Figure S1.
FIT2 is required in the maintenance of ER homeostasis.(A) Sequence alignment of FIT2. The mutation site in fitm-2(erm002) is highlighted in red. The black box outlines the conserved SGH sequence containing the predicted active residue His. (B) Colocalization of FITM-2::RFP and ER marker GFP::TRAM-1 in the hypodermis. 3D-SIM images. Scale bar, 10 µm. (C) Depiction of fitm-2(av41). Red dots show two stop codons on the N-terminus of FITM-2. (D) Representative images of fitm-2(av41) animals at the young adult stage carrying the qxIs439 (Phyp7::gfp::tram-1) transgene. The ER of WT animals shown in B was used for comparison. 3D-SIM images. Scale bar, 10 µm. (E) ER morphology of early embryos and oocytes in fitm-2(av41) animals carrying the ojIs23 (Ppie-1::gfp::sp12) transgene. Single confocal planes. Scale bar, 10 µm. (F) Relative mRNA level of FIT2 in control and FIT2-depleted COS-7 cells, indicating the knockdown efficiency. (G) ER morphology in control and FIT2-depleted COS-7 cells. Cells were transfected with control or FIT2 siRNA for 48 h, fixed, and stained with anti-calreticulin and anti–Climp-63 antibodies. Scale bar, 10 µm. (H) Representative images of wild-type and fitm-2 mutants at the young adult stage carrying the zcls4 (Phsp-4::gfp) transgene. Images were acquired under the same excitation light intensity. Scale bar, 100 µm. (I) Expression level of PDI and BIP in control and FIT2-knockdown COS-7 cells. (J) ER morphology in control and FIT2-HA–overexpressing COS-7 cells. Scale bar, 10 µm. (K) Bubble-like structures in COS-7, U2OS, and HEK293T cells overexpressing FIT2-HA. Single confocal planes. Scale bars, 10 µm. (L) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA antibody, as well as Oil Red. Arrows indicate bubble-like structures stained by Oil Red. Scale bar, 10 µm. (M) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA and anti-calreticulin antibodies. The insets show high-magnification details in the white squares. Scale bars: 10 µm (main panel) or 2 µm (inset). (N) Immunofluorescence assay using phalloidin (Dylight 405; Thermo Fisher Scientific) and antibodies targeting HA, septin 2, and α-tubulin in COS-7 cells transfected with FIT2-HA. Scale bar, 10 µm; 2 µm (inset). BIP, binding immunoglobulin protein; H. sapiens, Homo sapiens; PDI, protein disulfide isomerase; siControl, small interfering control; siFIT2, small interfering FIT2.

FIT2 is required in the maintenance of ER homeostasis. (A) Sequence alignment of FIT2. The mutation site in fitm-2(erm002) is highlighted in red. The black box outlines the conserved SGH sequence containing the predicted active residue His. (B) Colocalization of FITM-2::RFP and ER marker GFP::TRAM-1 in the hypodermis. 3D-SIM images. Scale bar, 10 µm. (C) Depiction of fitm-2(av41). Red dots show two stop codons on the N-terminus of FITM-2. (D) Representative images of fitm-2(av41) animals at the young adult stage carrying the qxIs439 (Phyp7::gfp::tram-1) transgene. The ER of WT animals shown in B was used for comparison. 3D-SIM images. Scale bar, 10 µm. (E) ER morphology of early embryos and oocytes in fitm-2(av41) animals carrying the ojIs23 (Ppie-1::gfp::sp12) transgene. Single confocal planes. Scale bar, 10 µm. (F) Relative mRNA level of FIT2 in control and FIT2-depleted COS-7 cells, indicating the knockdown efficiency. (G) ER morphology in control and FIT2-depleted COS-7 cells. Cells were transfected with control or FIT2 siRNA for 48 h, fixed, and stained with anti-calreticulin and anti–Climp-63 antibodies. Scale bar, 10 µm. (H) Representative images of wild-type and fitm-2 mutants at the young adult stage carrying the zcls4 (Phsp-4::gfp) transgene. Images were acquired under the same excitation light intensity. Scale bar, 100 µm. (I) Expression level of PDI and BIP in control and FIT2-knockdown COS-7 cells. (J) ER morphology in control and FIT2-HA–overexpressing COS-7 cells. Scale bar, 10 µm. (K) Bubble-like structures in COS-7, U2OS, and HEK293T cells overexpressing FIT2-HA. Single confocal planes. Scale bars, 10 µm. (L) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA antibody, as well as Oil Red. Arrows indicate bubble-like structures stained by Oil Red. Scale bar, 10 µm. (M) COS-7 cells were overexpressed with FIT2-HA and stained with anti-HA and anti-calreticulin antibodies. The insets show high-magnification details in the white squares. Scale bars: 10 µm (main panel) or 2 µm (inset). (N) Immunofluorescence assay using phalloidin (Dylight 405; Thermo Fisher Scientific) and antibodies targeting HA, septin 2, and α-tubulin in COS-7 cells transfected with FIT2-HA. Scale bar, 10 µm; 2 µm (inset). BIP, binding immunoglobulin protein; H. sapiens, Homo sapiens; PDI, protein disulfide isomerase; siControl, small interfering control; siFIT2, small interfering FIT2.

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