Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection

Journal: European journal of histochemistry : EJH

Article Title: Ultrastructure and immunohistochemical characterization of proteins concerned with the secretory machinery in goat ceruminous glands.

doi: 10.4081/ejh.2017.2828

Figure Lengend Snippet: Figure 6. Immunohistochemical staining for Rab3D detection in the ceruminous glands. a) Rab3D. b) Negative control for Rab3D using absorbed antibodies.

Article Snippet: The primary antibodies were used as follows: β-defensin 1 (DEF01-S; Biologo, Kronshagen, Germany), β-defensin 2 (DEF02-S; Biologo), VAMP-1 (ab3346; Abcam Plc., Cambridge, UK), VAMP-2 (ab70222; Abcam), VAMP-3 (ABIN675818; Antibodies-online, Inc., Atlanta, GA, USA), VAMP-4 (ab80989; Abcam), VAMP-7 (HPA036733; Atlas antibodies AB, Stockholm, Sweden), VAMP-8 (NBP1-20232; Novus Biologicals, Littleton, CO), syntaxin 2 (ADI-VAP-SV065; Enzo Life Sciences, Inc., Farmingdale, NY, USA), syntaxin 4 (HPA001330; Atlas antibodies), syntaxin 6 (ab56656; Abcam), SNAP-23 (10825-1- AP; Proteintech Group, Inc., Rosemont, IL, USA), Rab3D (12320-1-AP; Proteintech).

Techniques: Immunohistochemical staining, Staining, Negative Control

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Analysis of response of cell lines from the Dependency Map portal (DepMap) database to CRISPR-knockout (blue curve) or RNAi-mediated inhibition of NRAS -mRNA (green curve) shows that the vast majority of cell lines presented no dependency on NRAS -mRNA expression (dependency score 0, black dotted line). b Filtering for melanoma cell lines showed that specifically NRAS -mutant melanoma cells presented a strong vulnerability on NRAS -mRNA expression (dependency score ≤ -1, red dotted line). Dot plots represent all analyzed cell lines (black: non-melanoma, yellow: NRAS wild type melanoma, red: NRAS -mutant melanoma), highlighting that the dependent melanoma cell lines harbor NRAS mutations. c Subcellular mRNA enrichment analysis was done using qRT-PCR to compare the ratio of nuclear versus cytoplasmic mRNA levels of NRAS , GAPDH and B-ACTIN in D04 and MM415 cells. The data are presented as fold-change of nuclear to cytoplasmic ratio normalized to GAPDH ( n = 3) and show higher nuclear enrichment of NRAS-mRNA , when compared to reference genes. The error bars represent Standard Error (s.e.m.). d , e Representative images of RNA in situ hybridization (RNA-ISH) derived from d D04 and e MM415 cell pellets. Fluorescent signals are either produced by DAPI DNA staining to mark the nuclear regions (blue) or probes that stain the NRAS -mRNA (red). f Quantification of punctua per nucleus from fluorescent signals produced by probes that stain NRAS -mRNA in D04 and MM415 cells. The calculations included > 1000 cells per cell line. g Intronic (small bars) and exonic (large bars) regions of the NRAS gene (ENSG00000213281.5) as annotated in the Genecode database (V44). NRAS ASO target regions are highlighted in black and the codons Q61 and G12 are highlighted in red. h NRAS -mRNA (Genecode ID: ENST00000369535.5) secondary structure as predicted by the Minimum Free Energy (MFE) model. NRAS ASO target regions are highlighted in black, provided in additional cutout and zoom. Codon Q61 is highlighted in red. The ASO target regions represent stable and accessible structures.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: CRISPR, Knock-Out, Inhibition, Expressing, Mutagenesis, Quantitative RT-PCR, RNA In Situ Hybridization, Derivative Assay, Produced, Staining

