Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 localizes distinctly to LDs and the ER in SUM159 cells. (A) Overexpressed GFP-Rab18 localizes to LDs (LipidTox) (white arrowheads) and the ER (mCherry-ER3), and localization depends on GTP state (white arrows). SUM159 cells coexpressing mCherry-ER3 and GFP-tagged WT Rab18, GDP-bound Rab18(S22N) mutant, or GTP-bound Rab18(Q67L) mutant were incubated with OA for 0 or 18 h and imaged with spinning disk confocal. Scale bar 5 µm and for inlay 1 µm. (B) Rab18 localizes to the ER and LD structures. SUM159 cells co-expressing mCherry-ER3 and GFP-Rab18 were incubated with oleic acid for 18 h and imaged by SIM. Max projections of 1.25-µm stacks are shown. Scale bars, 1 µm. (C) Quantification of Rab18 signal distribution in SIM images. n = 5 fields. (D) Rab18 was detected in LD fractions and total cell lysates of SUM159 cells. LD fractions and cell lysates isolated from SUM159 cells after 18 h oleic acid were analyzed by mass spectrometry to detect proteins on LDs compared with total lysate. ND = not detected.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Mutagenesis, Incubation, Expressing, Isolation, Mass Spectrometry

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 deletion does not affect ER morphology. (A) Sequence analysis of Rab18 KO clones A and B. CRISPR/Cas9-mediated genome editing of the Rab18 locus introduces early stop codons at exons 4 (clone A) and 5 (clone B). (B) qPCR data reveal decreased Rab18 mRNA levels by 98 and 96% in Rab18KO-A and –B, respectively, compared with WT control. WT vs. Rab18KO-A* in gray, WT vs. Rab18KO-B* in black. (C) No Rab18 protein is detected in knockout clones by Western blot. Expression levels of Rab18 protein in WT and Rab18 KO cells were analyzed by Western blot with an antibody against endogenous Rab18. No detectable Rab18 protein was found in the Rab18KO-A or Rab18KO-B. (D) Rab18 peptide fragments were not detected by mass spectrometry in Rab18KO-A. WT SUM159 cell lysates and Rab18KO-A cell lysates were analyzed by mass spectrometry with sequence coverage of 68.4% for Rab18. (E) ER morphology in Rab18 KO clones is similar to WT cells. Cells were transfected with GFP-ERox to analyze general ER morphology. Separately, cells were fixed and probed with Reticulon 4 (Rtn4) antibody to visualize ER tubules. Scale bar 5 µm and for inlay 1 µm.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Sequencing, Clone Assay, CRISPR, Control, Knock-Out, Western Blot, Expressing, Mass Spectrometry, Transfection

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: LD biogenesis is not affected in Rab18 KO cells. (A) LD morphology is similar in Rab18KO clones and WT cells with oleic acid incubation. Representative images of WT and Rab18 KO cells prestarved for 5 h before addition of oleic acid and after 24 h of oleic acid incubation. Scale bar 5 µm and for inlay 1 µm. (B) Rab18 KO average BODIPY object area and number are slightly smaller than WT after 24 h oleic acid incubation. WT and Rab18 KO cells were incubated with oleic acid for indicated time points, fixed, and imaged by high-throughput microscopy. Average BODIPY object area and number per cell per field (>five cells per field to obtain representative measurements) were quantified per condition. n > 27 fields/point. WT vs. Rab18KO-A* in gray; WT vs. Rab18KO-B* in black. (C) Rab18KO and WT average object area vs. number 90% confidence intervals overlap at 2 and 24 h oleic acid incubation. Average object number plotted against average object area per cell per field with 2 and 24 h oleic acid. Ellipses represent 90% confidence intervals. (D, E) Rab18KO clones have similar synthesis of TG, PE, and PC as WT cells with oleic acid incubation. Incorporation of [ 14 C] oleate into triglycerides (TGs), phophatidylethanolamine (PE), and phosphatidycholine (PC) were measure over time. Lipids were extracted from cells and separated by TLC. Representative autoradiographs of three replicates per genotype are shown (D). (E) Quantified TG, PC, and PE levels are similar between Rab18KO clones and WT cells over time. TLC plates were developed and incorporation of [ 14 C] oleate into TGs, PE, and PC was quantified using Fiji. Data presented are normalized CPM to mg/ml protein and relative to WT at t = 0 h. n = 3.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Clone Assay, Incubation, High Throughput Screening Assay, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 deletion does not affect TG turnover. (A) LDs are degraded similarly in Rab18KO clones and WT cells with starvation. Representative images of WT, Rab18KO-A, and Rab18KO-B cells after 12 h OA loading ( t = 0) and 24 or 48 h of starvation. LDs stained with BODIPY 493/503 and nuclei with Hoechst. Scale bar 5 µm and inlay 1 µm. (B) Rab18KO average BODIPY object area and number are similar to WT after 48 h starvation. WT and Rab18KO cells were incubated with oleic acid for indicated time points as in A. Cells were fixed and imaged by high-throughput microscopy. Average BODIPY object area and number per cell per field (>5 cells per field) was quantified per condition. n > 7 fields. WT vs. Rab18KO-A * in gray; WT vs. Rab18KO-B * in black. (C) Rab18KO clones have similar synthesis of TG and PE and less PC than WT cells with starvation. WT and Rab18KO cells were incubated with [ 14 C] oleic acid for 18 h, followed by starvation for increasing time. Total lipids were extracted at each time point and separated by TLC to detect radiolabeled TG, PE, and PC levels. Representative autoradiographs of three replicates per genotype. (D) Quantified TG and PE levels are similar, and PC levels are less between Rab18KO clones and WT cells over time with starvation. TG, PC, and PE signals were quantified from TLC plates using Fiji. Data presented are normalized CPM to mg/ml protein and relative to WT at t = 0 h. n = 3 biological replicates. (E) Rab18KO clones have decreased free fatty acid release over time with starvation. The [ 14 C]-labeled free fatty acid release measured over time with starvation after WT; Rab18KO-A, and Rab18KO-B cells were incubated with [ 14 C] oleic acid for 18 h. n = 3 biological replicates.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Clone Assay, Staining, Incubation, High Throughput Screening Assay, Microscopy, Labeling

Journal: Journal of Lipid Research

Article Title: DFCP1 is a regulator of starvation-driven ATGL-mediated lipid droplet lipolysis

doi: 10.1016/j.jlr.2024.100700

Figure Lengend Snippet: DFCP1 Controls ATGL Localization in Cells. A: Representative images of U2OS control (C) and DFCP1 CRISPR KO cells (KO) expressing GFP-ATGL and treated with 200 μM OA for 20 h. Cells were then fed (top row) or starved (bottom row) for 4 h before treating with LipidTOX Deep Red for 30 min prior to imaging. B: Colocalization analysis (Pearson’s correlation coefficient) of GFP-ATGL with LDs in control (black) and KO (magenta) cells described in A. C: Western blot of LDs purified from control (C, black) and DFCP1 KO (KO, magenta) U2OS cells showing the abundance of ATGL relative to the established LD associated protein, Rab18, and normalized to the mean abundance of ATGL found in the control samples. D: Representative images of U2OS control and DFCP1 KO cells expressing the lipase-dead mutant (D166G) of ATGL (BFP-ATGL DG ). Cells were treated with OA and starved as in (A). E: Colocalization analysis of GFP-ATGL DG with LDs in control (black) and KO (magenta) cells described in (D). F: Representative western blots showing PLIN2 expression relative to 14-3-3 expression in fed and starved control (C, black) and DFCP1 KO (KO, magenta) clarified U2OS cell lysates. (G) Representative images of U2OS cells expressing GFP-ATGL and either BFP (left) or BFP-DFCP1 WT (right). Cells were treated with OA and starved as in (A). H: Colocalization analysis of GFP-ATGL with LDs in cells described in (G and K) that were transfected with GFP-ATGL and BFP (black), BFP-DFCP1 WT (WT, orange) or BFP-DFCP1 KA (KA, blue). I: Representative images of U2OS cells expressing the lipase-dead mutant (D166G) of ATGL (BFP-ATGL DG ) with either GFP (left) or GFP-DFCP1 WT (right). Cells were treated with OA and starved as in (A). J: Colocalization analysis of BFP-ATGL DG with LDs in cells described in I. K: Representative image of U2OS cells expressing BFP-DFCP1 KA . Cells were treated with OA and starved as in (A). L: Colocalization of GFP-ATGL with either BFP-DFCP1 WT (WT, orange) or BFP-DFCP1 KA (KA, blue) in the cells described in (K). The scale bars in whole-cell and inset images represent 10 and 2 μm, respectively. The statistical significance of the measurements in (B, E, H, J, and L) was determined using an unpaired parametric student t test on the indicated number of observations from two independent transfections. The statistical significance of the measurements in (C and F), was determined using an unpaired parametric student t test on 5 independent LD isolations for (C) and 3 independent experiments for each condition in (F) (all technical replicates are shown). Exact P -values are reported with exception to P > 0.05, which is not considered to be significant (n.s.).

