Journal: Molecular Medicine

Article Title: Involvement of the TNF-α/SATB2 axis in the induced apoptosis and inhibited autophagy of osteoblasts by the antipsychotic Risperidone

doi: 10.1186/s10020-022-00466-9

Figure Lengend Snippet: Risperidone causes an inhibition of autophagy and a promotion of apoptosis in femur tissues of mice. Mice were treated with 0, 0.3, 0.45, 0.6, 0.75 mg/kg of Risperidone. A representative micro-CT 3D and plan scan images of cross-sectional area of femur tissues. B statistical analysis of BMD in femur tissues of mice assessed by micro-CT. C statistical analysis of BMC in femur tissues of mice. D representative images of HE staining in femur tissues of mice. E Western blot analysis of autophagy-related proteins (LC3 II/I, Beclin1, and p62) expression in femur tissues of mice. The band intensity was assessed. F Western blot analysis of apoptosis-related proteins (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9). Different numbers represent different concentrations of Risperidone (mg/kg). G co-localization of LC3 II (red), LC3 I (red), Beclin1 (red), and p62 (red) with the osteoblast marker RUNX2 (green) by double-labeled immunofluorescence, wherein the nucleus is labeled by DAPI (blue). * p < 0.05, compared with the treatment of 0 mg/kg of Risperidone. The experiments are repeated 3 times

Article Snippet: According to the instructions of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, BioVision, Milpitas, CA, USA), Annexin-V-FITC, propidium iodide (PI) and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution were mixed with the Annexin-V-FITC/PI staining solution at a ratio of 1:2:50.

Techniques: Inhibition, Micro-CT, Staining, Western Blot, Expressing, Marker, Labeling, Immunofluorescence

Journal: Molecular Medicine

Article Title: Involvement of the TNF-α/SATB2 axis in the induced apoptosis and inhibited autophagy of osteoblasts by the antipsychotic Risperidone

doi: 10.1186/s10020-022-00466-9

Figure Lengend Snippet: Risperidone confers an inhibitory effect on differentiation and autophagy and a promotive effect on apoptosis of osteoblasts in vitro. A the mRNA expression of OPG, collagen I and RANKL in MC3T3-E1 cells determined by RT-qPCR. B – C Western blot analysis of differentiation-related protein (OPG, collagen I and RANKL) expression in MC3T3-E1 cells. The band intensity was assessed. D representative images of ALP staining in MC3T3-E1 cells. E, statistical results of ALP positive rate in MC3T3-E1 cells. F representative images of alizarin red S staining in MC3T3-E1 cells. G statistical results of integrated optical density (IOD) of alizarin red S in MC3T3-E1 cells. H representative images of autophagosome in MC3T3-E1 cells assessed by immunofluorescence. I Western blot analysis of autophagy-related protein (LC3 II/I, Beclin1, and p62) expression in MC3T3-E1 cells. The band intensity was assessed. J Western blot analysis of apoptosis-related protein (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9) expression in MC3T3-E1 cells. K apoptosis rate in MC3T3-E1 cells evaluated by flow cytometry. * p < 0.05, compared with MC3T3-E1 cells treated with normal culture medium. The cell experiments are repeated 3 times

Article Snippet: According to the instructions of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, BioVision, Milpitas, CA, USA), Annexin-V-FITC, propidium iodide (PI) and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution were mixed with the Annexin-V-FITC/PI staining solution at a ratio of 1:2:50.

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence, Flow Cytometry

Journal: Molecular Medicine

Article Title: Involvement of the TNF-α/SATB2 axis in the induced apoptosis and inhibited autophagy of osteoblasts by the antipsychotic Risperidone

doi: 10.1186/s10020-022-00466-9

Figure Lengend Snippet: Risperidone inhibits autophagy and accelerates apoptosis of osteoblasts via the TNF-α/SATB2 axis. A representative images of autophagosome in MC3T3-E1 cells assessed by immunofluorescence. B Western blot analysis of autophagy-related protein (LC3 II/I, Beclin1, and p62) expression in MC3T3-E1 cells. The band intensity was quantified. C Western blot analysis of apoptosis-related proteins (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9). The protein band was quantified. D apoptosis rate of MC3T3-E1 cells determined by flow cytometry. * p < 0.05, compared with MC3T3-E1 cells treated with si-NC. # p < 0.05, compared with MC3T3-E1 cells treated with Risperidone + si-NC. & p < 0.05, compared with MC3T3-E1 cells treated with Risperidone + si-TNF-α. The experiments are repeated 3 times

Article Snippet: According to the instructions of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, BioVision, Milpitas, CA, USA), Annexin-V-FITC, propidium iodide (PI) and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution were mixed with the Annexin-V-FITC/PI staining solution at a ratio of 1:2:50.

