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. Author manuscript; available in PMC: 2024 Mar 18.
Published in final edited form as: Nat Cell Biol. 2022 Feb 14;24(2):168–180. doi: 10.1038/s41556-022-00843-w

Fig. 6. ECM stiffness regulates fission via Spire1c- and Arp2/3-dependent peri-mitochondrial F-actin.

Fig. 6

a, Left: colocalization of transfected WT but not R62D monomeric mutant Flag-Actin with phalloidin in MCF10A-RAS cells. Centre: PLA (proximity ligation assay) between Flag-Actin and TOMM20. No probes: absence of secondary antibodies (ab). αTOMM20, αFLAG: only one primary ab was used. Cells were treated with YM alone and in combination with LatrunculinA (LATA) or Arp2/3 inhibitor (CK666). Scale bars = 2 μm (n=20). b, PLA between endogenous filamentous actin and TOMM20 in MCF10A-RAS cells treated as in a. αTOMM20; Bio-Phalloidin + αBio: only one primary ab was used as specificity control. Scale bars = 5 μm (n=20). c, Co-localization of F-Actin/mitochondria PLA with DRP1-GFP puncta in MCF10A-RAS cells. Scale bar = 5 μm. d, Quantification of endogenous DRP1 puncta in MCF10A-RAS cells cultured on soft or stiff Matrigel and treated with LatrunculinA (LATA) or Arp2/3 inhibitor (CK666) (n=35). e, Quantification of DRP1-GFP puncta in D2.0R cells treated as in a. Scale bars = 10 μm.

(n=35). f, Mitochondrial length in MCF10A-RAS cells expressing dominant-negative Spire1C isoforms (n=141). g, Quantification of mtROS in MCF10A-RAS cells expressing dominant-negative Spire1C isoforms and plated on stiff or soft hydrogels for 6 h. Normalized to median intensity in the control (n=4).

h, Quantification of mitochondrial ROS in MCF10A-RAS cells depleted of total INF2 (siINF2) or of the mitochondrial splice isoform (siINF2-CAAX), and treated with YM. Normalized to mean intensity in control (n=4). i,j, qPCR in MCF10A-RAS for total (i) or of the mitochondrial splice isoform (j) of INF2. mRNA expression data are relative to GAPDH levels normalized to control (n=4).

Images in a,b,c,e, are representative of at least two independent experiments with similar results. ‘n’ refers to number of cells (a,b,d,e) across two independent experiments each condition; number of mitochondria (f) from 10 cells across two independent experiments each condition; number of biologically independent samples (g-j) across two independent experiments each bar. Unpaired two-tailed Student’s t-tests in a and b; Dunnet’s tests in d-j Data are mean and single points. See Source Data Table 6.