Journal: Oncotarget

Article Title: Mevastatin blockade of autolysosome maturation stimulates LBH589-induced cell death in triple-negative breast cancer cells

doi: 10.18632/oncotarget.14868

Figure Lengend Snippet: Prior to mevastatin plus LBH589 (25 nM) treatment for 24 h, MDA-MB-231 ( A ) and MDA-MB-468 ( B ) cells were incubated with or without z-VAD (20 μM) for 2 h. Cell lysates were analyzed by Western blotting for Vps34, Beclin 1, LC3B, p62/SQSTM1, and NBR1, with GAPDH as a loading control. Relative p62/SQSTM1 expression (compared with GAPDH) was analyzed by qPCR. Student's t -test. * P < 0.05; ** P < 0.01 ( C ). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) and/or LBH589 (25 nM) for 24 h. Mevastatin reduced LBH589-dependent expression of Vps34 and Beclin 1 in a dose-dependent manner ( D ). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) for 24 h and probed for endogenous Vps34 and Beclin 1. Mevastatin abrogated Vps34-Beclin 1 complex formation dose-dependently in the presence of LBH589 ( E ). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) plus 25 nM LBH589. Immunoblot analyses were performed on immuoprecipitates as indicated. tfLC3 stable MDA-MB-231 cells were analyzed by confocal microscopy ( F ). MDA-MB-231_tfLC3 cells were treated with mevastatin (2 μM) and/or LBH589 (25 nM) for 24 h. Representative fluorescence images are shown. MDA-MB-231 cells transfected with flag-Rab7 (or pcDNA3.1) were treated with mevastatin (2 μM) and/or LBH589 (25 nM) for 48 h, and cell proliferation was measured. Student's t -test. ** P < 0.01; *** P < 0.001 ( G ). Mevastatin with or without LBH589 (25 nM) reduced prenylated Rab7 in a dose-dependent manner ( H ). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) and/or LBH589 (25 nM) for 24 hours. Cell lysates were immunoblotted for unprenylated and prenylated Rab7. Prior to treatment with mevastatin (2 μM) and LBH589 (25 nM) for 24 h, MDA-MB-231 cells were incubated with or without mevalonate (250 μM) for 6 h, or with or without compound C (2 μM) for 2 h ( I ). Cell lysates were immunoblotted for LC3B, p62/SQSTM1, and unprenylated and prenylated Rab7, with GAPDH as a loading control.

Article Snippet: Briefly, cDNA encoding mRFP-GFP-LC3 (tfLC3) was excised from ptfLC3 (Addgene, 21074) and sub-cloned into the lentiviral vector, pCDH1-MCS1-EF1-puro (System Bioscience, CD510A-1).

Techniques: Incubation, Western Blot, Control, Expressing, Confocal Microscopy, Fluorescence, Transfection

