EMBO/soda-nel huggingface dataset:
Cancer-secreted miRNAs regulate amino-acid-induced mTORC1 signaling and fibroblast protein synthesis (2020)
Figure 7

Figure 7 - RAGC levels in tumor stroma are inversely correlated with miR-105.

A,B. A breast cancer tissue array was analyzed for correlations among RagC, miR-105, and miR-204 in tumor stroma by IHC/ISH-determined scores. Representative IHC and ISH images (A) and correlations determined by Kendall's tau correlation tests (N=24 independent tumors; Kendall's tau and two-sided P value as indicated) (B) are shown. Bar=200 μm.

C,D. Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR-105 or empty vector were collected from NSG mice for RagC IHC (n=5 tumors for each group). The relative intensity of RagC stain in tumor stroma was quantified by ImageJ. Bar=50 μm. In (D), data are presented as mean ± SD. *P<0.05 (Student's t-test).

Expanded View Figure Legends
Ambra1 regulates autophagy and development of the nervous system (2007)
figf1

a, Detection of wild-type (+) and Ambra1 gene-trap transcripts (gt) from total E14.5 embryos analysed by northern blotting. Size marker, 28S ribosomal RNA. b, Immunoblot analysis of E14.5 embryo brain extracts using antibodies against Ambra1 (left panel) and &bgr;-gal (right panel). Specific bands (arrowheads) and unspecific signals (asterisk) are indicated. Molecular weights are measured in kilodaltons (kDa). The fusion protein can be revealed only by means of the more sensitive anti-&bgr;-gal antibody. c–f, Expression of Ambra1 in the mouse embryonic nervous system. &bgr;-gal-staining on whole-mount Ambra1+/gt mouse embryos at E8.5 (c), E11.5 (d) and on a cross-section of E11.5 spinal cord (e) is shown. Fb, forebrain; Hb, hindbrain; Mb, midbrain. f, Matching expression of the endogenous gene was revealed by whole-mount Ambra1 mRNA in situ hybridization analysis in E11.5 wild-type embryos. g–l, Electron scanning microscopy analysis of E11.5 (g) and E12.5 (h) Ambra1gt/gt embryos. Note the failure of the neural tube closure, the extensive midbrain/hindbrain exencephaly (Ex) with a closed telencephalon (T), and the lumbosacral spina bifida (Sb). i, j, E14.5 wild-type (i) and Ambra1gt/gt (j) embryos are characterized by prominent exencephaly. k, l, Histological analysis of E12.5 wild-type (k) and Ambra1gt/gt (l) embryos on cross-section. Note the absence of a normal ventricular system, the extensive overgrowth of the proliferative neuroepithelium in the diencephalon (Di) and spinal cord (Sc), and the enlarged fifth ganglia (VG) in the Ambra1gt/gt embryo. m–p, TUNEL (TdT-mediated dUTP nick end labelling) staining of E10.5 brain (m, n) and E9.5 spinal cord (o, p) in wild-type (m, o) and Ambra1gt/gt (n, p) embryo sections. q–t, Analysis of cell proliferation in wild-type (q, s) and Ambra1gt/gt (r, t) embryos on transverse sections of E8.5 cephalic neural folds in prospective hindbrain region (mitoses, arrows in q, r) and on sagittal sections of E10.5 forebrain (BrdU uptake, s, t). Scale bars: c, g, h, k, 500 &mgr;m; d, 1 mm; e, 150 &mgr;m; i, 2 mm; m, s, 400 &mgr;m; o, 78 &mgr;m; and q, 50 &mgr;m.
Ambra1 regulates autophagy and development of the nervous system (2007)
figf3