Techniques: Immunofluorescence, Western Blot, Expressing, Flow Cytometry

Journal: Molecular Medicine

Article Title: Involvement of the TNF-α/SATB2 axis in the induced apoptosis and inhibited autophagy of osteoblasts by the antipsychotic Risperidone

doi: 10.1186/s10020-022-00466-9

Figure Lengend Snippet: Risperidone represses differentiation and autophagy while enhancing apoptosis via the TNF-α/SATB2 axis in vivo. A, Western blot analysis of differentiation-related proteins (OPG, collagen I and RANKL) expression in femur tissues of mice. The band intensity was assessed. B, statistical results of alizarin red S staining in femur tissues of mice. C, Western blot analysis of apoptosis-related proteins (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9) in femur tissues of mice. The protein band was assessed. D, apoptosis rate in femur tissues in mice assessed by TUNEL staining. E, representative images of femur tissues in mice stained by immunohistochemistry and statistical results of LC3B positive rate. F, Western blot analysis of autophagy-related proteins (LC3 II/I, Beclin1, and p62) in femur tissues of mice. The band intensity was quantified and analyzed. * p < 0.05, compared with mice introduced with lentivirus expressing si-NC. # p < 0.05, compared with mice introduced with Risperidone + lentivirus expressing si-NC. & p < 0.05, compared with mice introduced with Risperidone + lentivirus expressing si-TNF-α. The experiments are repeated 3 times n = 6

Article Snippet: According to the instructions of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, BioVision, Milpitas, CA, USA), Annexin-V-FITC, propidium iodide (PI) and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution were mixed with the Annexin-V-FITC/PI staining solution at a ratio of 1:2:50.

Techniques: In Vivo, Western Blot, Expressing, Staining, TUNEL Assay, Immunohistochemistry

Journal: Molecular Medicine

Article Title: Involvement of the TNF-α/SATB2 axis in the induced apoptosis and inhibited autophagy of osteoblasts by the antipsychotic Risperidone

doi: 10.1186/s10020-022-00466-9

Figure Lengend Snippet: Schematic diagram of the mechanism by which Risperidone affects the autophagy and apoptosis of osteoblasts. Risperidone promotes TNF-α expression to represses SATB2 expression, thus inhibiting autophagy and enhancing apoptosis of osteoblasts, ultimately arresting osteoblast differentiation

Article Snippet: According to the instructions of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201-100, BioVision, Milpitas, CA, USA), Annexin-V-FITC, propidium iodide (PI) and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer solution were mixed with the Annexin-V-FITC/PI staining solution at a ratio of 1:2:50.

