control peptide cp  (Novus Biologicals)

Journal: British journal of haematology

Article Title: MUC1-C IS A TARGET IN LENALIDOMIDE RESISTANT MULTIPLE MYELOMA

doi: 10.1111/bjh.14801

Figure Lengend Snippet: A. Schema of the MUC1-C subunit with amino acid sequence of the intrinsically disordered cytoplasmic domain. Highlighted are the sites that interact with GSK3β and β-catenin, linking MUC1-C to the WNT signalling pathway. The MUC1-C CQC motif is necessary for MUC1-C homodimerization, nuclear import and oncogenic function. The MUC1-C CQC motif is the target of the cell-penetrating GO-203 peptide. CP-2 is a control peptide with a CQC→AQA alteration that does not interact with the cytoplasmic domain. CD, cytoplasmic domain; ED, extracellular domain; TM transmembrane domain. B and C. MM.1S (B) and RPMI8226 (C) cells left untreated (control; CTL) and treated with (i) 2.5 μM GO-203 alone each day for 72 h, (ii) 2 μM lenalidomide (LEN) alone for 72 h, or (iii) GO-203 combined with LEN for 72 h. GO-203/LEN-treated cells were also incubated in the presence of 5 mM GSH for 72 h. Lysates were immunoblotted with the indicated antibodies. D and E. MM.1S (D) and RPMI8226 (E) cells were treated with (i) the indicated concentrations of GO-203 alone each day for 72 h, (ii) the indicated concentrations of LEN alone for 72 h, and (iii) GO-203 combined with LEN for 72 h. Mean cell survival was assessed in triplicate by Alamar blue assays. Numbers 1–6 in the graphs (left) represent combinations listed in the tables (right). FA, fraction affected. The results are representative of 3 independent experiments.

Article Snippet: Cells were treated with the MUC1-C inhibitor GO-203 ([R] 9 -CQCRRKN), the inactive control peptide CP-2 ([R] 9 -AQARRKN) (AnaSpec, Fremont, CA, USA), LEN (SelleckChem, Houston, TX, USA) or glutathione (GSH) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Sequencing, Incubation

Journal: British journal of haematology

Article Title: MUC1-C IS A TARGET IN LENALIDOMIDE RESISTANT MULTIPLE MYELOMA

doi: 10.1111/bjh.14801

Figure Lengend Snippet: A. Drug-naïve MM.1S and lenalidomide (LEN)-resistant MM.1S/LR cells were left untreated (CTL) and treated with 10 μM LEN for 96 h. Percentage survival (mean±SD of 3 determinations) was assessed by Alamar blue assays (left). Cells were also incubated with PI/annexin V and analysed by flow cytometry (right). The results are expressed as the percentage (mean±SD of 3 determinations) of apoptotic/necrotic cells. B. Lysates from wild-type (WT) and LEN-resistant (LR) MM.1S cells were immunoblotted (IB) with indicated antibodies. C. Drug-naïve H929 and LEN-resistant H929/LR cells were left untreated (CTL) and treated with 10 μM LEN for 96 h. Percentage survival (mean±SD of 3 determinations) was assessed by Alamar blue assays (left). Cells were incubated with PI/annexin V and analysed by flow cytometry (right). The results are expressed as the percentage (mean±SD of 3 determinations) of apoptotic/necrotic cells. D. Lysates from wild-type (WT) and LEN-resistant (LR) H929 cells were immunoblotted with indicated antibodies. E and F. MM.1S/LR (E) and H929/LR (F) cells were left untreated (CTL) or treated with 4 μM GO-203 or CP-2 each day for 72 h. GO-203-treated cells were also incubated in the presence of 5 mM GSH for 72h. Lysates were immunoblotted with the indicated antibodies. These results are representative of 3 independent experiments. * p<0.01; # not significant.

Article Snippet: Cells were treated with the MUC1-C inhibitor GO-203 ([R] 9 -CQCRRKN), the inactive control peptide CP-2 ([R] 9 -AQARRKN) (AnaSpec, Fremont, CA, USA), LEN (SelleckChem, Houston, TX, USA) or glutathione (GSH) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Flow Cytometry

Journal: British journal of haematology

Article Title: MUC1-C IS A TARGET IN LENALIDOMIDE RESISTANT MULTIPLE MYELOMA

doi: 10.1111/bjh.14801

Figure Lengend Snippet: A. MM.1S/LR cells were left untreated (CTL) and treated with 4 μM GO-203 or CP-2 each day for 72 h. B. H929/LR cells were left untreated (CTL) and treated with 4 μM GO-203 or CP-2 each day for 72 h. Cells were incubated with propidium iodide/annexin V and analysed by flow cytometry. The results are expressed as the percentage (mean±SD of 3 determinations) of apoptotic/necrotic cells. C and D. MM.1S/LR (C) and H929/LR (D) cells were treated with (i) the indicated concentrations of GO-203 alone each day for 72 h, (ii) the indicated concentrations of LEN alone for 72 h, and (iii) GO-203 combined with LEN for 72 h. Mean cell survival was assessed in triplicate by Alamar blue assays. Numbers 1–6 in the graphs (left) represent combinations listed in the tables (right). FA, fraction affected. The results are representative of 3 independent experiments. * p<0.01.

