Figure 7.
Figure 7. NRZ complex acts as a downstream effector of Rab18 to control LD growth. (A–C) Increased supersized LDs and reduced mature LDs in NAG-deficient (NAG KO) and ZW10-deficient (ZW10 KO) 3T3-L1 preadipocytes. Bars: 10 µm; (insets) 2 µm. (B) The mean number of LDs in each diameter in A. (C) Quantification of the number of mature LDs per cell in A. Mean ± SD; n = 13 for control cells, n = 22 for NAG-deficient cells, n = 20 for ZW10-deficient cells; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (D) Reduced cellular TAG levels in NAG KO- and ZW10 KO 3T3-L1 preadipocytes. Three independent experiments were performed. Mean ± SD; n = 3; *, P < 0.05; **, P < 0.01 by one-way ANOVA with Tukey post hoc tests. (E) Increased ER stress in NAG KO cells after long-term OA treatment (400 µM). Cells treated with 1 µM TG for 6 h were used as a positive control. (F–H) Introduction of Cherry-Rab18 (red) into NAG KO cells did not restore its function in the accumulation of mature LDs. (F) Representative images of LD morphology. LDs were stained with Bodipy 493/503. Green, LDs; red, Cherry. Bars: 10 µm; (insets) 2 µm. (G) Quantification of the number of mature LDs in each cell Mean ± SD; n = 25 cells for vector-transfected cells, n = 27 cells for Rab18-transfected cells; NS, no significance by Mann-Whitney test. (H) Quantification of the size of the largest LD in each cell Mean ± SEM; n = 31 cells for vector-transfected cells, n = 33 cells for Rab18-transfected cells; NS, no significance by two-tailed t test. (I) Representative EM images showing LD morphology in control and NAG KO 3T3-L1 preadipocytes. Bars, 500 nm. (J) The total number of LDs in each cell treated with OA for 1 h was increased. Mean ± SD; n = 137 ROIs for control cells, n = 154 ROIs for NAG-deficient cells; ***, P < 0.001 by Mann-Whitney test. (K and L) Defective LD growth and maturation in NAG KO cells treated with OA for 8 h. Number of LDs (K) and histogram of the mean number of LDs (L) in each diameter in cells treated with OA for 8 h in K. Mean ± SD; n = 25 ROIs for control cells, n = 29 ROIs for NAG-deficient cells; ***, P < 0.001; NS, no significance by Mann-Whitney test. All experiments were performed at least twice.

NRZ complex acts as a downstream effector of Rab18 to control LD growth. (A–C) Increased supersized LDs and reduced mature LDs in NAG-deficient (NAG KO) and ZW10-deficient (ZW10 KO) 3T3-L1 preadipocytes. Bars: 10 µm; (insets) 2 µm. (B) The mean number of LDs in each diameter in A. (C) Quantification of the number of mature LDs per cell in A. Mean ± SD; n = 13 for control cells, n = 22 for NAG-deficient cells, n = 20 for ZW10-deficient cells; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (D) Reduced cellular TAG levels in NAG KO- and ZW10 KO 3T3-L1 preadipocytes. Three independent experiments were performed. Mean ± SD; n = 3; *, P < 0.05; **, P < 0.01 by one-way ANOVA with Tukey post hoc tests. (E) Increased ER stress in NAG KO cells after long-term OA treatment (400 µM). Cells treated with 1 µM TG for 6 h were used as a positive control. (F–H) Introduction of Cherry-Rab18 (red) into NAG KO cells did not restore its function in the accumulation of mature LDs. (F) Representative images of LD morphology. LDs were stained with Bodipy 493/503. Green, LDs; red, Cherry. Bars: 10 µm; (insets) 2 µm. (G) Quantification of the number of mature LDs in each cell Mean ± SD; n = 25 cells for vector-transfected cells, n = 27 cells for Rab18-transfected cells; NS, no significance by Mann-Whitney test. (H) Quantification of the size of the largest LD in each cell Mean ± SEM; n = 31 cells for vector-transfected cells, n = 33 cells for Rab18-transfected cells; NS, no significance by two-tailed t test. (I) Representative EM images showing LD morphology in control and NAG KO 3T3-L1 preadipocytes. Bars, 500 nm. (J) The total number of LDs in each cell treated with OA for 1 h was increased. Mean ± SD; n = 137 ROIs for control cells, n = 154 ROIs for NAG-deficient cells; ***, P < 0.001 by Mann-Whitney test. (K and L) Defective LD growth and maturation in NAG KO cells treated with OA for 8 h. Number of LDs (K) and histogram of the mean number of LDs (L) in each diameter in cells treated with OA for 8 h in K. Mean ± SD; n = 25 ROIs for control cells, n = 29 ROIs for NAG-deficient cells; ***, P < 0.001; NS, no significance by Mann-Whitney test. All experiments were performed at least twice.

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