Techniques: Expressing

Journal: Nature Communications

Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis

doi: 10.1038/s41467-026-69899-8

Figure Lengend Snippet: a Gene expression analysis of tumour samples from treatment-naïve and relapsed SCLC patients. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . b Single-sample Gene Set Enrichment Analysis (ssGSEA) of pathways in relapsed ( n = 18) versus treatment-naïve ( n = 81) SCLC tumours. Enrichment was assessed by normalized enrichment score (NES, one-sided), and significance was determined by FDR-adjusted q -value. c GSEA analysis of NE and non-NE gene signatures in relapsed and treatment-naïve samples; n = 18 relapsed patient samples and n = 81 treatment-naïve patient samples. d Volcano plot of ssGSEA analysis highlighting pathways involved in non-NE and NE SCLC tumours from the RNA-seq dataset of IMpower133 human SCLC cohort ( n = 271); e Bright-field images of different SCLC cell models and the corresponding immunoblot analysis assessing the expression of SCLC cell state markers. For H209/H209A, the samples derive from the same experiment, but different gels for ASCL1, c-Myc, another for MYCL, another for MYCN, another for NEUROD1, YAP1 and β-tubulin were processed in parallel. For H69/H69M, the samples derive from the same experiment but different gels for ASCL1 and MYCN, another for NEUROD1 and YAP1, another for MYCL, another for POU2F3, another for c-Myc, another for β-tubulin were processed in parallel; For H82/H82R, the samples derive from the same experiment but different gels for ASCL1 and MYCN, another for NEUROD1, YAP1 and β-tubulin, another for MYCL, another for c-Myc, another for POU2F3 were processed in parallel; For H209/H029M, the samples derive from the same experiment but different gels for ASCL1, another for YAP1, another for MYCL, another for MYCN, another for c-Myc, another for NEUROD1 and β-tubulin were processed in parallel. Blots are representative of n = 3 biologically independent experiments. f Polysome profiling analysis assessing translation activity across multiple models. The ratio of the area under curve (AUC) between polysome and monosome fractions was calculated using an in-house MATLAB script. Polysome profiles are representative of n = 3 biologically independent experiments for each cell model. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. g CCK-8-based cell viability analysis across multiple SCLC models. Cells were treated with carboplatin (Carbo) and etoposide (Etop) for 72 h at the indicated doses. The one-sided extra-sum of square F test was used for statistical analysis of n = 3 biologically independent experiments.

Article Snippet: The following chemicals (stored at −20 °C or −80 °C) were used for experiments where indicated: Carboplatin (Selleckchem #S1215, stock 10 mM), Etoposide (Selleckchem #S1225, stock 50 mM in DMSO), Harringtonine (Abcam #ab141941, stock 100 μg ml − 1 in DMSO), Cycloheximide (MCE, #HY12320, stock 100 mg ml − 1 in DMSO), GSK-126 (MCE, #HY13470, stock 10 mM in DMSO), ifebemtinib (Selleckchem #E1114, stock 10 mM in DMSO), SCH772984 (Selleckchem #S7101, stock 10 mM in DMSO), GSK2256098 (Selleckchem #S8523, stock 10 mM in DMSO), Cobimetinib (Selleckchem #S8041, Stock 10 mM in DMSO), Rapamycin (Selleckchem #S1039, stock 10 mM in DMSO).

Techniques: Gene Expression, RNA Sequencing, Western Blot, Expressing, Activity Assay, CCK-8 Assay

Journal: Nature Communications

Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis

doi: 10.1038/s41467-026-69899-8

Figure Lengend Snippet: a . Immunoblot analysis of translation initiation factor expression across multiple SCLC models. β-tubulin was used as a loading control. Blots are representative of n = 3 biologically independent experiments. For H69/H69M and H82/H82R, the samples derive from the same experiment but different gels for vimentin, another for eIF4E, another for eIF6, another for eIF4A1 and β-tubulin were processed in parallel; For H209/H209A, the samples derive from the same experiment but different gels for eIF4E, another for eIF6, another for eIF4A1, another for vimentin and β-tubulin were processed in parallel; For H209/H209M, the samples derive from the same experiment but different gels for eIF4E, another for eIF4A1, another for vimentin, another for eIF6 and β-tubulin were processed in parallel. b Quantitative PCR analysis of EIF6 mRNA expression across paired SCLC cell models. Error bars represent mean±s.d. from n = 3 or 4 independent experimental replicates, as indicated. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. c Representative micro-CT and H&E staining images of RPM mice showing lung tumour formation (yellow circles) following intranasal infection with Ad5-Cgrp-Cre. Each group was analysed by micro-CT and H&E staining in n = 3 biologically independent mice. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . d H&E and immunohistochemistry (IHC) staining for ASCL1 and EIF6 in RPM tumours at different stages. Each group was analysed in three biologically independent mice. e Multiplex immunofluorescence (mIF) analysis of human SCLC surgical samples. Images were analysed using QuPath to quantify the percentage of ASCL1⁺, NEUROD1⁺ and EIF6⁺ cells (top). Representative mIF and H&E images from the same tumour are shown below, illustrating the spatial distribution of ASCL1, NEUROD1 and EIF6. Insets show magnified regions of interest. mIF images are representative of n = 38 patient FFPE samples. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . f Pearson correlation analysis between the number of EIF6⁺ and ASCL1⁺ cells across 38 human SCLC tumours. g Pearson correlation analysis of EIF6 and ASCL1 expression levels in human SCLC samples from the IMpower133 cohort ( n = 271). The shaded area indicates the 95% confidence interval of the regression line. h Immunoblot analysis showing chemotherapy-induced EIF6 expression and key translation initiation factors across multiple SCLC cell models. β-tubulin was used as a loading control. Carbo, carboplatin (10 μM); Etop, etoposide (2 μM). Blots are representative of n = 3 biologically independent experiments performed on separate cell preparations with similar results. For H69, the samples derive from the same experiment but different gels for eIF4A1 and eIF4E, another for eIF6, another for vimentin and β-tubulin were processed in parallel. For the other two cells, the samples derive from the same experiment but different gels for eIF4A1 and eIF4E, another for eIF6, vimentin and β-tubulin were processed in parallel. i H69 xenograft tumours were harvested at four time points during chemotherapy (carboplatin 60 mg kg⁻¹ plus etoposide 12 mg kg⁻¹, i.p., weekly) (top). Individual tumour growth curves corresponding to each biopsy time point are shown below. Tumour growth curves are from n = 6 (vehicle), n = 6 (regression), n = 3 (residual) and n = 4 (regrowth) mice. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . j Quantification of EIF6 protein levels by immunoblotting in tumours collected at the four time points shown in ( i ). Error bars represent s.d. from n = 3 mice per group.

