Journal: Protein Science : A Publication of the Protein Society

Article Title: Lectin‐anticancer peptide fusion demonstrates a significant cancer‐cell‐selective cytotoxic effect and inspires the production of “clickable” anticancer peptide in Escherichia coli

doi: 10.1002/pro.4830

Figure Lengend Snippet: Production of rRDP215 as GST‐fusion protein in E. coli and the purification. (a) Anti‐his immunoblot analysis revealed expression of the rRDP215‐GST fusion but not of the GST‐rRDP215 fusion protein. The details of the fusion constructs are depicted in the Additional file 1: Figure . Soluble and insoluble protein fractions before induction (0 h) and 2 and 4 h after induction of the gene expression with IPTG were separated on a 4%–12% SDS‐gel. Proteins were transferred to a nitrocellulose membrane and were probed by mouse anti‐6x‐His tag monoclonal primary antibody and goat anti‐mouse secondary antibody‐HRP conjugate. The black arrow indicates rRDP215‐GST (MW calc ~30 kDa); M1, MagicMark XP Western Protein Standard (Thermo Fisher Scientific). (b) Coomassie stained SDS‐gel showing the purification of rRDP215‐GST by immobilized glutathione affinity chromatography. M2, molecular mass standards in kDa; Lane 1, clarified lysate; lane 2, flow through after loading clarified lysate on a glutathione agarose column; lane 3, column wash with reduced l ‐glutathione; lanes 4–8, elution fractions.

Article Snippet: Wash and elution steps were performed with PBS and 50 mM reduced l ‐glutathione (Cube Biotech GmbH) in PBS, respectively.

Techniques: Purification, Western Blot, Expressing, Construct, Gene Expression, SDS-Gel, Membrane, Staining, Affinity Chromatography

Journal: Journal of hazardous materials

Article Title: Sulfate supplementation affects nutrient and photosynthetic status of Arabidopsis thaliana and Nicotiana tabacum differently under prolonged exposure to cadmium.

doi: 10.1016/j.jhazmat.2022.130527

Figure Lengend Snippet: Fig. 7. GSH and total PCs content in shoot and root of A.thaliana plants exposed to low sulfate treatment in combination with cadmium stress. Lines represent medians and circles individual biological replicas (n = 4–6). Different letters indicate significant differences between treatments calculated by the non-parametric vdW test with the vdW post-hoc test (FDR-corrected, p < 0.05). CTR, plants cultivated in medium without Cd; +Cd, plants cultivated in medium supplemented with 4 µM CdCl2.

Article Snippet: Standard curves of GSH and PCs were generated by external standards of reduced GSH (Carbosynth; FG10640) and PC2–5 (KareBay Biochem; KP1372 to KP1375).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO preserves the stemness of CSCs. (A) mRNA expressions of PROM1, ALDH1, POU5F1, SOX2 and KLF4 were assessed in non‐CSCs and CSCs by qPCR. Mean ± SEM from 4 to 10 independent experiments. (B) GSEA snapshots of KEGG pathway enrichment analysis. (C) Genes involved in nitrogen, glucose, lipid, and amino acid metabolism were categorized into three groups, up‐regulation, down‐regulation and no regulation in CSC compared to non‐CSC. (D) Heatmap showed nitrogen metabolism‐related genes expressed in CSCs compared with non‐CSCs. (E) non‐CSCs were treated with DETA NONOate for 24 h and the concentrations of intracellular cGMP were determined by ELISA. (F) Non‐CSCs were treated with or without DETA NONOate (20 μM) for 24 h and detected for PROM1, ALDH1, POU5F1 and SOX2 mRNA expressions by qPCR. Mean ± SEM from 6 to 7 independent experiments. (G) Non‐CSCs were treated with or without DETA NONOate (20 μM) for 24 h and the wound healing assays were performed. Mean ± SEM from six independent experiments. (H) CSCs were treated with DETA NONOate for 24 h and the concentrations of intracellular cGMP were determined by ELISA. (I) CSCs treated with L‐NAME (100 μM) for 24 h were detected for expression of NOS2 by qPCR. Mean ± SEM from seven independent experiments. (J) mRNA levels of PROM1, ALDH1, POU5F1 and SOX2 were measured in CSCs with or without L‐NAME (100 μM) treatment for 24 h. Mean ± SEM from 6 to 8 independent experiments. (K) CSCs treated with or without L‐NAME (100 μM) for 7 days were detected for sphere formations. Representative images and Mean ± SEM from seven independent experiments were shown. Scale bar: 10 μm. (L) Ration of CSCs formation at first (G1) and second (G2) generation after L‐NAME (100 μM) treatment. Mean ± SEM from five independent experiments. (M) mRNA expression of NOS2 was assessed in non‐CSCs and CSCs by qPCR. Mean ± SEM from four independent experiments. (N) CSCs were exposed to L‐NAME (100 μM) in the presence or absence of the DETA NONOate (20 μM) for 24 h. mRNA expressions of indicated stem‐related genes were detected by qPCR. (O) CSCs conditioned with L‐NAME (100 μM), with or without the addition of DETA NONOate (20 μM), were assayed for sphere formation. Representative images and mean ± SEM from five independent experiments are shown. (P, Q) CSCs were treated with GSNO (200 μM) and 1400 W (100 μM) for 24 h and the concentrations of intracellular cGMP were determined by ELISA. (R) CSCs treated with 1400 W (100 μM) in the presence or absence of the GSNO (200 μM) were analysed for sphere formation. Mean ± SEM from 6 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 with paired t‐test (E, F, H–L, P, Q), unpaired t ‐test (A, M) and ANOVA plus Turkey's method (G, N, O, R).

Article Snippet: L‐NAME, 1400 W 2HCl and S‐Nitrosoglutathione (GSNO) was purchased from Selleck (L‐NAME, S2877; 1400 W, S8337; GSNO, E4443) and DETA NONOate, Mitomycin C and proteasome inhibitor MG132 were from MCE (DETA NONOate, HY‐136278; Mitomycin C, HY‐13316; MG132, HY‐13259).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO sustains CSC stemness through the activation of Notch1. (A) Immunoblot analysis of Notch1 and Hes1 protein in non‐CSCs and CSCs. (B) The protein expression of Notch1 was analysed in non‐CSCs treated with DETA NONOate (20 μM) for 24 h. (C) CSCs were treated with L‐NAME for 24 h and detected for Notch1 protein with immunoblots. (D, E) CSCs were treated with L‐NAME (100 μM)/1400 W (100 μM) in the presence or absence of the DETA NONOate (20 μM)/GSNO (200 μM) for 24 h. Protein expression of Notch1 and Hes1 were detected by immunoblot. (F–H) CSCs were exposed to L‐NAME (100 μM) with or without the reintroduction of NICD and were analysed for expression of Notch1 protein, stem‐related transcripts and spheres formation. Mean ± SEM from 5 to 9 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 with paired t ‐test (B, C), unpaired t ‐test (A) and ANOVA plus Turkey's method (D, G, H).

Article Snippet: L‐NAME, 1400 W 2HCl and S‐Nitrosoglutathione (GSNO) was purchased from Selleck (L‐NAME, S2877; 1400 W, S8337; GSNO, E4443) and DETA NONOate, Mitomycin C and proteasome inhibitor MG132 were from MCE (DETA NONOate, HY‐136278; Mitomycin C, HY‐13316; MG132, HY‐13259).

Techniques: Activation Assay, Western Blot, Expressing