a, AMBRA1 downregulation in 2FTGH cells using specific short interfering RNA (siRNA) oligoribonucleotides (1 and 2). AMBRA1 mRNA and protein levels were analysed by quantitative polymerase chain reaction (PCR, left) and western blotting (right), respectively. An unrelated oligoribonucleotide was used as the control. Tubulin was used as the protein-loading control. RL, relative levels. The arrowhead indicates the AMBRA1-specific band. b, Rapamycin-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were treated with rapamycin; after 48 h, the occurrence of autophagy was analysed by appearance of GFP–LC3 punctate staining or by LC3-I to LC3-II conversion (western blot in inset). Representative results are accompanied by a graph reporting data from three experiments. Scale bar, 20 &mgr;m. c, Nutrient-starvation-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were starved for 4 h and analysed for appearance of GFP–LC3 punctate staining. d, AMBRA1 overexpression increases basal and rapamycin-induced autophagy. 2FTGH cells were transduced with full-length AMBRA1 (FL), AMBRA1 fragments (F1–3), BECN1 and &bgr;-gal (negative control) encoding retroviruses. They were then stimulated with rapamycin or left untreated, and analysed by appearance of GFP–LC3 punctate staining. e, BECN1-induced autophagy requires AMBRA1. After AMBRA1 downregulation, GFP–LC3-expressing 2FTGH cells were transduced with a BECN1-encoding retrovirus. Occurrence of autophagy was analysed 48 h later by appearance of GFP–LC3 punctate staining. f, AMBRA1 downregulation reduces the amount of Vps34 associated to BECN1 during autophagy. Forty-eight hours after AMBRA1 downregulation with oligo 2, 2FTGH cells were either starved or cultured in standard medium for an additional 4 h. Protein extracts were immunoprecipitated (IP) using an anti-BECN1 antibody. Purified complexes and corresponding total extracts were analysed by western blotting with anti-Vps34 (left panels) and anti-BECN1 (right panels) antibodies. The graph indicates the signal intensity of BECN1-associated Vps34 as determined by densitometry. Values in a–f represent the mean ± s.d. of three experiments.
Ambra1 regulates autophagy and development of the nervous system (2007)
figf4

a, Alteration of both LC3 conversion and translocation in Ambra1gt/gt embryos. Left panel: immunoblot analysis of LC3 in E14.5 embryos. LC3-I to LC3-II conversion is reduced in mutant extracts (LC3-II/LC3-I ratio, as measured by densitometry, from left to right: 5%, 9.8%, 2.5%, 4.6%). B, body; H, head. Centre and right panels: sections from E10.5 Ambra1+/+;GFP–LC3 and Ambra1gt/gt;GFP–LC3 neuroepithelia were compared to evaluate GFP–LC3 subcellular distribution. Clusters of GFP–LC3 punctate structures (arrows) are indicated in wild-type sections. Images were optimized by deconvolution. Scale bar, 10 &mgr;m. b, Ubiquitin-positive cells in developing brains of Ambra1gt/gt embryos. Sagittal sections from E11.5 embryos were stained using an anti-ubiquitin antibody. Ubiquitin accumulates in numerous mutant neuroepithelial cells (shown at different magnification: upper panels, scale bar, 25 &mgr;m; lower panels, scale bar, 10 &mgr;m). Ambra1 mutant protein is visualized in the left panels by &bgr;-gal activity (light-blue staining). Counterstaining, haematoxylin blue. c, Rapamycin-induced autophagy is impaired in Ambra1gt/gt murine embryonic fibroblasts (MEFs). Cells dissected from embryos with different genotypes (A–D) were treated with rapamycin, and the occurrence of autophagy was analysed 48 h later by the appearance of GFP–LC3 punctate staining. Representative results are shown in the upper panels, whereas the lower panel shows the data from three experiments. Scale bar, 20 &mgr;m. d, Ultrastructural analysis by means of electron microscopy of ultrathin sections from wild-type and Ambra1gt/gt cultured MEFs. The arrows indicate autophagic vacuoles. Scale bar, 1.5 &mgr;m. Quantification of the mean number of autophagic vacuoles per cell in wild-type and Ambra1gt/gt cultured MEFs is shown in the graph. Values in c and d represent the mean ± s.d. of three experiments.
MOTS-c promotes phosphorodiamidate morpholino oligomer uptake and efficacy in dystrophic mice (2020)
Figure 2

Figure 2. Effect of MOTS-c on PMO uptake in dystrophic muscles in vitro and in vivo.

