbordered dots denote peptides differentially secreted in leaders and green dots denote peptides differentially secreted in singles. (F) Label-free quantification (LFQ) intensity quantification of Lama3, Lamb3, and Lamc2 peptides. # denotes an absence of signal detected (n = 3, ****P < 0.0001). (G) Protein levels of laminin-332 in leaders CM and singles CM. Total protein staining via Ponceau S was used as a loading control. (H) Laminin-332 immunofluorescence (IF) staining on invasive leaders and singles in collagen I 3D culture (10× and 63×). Scale bar: 100 μm for 10× image, 20 μm for 63× image, and 10 μm for 63× zoomed image. For all panels: mean ± SEM is shown. For G and H, three biological replicates were performed. Source data are available for this figure: SourceData F3.

Collectively invading cells overexpress and abundantly secrete laminin-332. (A) Integration of RNA sequencing log2 fold change values (x-axis) and mouse methylation array beta difference values (y-axis) for the leaders (L) versus singles (S) pair-wise comparison. Lama3, Lamb3, and Lamc2 are highlighted as red dots among gene transcripts with high mRNA transcription in leaders and significant hypomethylation at the promoter region compared to singles. (B) Raw mRNA counts for gene transcripts of the laminin-332 subunits from RNA sequencing analysis (n = 3, ****P < 0.0001). (C) Volcano plot of the leaders versus singles pair-wise comparison derived from RNA sequencing data highlighting Lama3, Lamb3, and Lamc2 (bordered red dots) as prominent DEGs. (D) Beta value comparison of distinct CpG loci within the promoter regions of Lama3, Lamb3, and Lamc2 between leaders and singles. (E) Volcano plot of differentially secreted peptides extracted via LC-MS/MS from CM from leaders and singles. Red, unbordered dots denote peptides differentially secreted in leaders and green dots denote peptides differentially secreted in singles. (F) Label-free quantification (LFQ) intensity quantification of Lama3, Lamb3, and Lamc2 peptides. # denotes an absence of signal detected (n = 3, ****P < 0.0001). (G) Protein levels of laminin-332 in leaders CM and singles CM. Total protein staining via Ponceau S was used as a loading control. (H) Laminin-332 immunofluorescence (IF) staining on invasive leaders and singles in collagen I 3D culture (10× and 63×). Scale bar: 100 μm for 10× image, 20 μm for 63× image, and 10 μm for 63× zoomed image. For all panels: mean ± SEM is shown. For G and H, three biological replicates were performed. Source data are available for this figure: SourceData F3.

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