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: Overexpression of circTRAPPC6B inhibited intracellular Mtb growth while activating autophagy in Mtb‐infected macrophages. (a, b) After transfection with circTRAPPC6B overexpressing plasmids or control plasmids for 24 h, THP‐1 macrophages (a) and macaque spleen macrophage (b) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (c, d) After transfection with siRNA against circTRAPPC6B (c) or plasmids overexpressing circTRAPPC6B (d) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs uninfected and untransfected control. ## P < 0.01 vs H37Rv‐infected but untransfected control. (e) After transfection with circTRAPPC6B overexpressing vectors or control vectors (NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. THP‐1 macrophages were incubated with anti‐LC3 for 2h at room temperature and then fluorescently labelled secondary Ab for 1 h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (f, g) Quantification assay of e. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs NC ( n = 3). MOI, multiplicity of infection; CFU, colony‐forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Over Expression, Infection, Transfection, Control, Western Blot, Expressing, Incubation, Immunofluorescence, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: miR‐874‐3p regulated intracellular Mtb growth and autophagy in Mtb‐infected macrophages. (a) After PBMCs were freshly isolated from blood by standard Ficoll density gradient centrifugation, miR‐874‐3p expression was analysed in human PBMCs from patients with 32 active TB and 31 HC by qRT‐PCR. U6 was used as a housekeeping gene for normalising changes in miRNA gene expression. Data are expressed as mean ± SEM. ** P < 0.01 vs HC. (b) Spearman’s correlation analysis between miR‐874‐3p and circTRAPPC6B expression in PBMCs of 32 TB patients. (c) After THP‐1 macrophages were infected with H37Rv at MOI = 1 at different time points (0, 1, 2, 3, 4, and 7 days), miR‐874‐3p expression was analysed by qRT‐PCR. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. ** P < 0.01, *** P < 0.001 vs 0 d. (d, e) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages (d) and macaque spleen macrophage (e) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (f) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. Then, THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (g, h) Quantification assay of f. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs miR‐NC. (i, j) After transfection with miR‐874‐3p mimics (i) or inhibitor (j) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, *** P < 0.001 vs uninfected and untransfected control; ### P < 0.001 vs H37Rv‐infected but untransfected cells. qRT‐PCR, quantitative real‐time polymerase chain reaction; PBMCs, peripheral blood mononuclear cells; TB, tuberculosis; HC, health control; MOI, multiplicity of infection; CFU, colony forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Isolation, Gradient Centrifugation, Expressing, Quantitative RT-PCR, Gene Expression, Transfection, Negative Control, Incubation, Immunofluorescence, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: circTRAPPC6B regulated autophagy in Mtb‐infected macrophages via miR‐874‐3p. After transfection with circTRAPPC6B‐overexpressing vectors (pHBAd‐cir), miR‐874‐3p mimics, or corresponding negative controls as indicated for 24 h, THP‐1 macrophages were infected with BCG at MOI = 10 or Mycobacterium H37Rv at MOI = 1 for 24 h. (a) Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in H37Rv‐infected THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001. (b) THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of BCG‐infected THP‐1 macrophages showing the change of LC3B puncta. (c) Quantification of b. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01. BCG, Bacillus Calmette‐Guérin; MOI, multiplicity of infection.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Transfection, Western Blot, Expressing, Incubation, Immunofluorescence

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Autophagy-dependent sensitization effects of PARP inhibitors on recurrent nasopharyngeal carcinoma treated with carbon ion and photon irradiation

doi: 10.3724/abbs.2025130

Figure Lengend Snippet: Olaparib combined with IR triggers multiple modes of cell death in HK-1 cells The cells were treated with C-ions (2 Gy) or X-rays (4 Gy) with or without olaparib (5 μM). (A,B) Bar graphs of apoptosis quantified by flow cytometry at 48 h and 72 h postirradiation for each treatment group, and the corresponding dot plot diagrams are shown in Supplementary Figure S4A. Bar graphs of cell necrosis (C,D) and ferroptosis (E,F) quantified by flow cytometry at 48 h and 72 h postirradiation. The data for 24 h postirradiation are shown in Supplementary Figure S4B-D. (G) Western blot analysis of LC3 at 24 h postirradiation, with β-actin as the loading control. (H) Representative TEM microphotographs of HK-1 cells in each group at 24 h postirradiation, with red arrows indicating double-membrane autophagosomes and single-membrane autophagolysosomes (scale bars: 2 μm and 500 nm). (I) Images acquired at 24 h postirradiation from stably expressing stubRFP-sensGFP-LC3 cells treated with olaparib and/or radiation (scale bar: 20 μm). Upon merging, autophagosomes displaying both RFP and GFP signals appeared as yellow puncta. Autophagolysosomes presented attenuated GFP signals due to their acidic environment and appeared as red foci of the RFP signal. (J,K) Autophagic vacuoles were labeled via CYTO-ID® staining at 48 h and 72 h after radiotherapy, and the fluorescence intensity was quantified via flow cytometry. The values were normalized to those of the nonirradiated vehicle-control group. The data for 24 h postirradiation are shown in Supplementary Figure S4E. (L) Representative images of SA-β-Gal staining at 72 h postirradiation. (M) SA-β-Gal-positive senescent cells, identified by blue staining, were counted in 10 random visual fields via an inverted microscope at 20× magnification. The results are expressed as the average number of positive cells per field. (N) Western blot analysis of P21 at 72 h postirradiation. β-actin served as the loading control. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Autophagic flux was assessed using a tandem fluorescently tagged LC3 probe ( GPL2001 ; GeneChem, Shanghai, China).

Techniques: Flow Cytometry, Western Blot, Control, Membrane, Stable Transfection, Expressing, Labeling, Staining, Fluorescence, Inverted Microscopy