(A) Measurement of PMO uptake in dystrophic muscle cells (H2K mdx cells) in the presence of MOTS-c (scale bar: 10 μm) (n=6; **p<0.001). PMO-M refers to PMO and MOTS-c (the same is for the rest unless otherwise specified).

(B) Tissue distribution of FITC-labeled PMO (50 mg/kg) mixed with MOTS-c (500 μg) in mdx mice 48 h after single intravenous injection. NC represents untreated mdx control. The abbreviation is the same as Figure 1D. Color scale represents the fluorescence intensity.

(C) Quantification of fluorescence intensity in body-wide tissues of mdx mice treated with PMO-M or PMO alone (n=3; *p<0.05). The comparison was conducted between PMO-M and PMO.

(D, E) Immunohistochemistry (D) and quantitative analysis (E) of dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 0.5 μg PMO mixed with MOTS-c (20 μg) (PMO-M) or PMO alone (scale bar: 100 μm) (n=3; **p<0.001). C57 means wild-type control C57BL/6.

(F) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO-M or PMO alone (n=3; *p<0.05). 0.5 µg and 2.5 µg total protein from C57BL/6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA muscles from C57BL/6 were used as normal controls (the same is for all Western blots unless otherwise specified).

(G) Systemic evaluation of MOTS-c (500 μg) and PMO at the dose of 50 mg/kg/week for 3 weeks in adult mdx mice intravenously. Tissues were examined two weeks after last injection. Immunohistochemistry for dystrophin- positive fibers in body-wide muscles of treated mdx mice (scale bar: 100 μm). C57 means wild-type control C57BL/6.

(H, I) Western blot (H) and quantitative analysis (I) for dystrophin expression in body-wide muscles from treated mdx mice (n=3; *p<0.05). 0.5 μg, 2.5 μg, 5 μg and 10 μg total protein from C57BL/6 and 20 μg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control.

Data information: Data were presented as mean±sem; two-tailed t test was used for statistical analysis. Exact p values are specified in Appendix Table S1.
MOTS-c promotes phosphorodiamidate morpholino oligomer uptake and efficacy in dystrophic mice (2020)
Figure 3

Figure 3. Investigation on the dose-dependent effect of MOTS-c on PMO activity in adult mdx mice.

(A) Diagram of dosing regimen for the dose-dependent effect of MOTS-c on PMO activity in mdx mice. i.v. refers to intravenous injection. PMO-M (+MOTS-c) means PMO-M supplemented with additional MOTS-c.

(B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (scale bar: 100 μm). TA refers to tibialis anterior.

(C, D) Western blot (C) and quantitative analysis (D) for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (n=3). 5 µg, 10 µg and 20 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.

(E, F) Immunohistochemistry (E) and quantitative analysis (F) of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline.

(G, H) Western blot (G) and quantitative analysis (H) of dystrophin expression in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) intravenously (n=3; *p<0.05). 0.5 µg, 1 µg, 2.5 µg, 5 µg and 10 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. Fold change refers to PMO-M relative to PMO alone.

Data information: Data were presented as mean±sem; two-tailed t test was used for statistical analysis. Exact p values are specified in Appendix Table S1.
MOTS-c promotes phosphorodiamidate morpholino oligomer uptake and efficacy in dystrophic mice (2020)
Figure 4

Figure 4. Long-term repeated administration of PMO-M in adult mdx mice. PMO-M was administered intravenously into adult mdx mice at the PMO dose of 12.5 mg/kg/week for 3 weeks followed by 12.5 mg/kg/month for 3 months and tissues were harvested two weeks after last injection.

(A) Diagram of dosing regimen for the long-term study of PMO-M in mdx mice. i.v. refers to intravenous injection.