Article Snippet: Cells were treated with the MUC1-C inhibitor GO-203 ([R] 9 -CQCRRKN), the inactive control peptide CP-2 ([R] 9 -AQARRKN) (AnaSpec, Fremont, CA, USA), LEN (SelleckChem, Houston, TX, USA) or glutathione (GSH) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Incubation, Flow Cytometry

Journal: British journal of haematology

Article Title: MUC1-C IS A TARGET IN LENALIDOMIDE RESISTANT MULTIPLE MYELOMA

doi: 10.1111/bjh.14801

Figure Lengend Snippet: A and B. The indicated wild-type (WT) and lenalidomide (LEN) resistant (LR) MM.1S (A) and H929 (B) cells were analysed for CD44 expression by flow cytometry. The results are expressed as relative CD44 levels (mean±SD of 3 determinations) as compared to that obtained for WT cells (assigned a value of 1). C and D. MM.1S/LR (C) and H929/LR (D) cells were left untreated (CTL) and treated with 4 μM GO-203 or CP-2 for 72 h. The results are expressed as relative CD44 levels (mean±SD of 3 determinations) as compared to that obtained for CTL cells (assigned a value of 1). The results are representative of 3 independent experiments. E. Primary CD138+ MM cells from Patient 1 (LEN-sensitive), Patient 2 (LEN-resistant) and Patient 3 (LEN-resistant) were left untreated (CTL) and treated with 5 μM GO-203 or CP-2 for 72 h. The results are expressed as relative CD44 levels (mean±SD of 3 determinations) as compared to that obtained for CTL cells (assigned a value of 1). F. Microarray gene expression data from GEO dataset GSE2658 (n=559) was RMA normalized and the correlation between MUC1 and CD44 expression in MM patients was assessed by Spearman correlation, where p<0.05 was considered as statistically significant. * p<0.01; ** p<0.05.

Article Snippet: Cells were treated with the MUC1-C inhibitor GO-203 ([R] 9 -CQCRRKN), the inactive control peptide CP-2 ([R] 9 -AQARRKN) (AnaSpec, Fremont, CA, USA), LEN (SelleckChem, Houston, TX, USA) or glutathione (GSH) (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Flow Cytometry, Microarray

Journal: Leukemia

Article Title: MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs

doi: 10.1038/leu.2017.163

Figure Lengend Snippet: MUC1 inhibition leads to decrease in PD-L1 expression. MUC1 was silenced in MOLM-14 and THP-1 AML cells using lentiviral shRNA hairpin against MUC1. As a control, MOLM-14 cells were infected with control shRNA. The cells were then evaluated for PD-L1 expression using (a) flow cytometry and (b) western blot analysis. (c) Tumor cells were obtained from patients with AML at diagnosis. The cells were then treated with 2.5 µm MUC1 inhibitor, GO-203 daily for 3 days. As a control the cells were treated with control peptide CP-3. Subsequently, the cells underwent western blot analysis for PD-L1 expression. Representative blots from patients AML1 and AML2 are shown (n =3). (d) A representative experiment demonstrating PD-L1 mRNA levels, in MUC1-silenced MOLM-14 and THP-1 cells evaluated using qPCR. Each condition was performed in triplicate (P<0.05; n=2).

Article Snippet: AML cells were treated once daily with 2.5 µ m MUC1-C inhibitor peptide (GO-203) or a control peptide (CP-3) (AnaSpec) for 3 days.

Techniques: Inhibition, Expressing, shRNA, Infection, Flow Cytometry, Western Blot

Journal: Leukemia

Article Title: MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs

doi: 10.1038/leu.2017.163

Figure Lengend Snippet: miR-34a and miR-200c regulate PD-L1 expression in AML cells. (a) The seed sequences of miR-34a and miR-200c on the 3′-UTR PD-L1, transcript variant 1, NM_014143.3, were identified by RegRNA: a regulatory RNA motifs and element web server. MUC1 was silenced in MOLM-14 cells using the CRISPR/Cas9 technology and in THP-1 cells using transduction with MUC1-specific shRNA. (b) Relative levels of miR-34a were detected in MOLM-14 and THP-1 cells using qPCR. miR-34a was overexpressed in MOLM-14 and THP-1 cells using lentiviral transduction. (c) PD-L1 levels were evaluated using western blot analysis in miR-34a overexpressed or control MOLM-14 and THP-1 AML cells. (d) Relative levels of miR-200c were detected in MOLM-14 and THP-1 cells using qPCR. MiR-200c was overexpressed in MOLM-14 cells using lentiviral transduction. PD-L1 levels were evaluated in control and miR-200c overexpressed cells using (e) western blotting and (f) flow cytometry. Tumor cells were obtained from PB or BM aspirated of patients with AML at diagnosis. The cells were then treated with 2.5 µm MUC1 inhibitor, GO-203 daily for 3 days. As a control the cells were treated with control peptide CP-3. (g) Subsequently, relative levels of miR-34a and miR200c were detected in these cells compared to controls using qPCR. Three patients were evaluated, the bar graphs represent patients AML2 and AML3 (P<0.05).

Article Snippet: AML cells were treated once daily with 2.5 µ m MUC1-C inhibitor peptide (GO-203) or a control peptide (CP-3) (AnaSpec) for 3 days.

Techniques: Expressing, Variant Assay, CRISPR, Transduction, shRNA, Western Blot, Flow Cytometry