Table 1. Overview of analyzed data.

Information is provided about the labeling method, the microscopy method, the total number of analyzed images, the number of analyzed cells, and the amount of focal adhesion marker expressing cells. Figs 2–4 show representative images for each experiment (see also related S1–S3 Figs).

Figure, Focal Adhesion Marker	HER2 labeling method, microscopy method, magnification	Analyzed Images	Analyzed cells	Focal adhesion marker positive cells	Comments
Fig 2A and 2B S1A and S1B Fig	Extracellular HER2 labeling.	4	31	9	Low transfection efficacy for eGFP-vinculin.
	Wide field fluorescence microscopy				Different focal areas for thick areas of cells impede colocalization analysis.
Vinculin	630x				Only for flattened areas at the cell edge colocalization could be analyzed. No overlapping expression was observed for vinculin and HER2.
Fig 2C	Extracellular HER2 labeling.	7	156	59	Colocalization analysis was impaired by different focal planes. No overlapping expression was observed for talin and HER2.
S1C and S1D Fig	Wide field fluorescence microscopy
Talin	400x
Fig 3A and 3B	Intracellular HER2.	10	64	34	TIRF analysis of intracellular labeled of HER2 confirmed HER2 expression at the cell-substrate interface. Talin was mainly expressed in HER2-low regions.
	TIRF
S2 and S3 Figs	1000x
Talin
Fig 3C	Intracellular HER2.	9	46	32	Control experiment omitting the HER2 antibody for intracellular labeling.
	TIRF
Talin	1000x				Almost no unspecific binding of strept-QDs was observed.
Fig 3D and 3E	Quantification of CFIs	5	16	16	HER2 expression intensities were decreased at talin positive spots.
Fig 4	Intracellular HER2 labeling	9	34	19	Wide field fluorescence microscopy revealed HER2 expression at the cell substrate interface. HER2 expression was heterogeneous with HER2-low and HER2-enriched regions. Talin expression was mainly observed in HER2-low regions.
	Wide field fluorescence
Talin	microscopy
	630x
Fig 4	Intracellular HER2 labeling	28	10	2	STEM revealed that HER2-QDs were mostly found outside talin-GFP positive regions.
Talin	STEM
	20,000x or 60,000x