Article Snippet: The following chemicals (stored at −20 °C or −80 °C) were used for experiments where indicated: Carboplatin (Selleckchem #S1215, stock 10 mM), Etoposide (Selleckchem #S1225, stock 50 mM in DMSO), Harringtonine (Abcam #ab141941, stock 100 μg ml − 1 in DMSO), Cycloheximide (MCE, #HY12320, stock 100 mg ml − 1 in DMSO), GSK-126 (MCE, #HY13470, stock 10 mM in DMSO), ifebemtinib (Selleckchem #E1114, stock 10 mM in DMSO), SCH772984 (Selleckchem #S7101, stock 10 mM in DMSO), GSK2256098 (Selleckchem #S8523, stock 10 mM in DMSO), Cobimetinib (Selleckchem #S8041, Stock 10 mM in DMSO), Rapamycin (Selleckchem #S1039, stock 10 mM in DMSO).

Techniques: Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Micro-CT, Staining, Infection, Immunohistochemistry, Multiplex Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis

doi: 10.1038/s41467-026-69899-8

Figure Lengend Snippet: a Enrichment of EMT and NE gene signatures following EIF6 knockdown in non-NE cell states. RNA-seq analysis is performed in n = 2 biologically independent samples. b Gene expression profiling of EMT- and NE-associated pathways upon EIF6 knockdown in non-NE cell states. RNA-seq analysis is performed in n = 2 biologically independent samples. c Immunoblot analysis of EMT-associated and NE proteins following EIF6 knockdown across multiple SCLC models. Blots are representative of n = 3 biologically independent experiments. For H69M, the samples derive from the same experiment but different gels for ZEB1, another for ZEB2, another for MYCN, another for YAP1, another for eIF6 and c-Myc, another for ASCL1, NEUROD1 and β-tubulin were processed in parallel; For H82R, the samples derive from the same experiment but different gels for ZEB2, ZEB1, MYCN and c-Myc, another for YAP1 and ASCL1, another for NEUROD1, eIF6 and β-tubulin were processed in parallel. For H209A, the samples derive from the same experiment, but different gels for ZEB2, ZEB1, MYCN and c-Myc, another for YAP1 and ASCL1, another for eIF6, another for NEUROD1 and β-tubulin were processed in parallel. d H69 cells with EIF6 knockdown were treated with HGF (40 ng mL⁻¹) for two weeks. Bright-field images show adherent growth (top). Attached colonies were stained with crystal violet for quantification ( n = 3 biologically independent experiments). Scale bar, 100 μm. Error bars represent s.d. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. e Re-expression of shRNA-resistant wild-type EIF6 using pCDH lentivirus restores HGF-induced cell attachment in EIF6 knockdown cells. Top: immunoblot showing EIF6 knockdown and re-expression efficiency. Bottom, quantification of crystal violet–stained colonies ( n = 4 independent experiments). Error bars represent s.d. Statistical significance was assessed using a two-sided unpaired Wilcoxon rank-sum test. f Representative images of H69 cells with EIF6 knockdown and re-expression after HGF treatment (40 ng mL⁻¹) for three weeks. Bright-field images (left) and crystal violet–stained colonies (right) are shown. Scale bar, 20 μm. g Live/dead cell analysis of shNTC and shEIF6 H69M cells treated with or without EP (carboplatin 10 μM and etoposide 2 μM) for 72 h, measured by flow cytometry. Data are mean ± SEM from n = 3 independent experiments. Statistical significance was determined using a two-sided unpaired Wilcoxon rank-sum test. h Chemotherapy sensitivity of subcutaneous xenografts derived from shNTC or shEIF6 H69 cells. Tumour volume changes were assessed after three chemotherapy cycles ( n = 13 tumours per group). Response categories were defined as CR (−100%), PR (−30% to 0%) and PD (>20%). Lowercase letters indicate tumours used for immunohistochemistry in i . Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h . i Representative immunohistochemistry images of ASCL1, NEUROD1, POU2F3, YAP1 and EIF6 in corresponding tumours from h . Scale bars, 50 μm.

