Fig. 5: Lamin A/C phosphorylation at Ser 301 is a signature of CAFs.

A IF analysis of CAFs and matched normal fibroblasts (HDFs) from the same patient showing phospho-Ser301 (pSer301) lamin A/C (red) and total lamin A/C (yellow). Representative images (left) and quantification of pSer301 lamin A/C fluorescence intensity (right), mean value as black bar. Two-tailed unpaired t-test, n = 50 cells per condition. Scale: 20 µm. Similar analysis in Supplementary Fig. 5A. B CAFs treated with the AR agonist Ostarine (10 μM) or DMSO for 48 h. IF with antibodies against AR (green) and pSer310 lamin A/C (yellow). Representative images and quantification of pSer310 lamin A/C signal, mean value as black bar. Two-tailed unpaired t-test, n = 51 cells per condition. Scale: 20 µm. C IF analysis of skin SCC-associated fibroblasts versus distal skin fibroblasts with antibodies against pSer301 lamin A/C (red), pan-cytokeratin (gray), and anti-PDGFRa (green). High magnification images shown, low magnification images are shown in Supplementary Fig. 5D. Scale: 5 µm. D IF analysis of skin SCC-associated fibroblasts vs. distal skin fibroblasts with antibodies against pSer301 lamin A/C (red) and vimentin (gray). High magnification images shown, low magnification images are shown in Supplementary Fig. 5E. Scale: 5 µm. E IF analysis of skin SCC-associated fibroblasts with antibodies against pSer301 lamin A/C (red), α-SMA (green), TNC (orange), and FAP (green; right). High magnification images shown, low magnification images are shown in Supplementary Fig. 5F. Scale: 5 µm. F Quantification of pSer301 in PDGFRa+ and vimentin+ fibroblasts from SCC-associated vs. distal skin, and pSer301 in FAP, α-SMA, and TNC positive fibroblasts from SCC-associated regions, mean value as black bar. Two-tailed paired t-test, for PDGFRa+ cells n = 51, vimentin+ cells n = 47 cells. G 3D volumetric surface reconstruction of confocal IF images of the SCC lesion and unaffected flanking skin as shown in Fig. 2E with antibodies against phospho-S301 (red) and total lamin A/C (yellow) and vimentin (purple), using the Imaris Surface tool, Manual mode. For each individual sample, threshold parameters were tested and set based on the weakest positive signal. H PLA of skin SCC-associated fibroblasts versus fibroblasts in distant unaffected skin with antibodies against lamin A/C and PPP1CA/B (red puncta), with concomitant IF with anti-vimentin antibodies for cell type identification (green). Shown are representative images (left) and quantification (right) of PLA signal in vimentin-positive cells in matched pairs of skin SCC-associated versus distal skin fibroblasts from three different patients, with mean value as red bar. Two-tailed unpaired t-test, for patient-1, n = 60 cells; for patient-2, n = 30 cells, and for patient-3; n = 37 cells per condition. Scale: 30 µm. Source data for individual graphs are in the Source Data file.