(B, C) Immunohistochemistry (B) and quantitative analysis (C) of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test).

(D) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO alone. 0.5 µg, 2.5 µg, 5 µg, and 15 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded.

(E) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05; two-tailed t test).

(F) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO-M (n=4) or PMO alone (n=3), untreated mdx controls (n=3) and C57BL/6 (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).

(G) Measurement of muscle endurance with the running wheel test for wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). Muscle functional tests were conducted two weeks after last injection.

Data information: Data were presented as mean±sem. Exact p values are specified in Appendix Table S1.
MOTS-c promotes phosphorodiamidate morpholino oligomer uptake and efficacy in dystrophic mice (2020)
Figure 5

Figure 5. Morphological assessment of mdx mice treated with long-term repeated administration of PMO-M or PMO.

PMO-M was administered intravenously into adult mdx mice at the PMO dose of 12.5 mg/kg/week for 3 weeks followed by 12.5 mg/kg/month for 3 months and tissues / blood were harvested 2 weeks after last injection.

(A) Measurement of serum creatine kinase (CK) levels in wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).

(B) Re-localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar: 100 μm). The arrowheads point to identical myofibers. C57 means wild-type C57BL/6; mdx represents untreated mdx controls.

(C) Measurement of body-weight changes of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3).

(D) Quantitative analysis of centrally nucleated fibres (CNFs) in triceps and quadriceps of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman- Keuls test).

(E) Measurement of cross-sectional area (CSA) of muscle fibres from mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, two-tailed t test).

Data information: Data were presented as mean±sem. Exact p values are specified in Appendix Table S1.
MOTS-c promotes phosphorodiamidate morpholino oligomer uptake and efficacy in dystrophic mice (2020)
Figure 6

Figure 6. Evaluation of toxicology and inflammation in mdx mice treated with long-term repeated doses of PMO-M or PMO.

PMO-M was administered intravenously into adult mdx mice at the PMO dose of 12.5 mg/kg/week for 3 weeks followed by 12.5 mg/kg/month for 3 months and tissues / blood were harvested 2 weeks after last injection.

(A, B) Measurement of serum indices including liver enzymes (AST, ALT and GGT) (A) and kidney markers (CREA and UA) (B) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test).

(C) Morphological examination of liver and kidney from wild-type C57BL/6 (C57), untreated mdx controls (mdx) and treated mdx mice (scale bar: 100 μm).

(D, E) Immunohistochemistry (D) and quantification (E) of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test).

Data information: Data were presented as mean±sem. Exact p values are specified in Appendix Table S1.

Expanded View (EV) Figure legends
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 1

Figure 1. The TRAPPII complex binds preferentially to the nucleotide-free form of Rab18.

A. Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg++ plus 0.1 mM GTPγS, GDP or 2 mM EDTA and immunoprecipitated with anti-Myc antibody. Rab18-HA co-immunoprecipitated with Myc-TRAPPC9 when it was nucleotide free (+EDTA).

B. A mutant of Rab18 unable to bind nucleotide, N122I, was co-immunoprecipitated with Myc-TRAPPC9 by anti-Myc antibody.

C. HA-tagged Rab1 and Rab18, but not Rab2, could co-immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex.

D. GST-Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST-Rab18 was pre-loaded with GTPγS or GDP, or stripped off nucleotide by EDTA. Lysates from 293T cells were subjected to GST-Rab18 pull-down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII, but not TRAPPIII complex was co-isolated with GST-Rab18, most strongly with Rab18 in nucleotide-free state.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 2

Figure 2. TRAPPII complex stimulates guanine nucleotide exchange on Rab1 and Rab18.

A. TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12, and the presence of various co-precipitating TRAPP subunits was detected by immunoblotting.

B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins.

E-G. Nucleotide exchange reactions measured by time-dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the 3H-GDP release experiments, the reactions were incubated for 30 minutes at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding.