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Using qRT-PCR to compare RNA levels in D04 and MM415 cells that were either treated with NRAS ASO-1 or NRAS ASO-2, showed a robust reduction of NRAS -mRNA levels after 6, 24, 48, and 72 hours, when compared to treatment with non-targeting Control ASO. Final oligonucleotide concentration was 100 nM; error bars represent s.e.m. ( n = 3). b , c Representative images of RNA in situ hybridization (RNA-ISH) derived from pellets of b D04 or c MM415 cells, either treated with NRAS ASO-1, or Control ASO. Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange). NRAS ASO-1 treatment strongly reduced NRAS -mRNA levels in the cytoplasm and nucleus of the cells and NRAS protein expression. Final oligonucleotide concentration was 100 nM and treatment period lasted for 24 h. d Immunoblotting showing a strong decrease in NRAS protein levels 1 day after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−66%) and MM415 (−87%) cell lysates. B-ACTIN served as loading control and normalization parameter. e Immunoblotting showing a decrease in p-ERK1/2 protein levels 2 days after NRAS ASO treatment compared to Control ASO treatment in D04 (−50%) and MM415 (−50%) cell lysates, while total ERK1/2 levels were not altered significantly. GAPDH served as loading control and normalization parameter. f Immunoblotting showing a decrease in p-S6 protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (−70%) and MM415 (−71%) cell lysates, while total S6 levels were not altered significantly. g Immunoblotting showing a small increase in p-AKT protein levels 2 days after NRAS ASO-1 treatment compared to Control ASO treatment in D04 (+18%) and MM415 (+12%) cell lysates. Total AKT levels were not altered significantly. Final oligonucleotide concentration was 100 nM. h A simplified illustration depicting key signaling pathways in NRAS -mutant melanoma, emphasizing the activation of crucial proteins contributing to cellular survival. Through transcription, the mutations in the NRAS gene are carried over to the NRAS -mRNA, which is translated into the constitutively active mutant NRAS protein, initiating downstream signaling cascades. This activation prompts the RAF kinase (not shown) to activate MEK, which, in turn, activates ERK. ERK signaling influences the activation of S6 ribosomal protein and translocates to the nucleus, regulating transcription and supporting cellular proliferation. S6 plays a pivotal role in translation, facilitating protein synthesis. The activation of this signaling pathways enhances cellular survival in NRAS -mutant melanoma. Phosphorylation-dependent activation steps are denoted by (P). i A simplified illustration highlighting the impact of NRAS ASO treatment: NRAS ASOs reduce NRAS -mRNA levels in both the cytoplasm and nucleus. This reduction is followed by decreased NRAS protein levels and the inhibition of MAPK-pathway signaling activity, as evidenced by diminished p-ERK and p-S6 protein levels. The pathway is essential for the NRAS-mutant cancer cells’ ability to proliferate and survive.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Quantitative RT-PCR, Control, Concentration Assay, RNA In Situ Hybridization, Derivative Assay, Produced, Staining, Expressing, Western Blot, Protein-Protein interactions, Mutagenesis, Activation Assay, Phospho-proteomics, Inhibition, Activity Assay

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000002), MM415 ( p = 0.00002), WM1366 ( p = 0.0005), Sk-Mel-2 ( p = 0.00001), VMM39 ( p = 0.00004), WM3060 ( p = 0.003), NZM40 ( p = 0.0006), WM3629 ( p = 0.0008), and the primary derived cell line Hs852T ( p = 0.000006). b Treatment with NRAS ASO-2 caused significant inhibition of cell growth in the NRAS -mutant melanoma cell lines D04 ( p = 0.000004) and MM415 ( p = 0.0001). The antiproliferative outcomes are similar when compared to treatment with NRAS ASO-1. c NRAS ASO treatment did not cause significant antiproliferative effects in primary human melanocytes (PHM, p = 0.33), primary human liver cells (Hs775li, p = 0.29), human colon cells (FHC, p = 0.29), and BRAF-mutant melanoma cells (Sk-Mel-28, p = 0.13). d NRAS ASO treatment significantly inhibited colony formation in the D04 ( p = 0.0017) and MM415 ( p = 0.008) cell lines compared to treatment with non-targeting Control ASOs. Treatment period was 7 days (50 nM final oligonucleotide concentration, n = 3). e Representative images of D04 colonies in 6 cm dishes after ASO treatment. f Dot plot graph of flow cytometric analysis of PI and Annexin V staining after 1 day of ASO-treatment (100 nM) shows increased apoptotic cell death in D04-cells treated with NRAS ASO (15,780 total events) compared to Control ASO treatment (44,285 total events). g Distribution of overall cell populations from panel f ) in regards of their apoptotic state. Bars represent the percentage of vital (Q2), early apoptotic (Q3), late apoptotic (Q4) and dead (Q1) cells. h NRAS ASO-mediated induction of apoptosis was confirmed by measurement of significantly increased activity levels of the apoptosis markers Caspase-3 & -7 after 1 day of treatment with either NRAS or Control ASOs (100 nM) in the D04 ( p = 0.002) and MM415 ( p = 0.0002) cell lines ( n = 4). i Treatment with NRAS ASO−1 caused significant inhibition of cell growth in the NRAS -mutant multiple myeloma (MM) cell line H929 ( p = 0.0005), and small cell lung cancer (SCLC) cell line SW1271 ( p = 0.0001). j Significant tumor growth reduction was observed when comparing treatment groups for subcutaneous systemic treatment with either NRAS ASO (X) or Control ASO (O) in mouse models carrying xenografts of the D04 melanoma cell line (3 × 200 µg ASO/week, n = 6, days of measurement and p -values: −3 –0.38, 1 –0.27, 3 –0.02, 5 –0.04, 8 –0.05, 10 – 0.02, 12 –0.06, 15 –0.02, 17 – 0.02, 19 – 0.03). At the endpoint of the experiment (day 19), the average tumor size in the NRAS ASO treatment group was 48% smaller compared to control. k NRAS -mRNA levels were significantly reduced (0.68-fold, s.e.m = 0.03, p = 0.0003) in tumors of the NRAS ASO treatment group compared to the Control ASO treatment group at the end of study period. Tumors were harvested at end of treatment period; gene expression was normalized to Β-ACTIN expression and NRAS -mRNA expression in NRAS ASO treated tumors was normalized to expression in Control ASO treated tumors ( n of each group = 5). l No significant weight changes were observed between the NRAS ASO (X) and Control ASO (O) groups at any time-point (days of measurement and p -values: -3 – 0.3, 1 – 0.36, 3 – 0.33, 5 – 0.46, 8 – 0.43, 10 – 0.5, 12 – 0.47, 15 – 0.49, 17 – 0.48, 19 – 0.49). m Blood of mice that either received a dose of NRAS ASO-1 (200 µg/injection), or ASO-free PBS was drawn 24 hours after injection and analyzed for parameters of liver function (Serum transaminases – ALT, AST, bilirubin - TBIL, direct (conjugated) bilirubin - DBIL, total protein - TP, albumin - ALB, and alkaline phosphatase – ALKP). All growth and weight curves are presented as polynomial trend lines (order: 2). Data in ( a – c , i ) were normalized to treatment with non-targeting Control ASO, final oligonucleotide concentration was 50 nM, treatment period was 5 days ( n = 3). The error bars in a – d ), h , i , m ) represent s.d., in j − l they represent s.e.m. Significance is shown as p -values calculated by Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Inhibition, Mutagenesis, Derivative Assay, Control, Concentration Assay, Staining, Activity Assay, Gene Expression, Expressing, Injection

