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. 2022 Oct 19;221(12):e202203139. doi: 10.1083/jcb.202203139

Figure S5.

Figure S5.

CFS1 does not colocalize with VPS3, VPS39, or VAMP711, but partially colocalizes with FREE1 and ALIX. (A) Confocal microscopy images of Arabidopsis root epidermal cells co-expressing pVPS3::mGFP-VPS3 with pUBQ::mCherry-CFS1. 5-d-old Arabidopsis seedlings were incubated in either control, nitrogen-deficient (−N) or 150 mM NaCl-containing 1/2 MS media before imaging. Representative images of 10 replicates are shown. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 5 μm. Inset scale bars, 2 μm. (B) Quantification of confocal experiments in A showing the Mander’s colocalization coefficients between mCherry-CFS1 and mGFP-VPS3. M1, fraction of mGFP-VPS3 signal that overlapped with the mCherry-CFS1 signal. M2, fraction of mCherry-CFS1 signal that overlapped with the mGFP-Vps3 signal. Bars indicate the mean ± SD of 10 replicates. (C) Confocal microscopy images of Arabidopsis root epidermal cells co-expressing pVPS39::VPS39-mGFP with pUBQ::mCherry-CFS1. 5-d-old Arabidopsis seedlings were incubated in either control, nitrogen-deficient (−N) or 150 mM NaCl-containing 1/2 MS media before imaging. Representative images of 10 replicates are shown. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 5 μm. Inset scale bars, 2 μm. (D) Quantification of confocal experiments in C showing the Mander’s colocalization coefficients between mCherry-CFS1 and VPS39-mGFP. M1, fraction of VPS39-mGFP signal that overlapped with the mCherry-CFS1 signal. M2, fraction of mCherry-CFS1 signal that overlapped with the VPS39-mGFP signal. Bars indicate the mean ± SD of 10 replicates. (E) Confocal microscopy images of Arabidopsis root epidermal cells co-expressing pUBQ::YFP-VAMP711 with pUBQ::mCherry-CFS1. 5-d-old Arabidopsis seedlings were incubated in either control, nitrogen-deficient (−N) or 150 mM NaCl-containing 1/2 MS media before imaging. Representative images of 10 replicates are shown. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 5 μm. Inset scale bars, 2 μm. (F) Quantification of confocal experiments in E showing the Mander’s colocalization coefficients between mCherry-CFS1 and YFP-VAMP711. M1, fraction of YFP-VAMP711 signal that overlapped with the mCherry-CFS1 signal. M2, fraction of mCherry-CFS1 signal that overlapped with the YFP-VAMP711 signal. Bars indicate the mean ± SD of 10 replicates. (G) Representative confocal microscopy images showing the colocalization of p35S::GFP-FREE1 and pUBQ::mCherry-CFS1 in Arabidopsis root epidermal cells. 5-d-old Arabidopsis seedlings were incubated in either control or 150 mM NaCl-containing 1/2 MS media for 1 h before imaging. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 5 μm. Inset scale bars, 2 μm. (H) Representative confocal microscopy images showing the colocalization of pALIX::GFP-3Gly-ALIX and pUBQ::mCherry-CFS1 in Arabidopsis root epidermal cells. 5-d-old Arabidopsis seedlings were incubated in either control or 150 mM NaCl-containing 1/2 MS media for 1 h before imaging. Area highlighted in the white-boxed region in the merge panel was further enlarged and presented in the inset panel. Scale bars, 5 μm. Inset scale bars, 2 μm.