H-I. Anti-Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc-TRAPPC9 were used as the GEF for GST-Rab1 or GST-Rab18 in 30 min incubations at 37°C with [35S]GTPγS.

Error bars = S.D.; n =3 (for all experiments shown in B-I)
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 3

Figure 3. Loss of TRAPPC9 function resulted in aberrantly large lipid droplets.

A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel. Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation. Bar =10 μm.

B. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual. Bar = 10 μm.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 4

Figure 4. Recruitment of Rab18 onto small lipid droplets was defective in TRAPPII deleted cells.

A. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493/503. Bar = 10 μm.

B. Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Bar = 10 μm.

C. DsRed-Rab18 was transfected into wildtype (top panels and insets) or TRAPPII deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with bodipy 493/503 (green). Bar = 10 μm.

D. Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation. Fractions 1 and 2 were enriched with LDs. The presence of Rab18, TRAPPC9, TRAPPC2, LD marker TIP47, and ER marker Calnexin were detected by SDS-PAGE followed by immunoblotting.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 5

Figure 5. Release of NEFA at various time points by 293T cells with the indicated genotypes or treatments.

Control siRNA depletion (siCtrl) was conducted with siRNA oligoes specific to a firefly luciferase sequence; siCOPB = depletion of β-COP; siRab18 = depletion of Rab18. C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted. N= 3, Error bars = S.D.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 6

Figure 6. Interaction between COPI and Rab18 is TRAPPII dependent.

A. Myc-TRAPPC9 directly interacted with FLAG-γ-COP. The presence of FLAG-γ-COP in immunoprecipitations of Myc-TRAPPC9 and Myc-TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting.

B,C. Co-immunoprecipitation between FLAG-γ-COP and HA-Rab18 in HEK293T cells with indicated genetic backgrounds. The presence of FLAG-γ-COP in immunoprecipitates of HA-Rab18 (and reciprocal IP in C) was investigated by immunoblotting.

D. The interaction between endogenous COPI and Rab18 was TRAPPII dependent. Endogenous Rab18 was immunoprecipitated by anti-Rab18 antibody from lysates of indicated genetic background. The presence of γ-COP and various TRAPP subunits were detected by their specific antibodies as indicated.

E. Co-immunoprecipitation between TRAPPII and HA-Rab18 in control (siFFL) and γ-COP (si-γ-COP) siRNA depleted cells. TRAPPII was precipitated with transfected Myc-TRAPPC10 and the presence of HA-Rab18 was determined by immunoblotting.

F. Co-immunoprecipitation between FLAG-γ-COP and TRAPPII in control (siFFL) and Rab18 (si-Rab18) siRNA depleted cells. TRAPPII was precipitated with Myc-TRAPPC10 and the presence of FLAG-γ-COP was determined by immunoblotting. The molecular weight of endogenous Rab18 overlaps with IgG light chain, and therefore, the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 7

Figure 7. Differential effect of COPI inhibition by siRNA depletion and BFA on Rab18 recruitment onto LD surface.

A. Huh-7 cells were depleted with control siRNA specific to firefly luciferase sequence (top panels) or with siRNA specific to γ-COP (bottom panels). Endogenous Rab18 (red) and LD (green) were stained and visualized. Bar = 10 μm.

B. Huh-7 cells were treated with or without BFA for 6 hours before staining for Rab18 (red) and LD (green). Bar = 10 μm.

C. TRAPPC9 were detected on Golgi but not LD surface. Huh-7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Bar = 10 μm.
COPI-TRAPPII activates Rab18 and regulates its lipid droplet association (2016)
Figure 8

Figure 8. Time-dependent relocations of COPI, TRAPPII and Rab18 onto LD surface upon oleic acid incubation.

Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.

A. In the merged images, bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re-colored to green for easy visualization. Bar = 10μm.

B. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ-COP in blue. In the colocalization between Rab18 and γ-COP on the right side, the γ-COP signal was re-colored to green for easy visualization. Bar = 10μm.