ent  (Proteintech)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Phosphorylation disrupts the interaction between the intrinsically disordered region of the oncogenic NDRG1 and lipid vesicles

doi: 10.1002/pro.70510

Figure Lengend Snippet: (a) Co‐sedimentation analysis of unmodified and phosphorylated NDRG1*C and liposomes with different lipid composition. (b) Circular dichroism of NDRG1*C (upper panel) and P‐NDRG1*C (bottom panel) in solution (black), and in the presence of DMPC (blue) and DMPG (red)‐based liposomes. Filled circles represent experimental points, solid lines are the fits obtained using BestSel. (c) FTIR spectra of NDRG1*C (upper panel) and P‐NDRG1*C (bottom panel) dried on ATR crystal (black), and in the presence of DMPC (blue) and DMPG (orange)‐based liposomes. Solid lines represent the experimental data, dashed lines represent the 2nd derivative of the FTIR spectra. (d) CW‐EPR spectra of the S336C Proxyl NDRG1*C in its unmodified and phosphorylated states and in the absence and in the presence of DMPC and DMPG‐based liposomes. (e) Intensity ratios of the peaks in the CACO spectra obtained at 700 MHz (16.4 T) by 13 C direct detection at pH 7.5 for NDRG1*C, in the absence and presence of DMPG‐based liposomes. The sequence of the protein is reported over the plots: for clarity, the protein sequence containing the phosphorylation sites is enclosed in a box, the sequence with α‐helical propensity is represented in bold, and the repeated sequences are colored in cyan, green, and blue. (f) Thermodynamic parameters (Δ G , blue; Δ H , green; − T Δ S , red) obtained from the fit of the binding isotherms obtained by ITC represented in Figure and reported in detail in Table .

Article Snippet: DMPG (1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol), sodium salt) and DMPC (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine) were purchased from Avanti Polar Lipids.

Techniques: Sedimentation, Liposomes, Circular Dichroism, Sequencing, Phospho-proteomics, Binding Assay

Journal: Advanced Science

Article Title: Super‐Enhancer‐Driven SOX4/SMAD3 Mediate Membrane Remodeling by Regulating Phospholipid Metabolism to Accelerate Leukemia Progression

doi: 10.1002/advs.202512332

Figure Lengend Snippet: SOX4/SMAD3 regulated saturated phospholipid synthesis to mediate AXL localization and activation. (A) GO pathway enrichment analysis of the downregulated lipid metabolism‐associated genes following knockdown of SOX4 and SMAD3 in LAMA‐84 cells. (B) Distribution of changes in each subclass of the lipid metabolome upon knockdown of SOX4 or SMAD3 in LAMA‐84 cells. (C) Volcano plots showing differentially expressed phospholipids upon knockdown of SOX4 or SMAD3. Each dot represents a phospholipid. (D) Repartition of PL‐SFAs, PL‐MUFAs and PL‐PUFAs in LAMA‐84 cells transfected with shRNA targeting SOX4, SMAD3, or vector control. (E) Heatmap visualization of specific saturated and monounsaturated phospholipid species altered in LAMA‐84 cells expressing indicated shRNA or vector, highlighting the marked reduction of PC(16:0/16:0) (DPPC) and PC(16:0/18:1) (POPC). (F) The relative cell viability of SOX4/SMAD3‐deficient LAMA‐84 cells following treatment with DPPC or POPC liposomes. (G) Representative flow cytometry histogram of surface CD71 expression in LAMA‐84 cells expressing empty vector and SOX4/SMAD3 shRNA. Cells were treated with vehicle control or DPPC liposomes. (H) FRAP images in LAMA‐84 cells transfected with shRNA targeting SOX4, SMAD3, or empty vector. Cells were treated with vehicle control or DPPC liposomes. Images are taken over a 30 s time course and arrows indicate location of photobleaching. n = 10. Scale bar, 5 µm. (I) FRAP recovery curves following treatment with SOX4/SMAD3 shRNA or vector in LAMA‐84 cells. Cells were treated with vehicle control or DPPC liposomes. n = 10. (J) Immunofluorescence staining of AXL localization (green) in LAMA‐84 cells with SOX4/SMAD3 shRNA or vector. DPPC liposomes was added to rescue membrane localization. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. (K) Western blot analysis of the total and phosphorylation levels of AKT, STAT5 and ERK upon knockdown of SOX4 or SMAD3 in LAMA‐84 cells. Cells were treated or untreated with DPPC. Data are presented as mean ± s.d. of three replicates. Two‐way ANOVA Dunnett's multiple comparisons test were performed. The experiments in panels (F–K) were repeated three times independently with similar results, and the results of 1 representative experiment are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant. See also Figure .

Article Snippet: The phospholipids DPPC(PC16:0/16:0) (1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine) and POPC (PC16:0/18:1) (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) (Avanti Polar Lipids) in chloroform were initially evaporated under a nitrogen stream, followed by completely drying under high vacuum.

Techniques: Activation Assay, Knockdown, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Liposomes, Flow Cytometry, Immunofluorescence, Staining, Membrane, Western Blot, Phospho-proteomics