Skip to main content
. 2021 May 10;12:2616. doi: 10.1038/s41467-021-22771-3

Fig. 2. MAM disruption by FUNDC1 deficiency reduces angiogenesis in vitro and in vivo.

Fig. 2

a HUVECs were transfected with scrambled (scr) siRNA or FUN14 domain-containing 1 (FUNDC1) siRNA for 24 h. The protein levels of FUNDC1 were determined by western blot assays (n = 5 independent experiments). b HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h. Representative confocal images are shown. Quantification of endoplasmic reticulum (ER)–mitochondria contacts using Pearson’s coefficient. (n = 5 independent experiments). Scale bars, 10 µm. c HUVECs transfected with Scr siRNA or FUNDC1 siRNA were plated onto matrigel. Representative images of tube formation are shown (left). Tube length/field in 10 random microscopic fields per group was measured by NIH ImageJ and statistically analyzed (n = 5 independent experiments) (right). Scale bar, 100 µm. d HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h. Three-dimensional spheroids were generated (left), and representative images of spheroid-sprouting were analyzed (n = 5 independent experiments) (right). Scale bar, 100 µm. e Representative confocal images of a P5 retina lobe stained with IsoB4 showing reduced number of sprouts (yellow stars) at the vascular front of FUNDC1f/YCdh5+ retinas compared with FUNDC1f/YCdh5− retinas. (n = 5 mice/group). Scale bars, 500 mm. f Matrigel plugs implanted into FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice for 10 days. Quantification of hemoglobin (Hb) extracted from matrigel plugs from FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice (n = 5 mice/group). g CD31 immunostaining of matrigel plugs implanted into FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice for 10 days (left). Quantification of the relative CD31-positive area (fold-over control) (right). (n = 5 mice/group). Scale bar, 100 µm. h Representative images showing blood flow reperfusion assessed by Doppler laser ultrasound on day 10 after ischemic injury. (n = 5 mice/group). The ratio of ischemic/non-ischemic perfusion was quantitatively analyzed. i Representative images showing anti-CD31 immunostaining in gastrocnemius muscle on day 10 after femoral artery ligation. (n = 5 mice/group). Scale bar, 100 µm. j, k Tumors following subcutaneous injection of Lewis lung carcinoma (LLC) tumor cells into the abdomen of FUNDC1f/YCdh5− and FUNDC1f/YCdh5+ mice. After 28 days, tumors were harvested and quantified. Scale bar, 100 µm. j Representative images of tumors (n = 5 mice/group). k Immunostaining of LLC tumor sections and quantification of relative CD31-positive areas. (n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using two-tailed t-tests to compare two groups. *p < 0.05 vs Scr siRNA or Fundc1f/YCdh5−. All values are mean ± S.D.