Journal: Cell death & disease

Article Title: Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

doi: 10.1038/s41419-021-04154-0

Figure Lengend Snippet: Fig. 8 Effects of S63845 and imatinib administered as single drug or in combination on the colony forming capacity of primary human CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase or from the bone marrow of healthy donors. Relative colony forming capacity of CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase (left panel; n = 6 for all S63845 concentrations) or from the bone marrow (BM) of healthy donors (right panel; n = 4 for all S63845 concentrations) in the absence or presence of 1 μM imatinib (IM) and/or the indicated concentrations of S63845. Data represent mean with range derived from four (CD34+ normal BM cells) to six (CD34+ CML cells) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Patient no. 1: ⚫; patient no. 2: ◼; patient no. 5: ◆; patient no. 6: ○; patient no. 8: △; patient no. 9: ▽; healthy donor no. 1 ⚫; donor no. 2: ◼; donor no. 3: ▲; donor no. 4: ▼.

Article Snippet: Primary human normal bone marrow CD34+ hematopoietic stem/ progenitor cells were obtained from American Type Culture Collection (Cat. No.: PCS-800-012) or from Lonza (Cat. No.: 2M-101), and kept in culture for 48 h in StemSpan SFEM II Medium supplemented with StemSpan CD34+ Expansion Supplement and 175 nM UM171 (all from STEMCELL Technologies) before starting the colony forming assay.

Techniques: Derivative Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Targeting MS4A4A: A novel pathway to improve immunotherapy responses in glioblastoma

doi: 10.1111/cns.14791

Figure Lengend Snippet: Inhibition of GBM cancer cell development by suppressing M2 macrophage infiltration through activation of TAM‐iron death pathway. (A) Flow cytometry was used to determine the proportion of M2 macrophages. (B) BMDMs were transfected with lentivirus for knockdown of MS4A4A or negative control plasmid, then induced into M2 phenotype using IL‐4 (20 ng/mL) and IL‐13 (20 ng/mL). qRT‐PCR was used to measure the interference efficiency of MS4A4A and expression of M2 markers (Mgl2, Arg1, and Tgfb1) and M1 marker iNOS. (C) THP‐1 human monocytic cell line was used to establish an overexpression cell line of MS4A4A, followed by differentiation into M0 macrophages using PMA (50 ng/mL). M0 macrophages were then polarized into M2 phenotype using IL‐4 (20 ng/mL) and qRT‐PCR was used to measure expression levels of MS4A4A and M2 markers (CD163, ARG1, and TGFB1) and M1 marker CD86. (D) PBMC cells were used to construct a cell line overexpressing MS4A4A. (E) Procedure for testing macrophage inhibition function: Mouse BMDMs and spleen cells were mixed with CT2A/GL261 cells, treated with MS4A4A antibody, and CD8 + T cells were sorted using flow cytometry and Ki‐67 expression was measured. (F) Analysis of Ki‐67 expression on designated CD8 + T cells using flow cytometry ( n = 3). (G) Apoptosis of CT2A cells was detected using fluorescence‐activated cell sorting (FACS). (H) Iron content in tumor‐associated macrophages (TAMs) of sh‐NC and sh‐MS4A4A groups. (I) Reactive oxygen species (ROS) content in TAM cells of sh‐NC and sh‐MS4A4A groups. (J) Expression of iron death‐related proteins in TAM cells of sh‐NC and sh‐MS4A4A groups. (K) Electron microscopy analysis of TAM cells in sh‐NC and sh‐MS4A4A groups. * p < 0.05, and all cell experiments were repeated three times.

Article Snippet: To induce differentiation into M0 macrophages, THP‐1 cells were incubated with 50 ng/mL of phorbol 12‐myristate 13‐acetate (PMA) from InvivoGen (USA) for 48 h. To further polarize M1, cells were incubated with 100 ng/mL lipopolysaccharide (LPS, L2630, Sigma‐Aldrich, USA) and 20 ng/mL recombinant human interferon‐γ (Recombinant Human IFN‐γ, AF‐300‐02, PeproTech, USA) for 48 h. As a control experiment, we conducted the same incubation treatment in PBMC cells (PCS‐800‐011, ATCC, USA) as in THP‐1 cells.

Techniques: Inhibition, Activation Assay, Flow Cytometry, Transfection, Knockdown, Negative Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Marker, Over Expression, Construct, Fluorescence, FACS, Electron Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein Rab27A in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Functional Assay, MTT Assay, Cell Culture, Transmission Assay, Electron Microscopy, Derivative Assay, Concentration Assay, Imaging, Flow Cytometry, Staining, Fluorescence, Microscopy, Incubation, Western Blot, Colony Assay, Co-Culture Assay, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Rab27A regulates EV release and histone H3.2 (H3C14) protein excretion in GCB‐resistant bladder cancer cells . (A) RT‐qPCR analysis was used to evaluate Rab27A knockdown efficiency in T24GCB and 5637GCB cells following siRab27A transfection. (B) Representative fluorescence microscopy images of T24GCB‐Vector and T24GCB‐siRab27A cells stained with H3C14‐FITC, Rab27A‐APC and DAPI. Scale bar: 20 µm. (C) Nanoparticle tracking analysis (NS300) of EVs released from T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells revealed particle size distribution and concentration. (D) MTT assay was used to evaluate cell viability of T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (E) Representative electron microscopy images show MVBs and ILVs in T24GCB‐Vector, T24GCB‐siRab27A and 5637‐Vector, 5637GCB‐siRab27A cells. (F, G) Quantification of MVBs per image (F) and ILVs per MVB (G) from panel L. (H, I) Colony formation assays were used to evaluate clonogenicity of T24GCB and 5637GCB cells transfected with Vector or siRab27A over 10 days ( n = 3 per group). (J) Western blot analysis of Rab27A, H3C14, CNT3 and TK1, as well as EV‐associated proteins (CD9, CD63, CD81, TSG101 and Alix) in T24GCB and 5637GCB cells transfected with Vector or siRab27A. (K) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB and 5637GCB cells treated with DMSO, neticonazole (1 µM), or ketoconazole (1 µM) for 24 h. (L, M) Colony formation assays in T24GCB and 5637GCB cells treated with DMSO, neticonazole, or ketoconazole (1 µM each) ± GCB (0.001 µM) for 7 days ( n = 3 per group). (N) Western blot analysis of Rab27A, H3C14, Alix, p‐ERK1/2, total ERK1/2 and BCL‐2 in T24GCB and 5637GCB cells after 24 h treatment with DMSO, neticonazole, or ketoconazole. (O) RT‐qPCR analysis was used to evaluate CNT3 knockdown efficiency in T24GCB and 5637GCB cells after siCNT3 transfection. (P) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB‐Vector, T24GCB‐siCNT3, and 5637GCB‐Vector, 5637GCB‐siCNT3 cells. (Q) MTT assay was used to evaluate viability of T24GCB‐Vector, T24GCB‐siCNT3 and 5637GCB‐Vector, 5637GCB‐siCNT3 cells treated with GCB (0–3 µM) for 48 h ( n = 6 per group). (R) Western blot analysis of CNT3, H3C14, Rab27A, EV markers (CD9, CD63, CD81, Alix and TSG101) and anti‐apoptotic proteins (BCL‐2 and BCL‐XL) in T24GCB and 5637GCB cells transfected with Vector or siCNT3. All data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Knockdown, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Staining, Concentration Assay, MTT Assay, Electron Microscopy, Western Blot, Derivative Assay, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction