Figure S4.
Tubule-forming proteins are required in the maintenance of LD homeostasis in HepG2 cells.(A) The levels of FIT2 and FIT2-related tubule-forming proteins were determined by Western blotting or real-time PCR as indicated after HepG2 cells were transfected with siRNAs for 48 h. (B) Western blotting of Rtn4 in WT and Rtn4-KO HepG2 cells. (C) LipidTOX staining of LDs and quantification of number of LDs per cell, as well as LD size, in WT and Rtn4 KO HepG2 cells without (−OA) or with (+OA) OA treatment. Colorized pictures were acquired using Imaris software. Different colors indicate different LD diameters, as labeled. n = 50–76 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. (D) The level of Climp-63 was determined by Western blotting after HepG2 cells were transfected with the indicated siRNAs for 48 h. (E) As in C, except in Control and Climp-63–depleted HepG2 cells. For groups without OA treatment, n = 76 and 74 cells, total LD numbers >2,000. For groups with OA treatment, n = 45 and 51 cells, total LD numbers >2,000. Mann–Whitney test; NS, P > 0.05. Scale bar, 10 µm. (F) Levels of LD-related proteins in HepG2 cells transfected with the indicated siRNAs. (G) Septin 2 KO (SEPT2 KO) HepG2 cells were generated using CRISPR/Cas9, two single clones (#10 and #16) picked, and the protein level of septin 2 in WT and SEPT2 KO cells measured by immunoblotting (IB). (H–J) The knockdown efficiencies of siRNAs targeting septin 6, 7, and 9 were tested by Western blotting. (K) As in Fig. 5 D, LipidTOX staining and Imaris 3D images of OA-treated HepG2 cells transfected with the indicated siRNAs. The numbers and sizes of the LDs in each group were determined by Imaris surface analysis. n = 50–76 cells/group, n = 74–101 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. siClimp-63, small interfering Climp-63; siControl, small interfering Control; si FIT2, small interfering siFIT2; siREEP5, small interfering REEP5; siRtn4, small interfering Rtn4; siSEPT, small interfering SEPT. Error bars represent SEM.

Tubule-forming proteins are required in the maintenance of LD homeostasis in HepG2 cells. (A) The levels of FIT2 and FIT2-related tubule-forming proteins were determined by Western blotting or real-time PCR as indicated after HepG2 cells were transfected with siRNAs for 48 h. (B) Western blotting of Rtn4 in WT and Rtn4-KO HepG2 cells. (C) LipidTOX staining of LDs and quantification of number of LDs per cell, as well as LD size, in WT and Rtn4 KO HepG2 cells without (−OA) or with (+OA) OA treatment. Colorized pictures were acquired using Imaris software. Different colors indicate different LD diameters, as labeled. n = 50–76 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. (D) The level of Climp-63 was determined by Western blotting after HepG2 cells were transfected with the indicated siRNAs for 48 h. (E) As in C, except in Control and Climp-63–depleted HepG2 cells. For groups without OA treatment, n = 76 and 74 cells, total LD numbers >2,000. For groups with OA treatment, n = 45 and 51 cells, total LD numbers >2,000. Mann–Whitney test; NS, P > 0.05. Scale bar, 10 µm. (F) Levels of LD-related proteins in HepG2 cells transfected with the indicated siRNAs. (G) Septin 2 KO (SEPT2 KO) HepG2 cells were generated using CRISPR/Cas9, two single clones (#10 and #16) picked, and the protein level of septin 2 in WT and SEPT2 KO cells measured by immunoblotting (IB). (H–J) The knockdown efficiencies of siRNAs targeting septin 6, 7, and 9 were tested by Western blotting. (K) As in Fig. 5 D, LipidTOX staining and Imaris 3D images of OA-treated HepG2 cells transfected with the indicated siRNAs. The numbers and sizes of the LDs in each group were determined by Imaris surface analysis. n = 50–76 cells/group, n = 74–101 cells/group, total LD numbers >2,000. Mann–Whitney test; ***, P < 0.001. Scale bar, 10 µm. siClimp-63, small interfering Climp-63; siControl, small interfering Control; si FIT2, small interfering siFIT2; siREEP5, small interfering REEP5; siRtn4, small interfering Rtn4; siSEPT, small interfering SEPT. Error bars represent SEM.

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