Fig 1: NDRG1 is required for normal breast cancer cell proliferation, viability and morphology. a Immunoblots of NDRG1 and GAPDH loading control after selection of four stable lentiviral transduced shRNA expressing cell lines. A green fluorescent protein (GFP) targeting shRNA serves as negative control, and silencing was verified at 1% O2. b Cell proliferation assays: cell number was compared by comparing NDRG1 silenced cell numbers to control at the times indicated: ***p < 0.0001, N = 3/group - see Additional file 1: Figure S5. c Cleaved caspase 3 (CC3) immunofluorescence analysis of stable shRNA expressing SKBR3 cells at 1 week post selection. CC3 positive area was normalized to total cell area and each population compared: shNDRG1_1 p = 2.5 × 10− 6, shNDRG1_2 p = 1.2 × 10− 4, scale = 100 μm, N = 12/group. d Individual frames from a 48 h live cell microscopy analysis of SKBR3 cells expressing the indicated shRNAs: N = 3/group, see Additional files 3, 4, 5, 6, 7 and 8: Movies S1–S6. e Mitotic cell fractions comparing the percentage of histone H3 phsopho-serine10 positive cells in the indicated shRNA expressing cell lines: shNDRG1_1 p = 0.02, shNDRG1_2 p = 0.06, N = 12/group. f Comparison of individual cell two-dimensional area based on phalloidin stain (p < 0.001, Mann-Whitney U test, N = > 650 cells each group). Log2 two-dimensional area is shown. All bar graphs represent means +/− SD. Comparisons were by two-sided Student’s t test unless indicated otherwise
Fig 2: SGK1, NDRG1, and NR3C1 basal expression patterns in neurons and oligodendrocytes in the PFC of D2 mice.NDRG1, NR3C1 and SGK1 were co-localized with the neuronal marker MAP2 and the oligodendrocyte marker CNPase. DAPI staining was used as a nuclear marker (right columns). Panels show (from left to right): (a) SGK1 staining, MAP2 staining, co-localization; (b) NDRG1 staining, MAP2 staining, co-localization; (c) NR3C1 staining, MAP2 staining, co-localization; (d) SGK1 staining, CNPASE staining, co-localization; (e) NDRG1 staining, CNPASE staining, co-localization; (f) NR3C1 staining, CNPASE staining, co-localization. Scale bar equals 50 µm.
Fig 3: Expression of total NDRG1 (green) and pNDRG1 (Thr346) (red) in cultured Schwann cells (a-d). In some of the cells, there was a granular nuclear signal (arrow) (b). Even though the distinct, punctate structures were overlaying the nucleus in many instances, they are actually located in the cytoplasm (arrowheads) (b). Bar 20 μm
Fig 4: Expression of NDRG1 in epithelia. Strong signal is present in the epithelium of the colon (a) and jejunum (b) of both the control and NDRG1mut/mut Alaskan malamute, whereas NDRG1 staining in the epithelial cells of the bile ducts in the liver was only detected in the control dog (c). NDRG1 is also present in the endometrium (d) and the epithelium of the prostate (e). In the lungs (f), the club cells (arrows) of the bronchiolar epithelium display a more intense signal than the surrounding epithelial cells. In addition, signal from the endothelium (arrowhead) can be seen. Note that except for the lack of signal in the bile ducts, the NDRG1 staining in the NDRG1mut/mut Alaskan malamute is similar to the controls. The extensive yellow-brown granules in the bile ducts of the NDRG1mut/mut Alaskan malamute is interpreted as pigment deposits. Bar 50 μm
Fig 5: Fluorescent fatty acid tracers reveal NDRG1 suppresses fatty acid incorporation Into neutral lipids and storage in lipid droplets. a Fluorescence micrographs of stable NDRG1 SKBR3 +/− NDRG1 depletion fed the 16-carbon boron-dipyrromethene (BODIPY) palmitate (BODIPY C16–100 μM, 16 h), fixed. Lipid droplets and nuclei counterstained with LipidTox Deep Red and Hoechst, respectively. b Quantification of total BODIPY C16 uptake in SKBR3 cells +/− NDRG1 depletion. Graphs represent average total signal per cell at indicated tracer concentrations. c Quantification of lipid droplet specific BODIPY C16 by segmentation of small granules. d Relative distribution of BODIPY C16 signal representing droplet/whole cell signal. e LipidTox stained lipid droplet numbers represented as objects per cell. f Comparison of lipid droplet intensities of starved and fed controls at day 3 in stable BODIPY 558/568 C12 loaded MCF7 cells. Complete medium versus Hank’s buffered saline (HBSS). g BODIPY 493/503 counterstaining of NDRG1-FLAG overexpressing and control cells fed complete medium or HBSS for 3 days. h Analysis of cell survival due to starvation in stable BODIPY 558/568 C12-fed MCF7 cells transduced with NDRG1-FLAG or empty vector after 3 days of culture in HBSS. i, j Time course of changes in lipid droplet formation and cell count due to HBSS starvation in stable MCF7 cells transduced with the indicated constructs. Values represent HBSS/complete medium. All bar graphs represent mean +/− SD and comparisons were by two-sided t test. N = 12/group for all. Scale bars = 25 μm. CTRL, control
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