Article Snippet: The anti-Rab18 (A2812) antibody was purchased from ABClonal.

Techniques: Control, CRISPR, Expressing, Imaging, Western Blot, Purification, Mutagenesis, Transfection

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: Correlation of RAB18 expression with clinicopathological parameters

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: RAB18 expression increased in HCC and indicated poor prognosis. A. Representative IHC images of RAB18 expression in HCC tissues and matched adjacent non-tumor tissues. Scale bar: 100 μm. B. RAB18 expression levels were compared among HCC and adjacent non-tumor tissue specimens, P = 0.003. C. Kaplan-Meier curves of 93 HCC patients according to RAB18 expression level. D. HCC and survival of HCC patients were correlated with RAB18 expression based on the TCGA database.

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: Univariate analysis of factors associated with survival

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: Multivariate analysis of factors associated with survival

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: Knockdown of RAB18 significantly decreased HCC cell proliferation in vitro. (A) RAB18 expression level in hepatic cell lines (Huh7, SMMC7721, Hep-3B, BEL-7402, and LO2) was determined by qRT-PCR and western blot analyses. (B) Expression levels of RAB18 were measured by qRT-PCR and western blot in HCC cells treated with LV-shRAB18 or LV-Scramble. (C, D) The effect of RAB18 on in vitro proliferation was analyzed by MTT (C), EdU incorporation assay (D), and colony-forming assay (E). (F) Western blot analysis was performed to detect the expression of CCND1, P21, and P27 in shRAB18 cells; GAPDH was used as a loading control.

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Western Blot

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: Decreased RAB18 expression inhibited the migration and invasion of HCC cells in vitro. A. Invasion capability of RAB18 siRNA-transfected cells was assessed by transwell assay. B. Migration of RAB18 siRNA-transfected cells was measured by wound-healing assays. C. Western blot analysis of the EMT-related markers E-cadherin, N-cadherin, vimentin, and snail after RAB18 siRNA transfection in HCC cells.

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: Expressing, Migration, In Vitro, Transfection, Transwell Assay, Western Blot

Journal: American Journal of Translational Research

Article Title: RAB18 promotes proliferation and metastasis in hepatocellular carcinoma

doi:

Figure Lengend Snippet: RAB18 augmented the proliferation and metastasis of HCC cells in vivo. A. The image of dissected tumors from nude mice with subcutaneous injection of SMMC7721-shRAB18 and control cells. B. The growth curves and weights of tumors derived from nude mice with subcutaneous injection of SMMC7721-shRAB18 and control cells. Tumor volume was measured with Vernier calipers every 3 days for 2 weeks, and the tumor wet weight was determined on day 18. Mean ± SD (n = 5). *P < 0.05. C. Tumors tissues were stained with H&E or antibodies against RAB18, Ki67, E-cadherin, and vimentin, Scale bar: 100 μm (bottom). D. Representative fluorescence imaging of the lungs. After 6 weeks, mice were sacrificed, and lungs were collected, *P < 0.05 based on Student’s t test. Scale bar: 5 mm. E. Lung metastases were confirmed by H&E staining, scale bar: 100 μm.

Article Snippet: The antibodies used were as follows: rabbit anti-RAB18 (1:200, Abcam, Cambridge, UK), anti-Ki67 (1:100, Abcam, Cambridge, UK), E-cadherin (1:500, CST, Boston, MA) and Vimentin (1:500, CST, Boston, MA).

Techniques: In Vivo, Injection, Derivative Assay, Staining, Fluorescence, Imaging