Journal: Communications Medicine

Article Title: Suppression of NRAS -mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

doi: 10.1038/s43856-025-00932-5

Figure Lengend Snippet: a Schematic illustration of HT-KAM analysis of the phosphor-catalytic activity of kinases. D04 and MM415 cells were either treated with NRAS or Control ASOs (50 nM, 1 day). Cells were lysed, and protein lysate was investigated for peptide-associated phosphorylation activity of kinases. b Comparison of kinase activity in treatment groups (NRAS ASO VS. Control ASO) showed that kinase activity of several kinases was significantly upregulated in the D04 and MM415 cell lines upon NRAS ASO treatment. Kinases are ranked by their relative increase of activity (from bottom to top). The top 3 kinases with strongest shift in activity increase are MAP2K1 (MEK1), FGFR2, and CDK4. The RET kinase activity shift is shown as a representative example for kinases that were downregulated in activity. c QRT-PCR analysis showing elevated NRAS -mRNA levels in D04 and MM415 cells after three days of drug-induced Inhibition of MEK (MEKi), using the small molecule inhibitor Trametinib (20 nM or 40 nM), when compared to control, treated with DMSO ( n = 3). d QRT-PCR analysis showing elevated NRAS -mRNA levels in the MEKi resistant cell lines D04RM and MM415RM, which were chronically exposed to Trametinib, when compared to their paternal treatment naïve cell lines D04 and MM415 ( n = 3). Error bars in panel ( c , d ) represent s.e.m. e Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the MEKi resistant NRAS mutant melanoma cell lines D04RM (p = 0.011), MM415RM ( p = 0.001), WM3629RM ( p = 0.0002), and Sk-Mel-2RM ( p = 0.015). Data were normalized to treatment with non-targeting Control ASO; treatment period was 5 days, final oligonucleotide concentration was 50 nM, and error bars represent s.d. ( n = 3). f – i Dual treatment with 20 nM of NRAS ASO and Trametinib (Tram, 0.5 nM −25 nM) caused robust synergistic effects in D04 ( f , g ) and MM415 ( h , i ) cells after 3 ( f , h ) and 5 ( g , i ) days of treatment ( n = 2). Dose response curves show NRAS ASO treatment (blue), trametinib treatment (yellow) and dual treatment (red). Synergism of dual cell growth inhibition is shown as bar graphs and determined by the HSA synergy score.

Article Snippet: Fluorescent signals were produced by DAPI DNA staining to mark the nuclear regions (blue), probes that stain the NRAS -mRNA (red), and two different antibodies that stain for NRAS protein (ProteinTech 10724-1-AP – green, LsBio LS-C174539 – orange).

Techniques: Activity Assay, Control, Phospho-proteomics, Comparison, Quantitative RT-PCR, Inhibition, Mutagenesis, Concentration Assay

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