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. 2022 May 19;13:2763. doi: 10.1038/s41467-022-30388-3

Fig. 4. ER accumulates at the ventral and dorsal nuclear region during nuclear positioning without interfering with the connection of dorsal actin with nuclear envelope TAN lines.

Fig. 4

a Representative epifluorescence images of scramble or Climp-63 siRNA treated fibroblasts expressing GFP-mini-N2G and stained with phalloidin (F-actin). Arrows show colocalization of Nesprin-2G with dorsal actin cables. b Quantification of number of TAN lines per nuclei in cells treated with scramble (grey) or Climp-63 (green) siRNA as in a. c Quantification of the percentage of nuclei with TAN lines in cells treated with scramble (grey) or Climp-63 (green) siRNA as in a. d Representative epifluorescence images of wound-edge cells after LPA stimulation. Cells were stained as follows: F-actin (Phalloidin, red), DNA (DAPI, blue). Nuclear region highlighted with a yellow dashed box. Left: Ventral focal plane. Right: Dorsal focal plane. e Quantification of the number of nuclear ventral stress fibers per nuclei in cells treated with scramble (grey) or Climp-63 (green) siRNA as in d. f Quantification of the number of nuclear dorsal actin cables per nuclei in cells treated with scramble (grey) or Climp-63 (green) siRNA as in d. g Fluorescence kymograph of nuclear movement and ventral ER accumulation in wound-edge GFP-KDEL-expressing cells treated with scramble (top) or Climp-63 (bottom) siRNA upon LPA stimulation. h Quantification of ventral ER accumulation during LPA stimulation in scramble (grey) or Climp-63 (green) siRNA treated cells, represented in g. Significance (Two-tailed unpaired t-test) was calculated between scramble and Climp-63 siRNAs for each time point. i Quantification of perinuclear ER accumulation (grey line) and the nuclear movement over time (blue line), during LPA stimulation of cells represented in g. The grey line corresponds to the grey line in h. j Fluorescence kymograph of nuclear movement and dorsal ER accumulation in wound-edge GFP-KDEL-expressing cells treated with scramble (top) or Climp-63 (bottom) siRNA upon LPA stimulation. k Quantification of dorsal ER accumulation during LPA stimulation in scramble (grey) or Climp-63 (green) siRNA treated cells, represented in j. Experiments were repeated 3 times. Scale bars: a, d 5 µm; g, j 20 µm. Error bars, SEM. **p < 0.01, *p < 0.05.