Article Snippet: The following chemicals (stored at −20 °C or −80 °C) were used for experiments where indicated: Carboplatin (Selleckchem #S1215, stock 10 mM), Etoposide (Selleckchem #S1225, stock 50 mM in DMSO), Harringtonine (Abcam #ab141941, stock 100 μg ml − 1 in DMSO), Cycloheximide (MCE, #HY12320, stock 100 mg ml − 1 in DMSO), GSK-126 (MCE, #HY13470, stock 10 mM in DMSO), ifebemtinib (Selleckchem #E1114, stock 10 mM in DMSO), SCH772984 (Selleckchem #S7101, stock 10 mM in DMSO), GSK2256098 (Selleckchem #S8523, stock 10 mM in DMSO), Cobimetinib (Selleckchem #S8041, Stock 10 mM in DMSO), Rapamycin (Selleckchem #S1039, stock 10 mM in DMSO).

Techniques: Knockdown, RNA Sequencing, Gene Expression, Western Blot, Staining, Expressing, shRNA, Cell Attachment Assay, Cell Analysis, Flow Cytometry, Derivative Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Eukaryote initiation factor 6 modulates small-cell lung carcinoma plasticity via the integrin-FAK signaling axis

doi: 10.1038/s41467-026-69899-8

Figure Lengend Snippet: a Immunoblot analysis of FAK phosphorylation status across multiple SCLC cell models. β-tubulin was used as a loading control. For H209/H209A, the samples derive from the same experiment, but different gels for FAK, another for p-FAK and β-tubulin were processed in parallel. For H209/H209M, the samples derive from the same experiment, but different gels for p-FAK, another for FAK and β-tubulin were processed in parallel. For H69 and H82 models, the samples derive from the same experiment, but different gels for p-FAK, another for FAK and β-tubulin were processed in parallel. Blots are representative of n = 3 biologically independent experiments. eIF6 knockdown in non-NE cell states leads to MAPK pathway downregulation, with the gene expression profiling showing reduced expression of multiple pathway components. H69M cells, RNA-seq analysis is performed in n = 2 biologically independent samples ( b ) and H209A cells, RNA-seq analysis is performed in n = 2 and 4 biologically independent samples ( c ). d Immunoblot analysis of the gene expression and pathway enrichment findings, demonstrating reduced ERK1/2 phosphorylation upon eIF6 knockdown in H69M and H209A cells. For H69/H69M, the samples derive from the same experiment but different gels for p-FAK, p-ERK, another for FAK, another for p-AKT, another for c-Myc, another for eIF6 and β-tubulin were processed in parallel. For H209/H209A, the samples derive from the same experiment, but different gels for p-FAK, p-ERK and c-Myc, another for eIF6, another for FAK, p-AKT and β-tubulin were processed in parallel. Blots are representative of n = 3 biologically independent experiments. e . CCK-8 cell survival assay following combination treatment with GSK2256098 (10 μM, GSK) and varying concentrations of carboplatin/etoposide (EP) for 72 h across three different SCLC cell models. The treatment response curve of n = 4 biologically independent experiments was shown. f Half-maximal inhibitory concentration (IC50) of carboplatin/etoposide (EP) in H69 and H69M cell lines. Statistical significance was assessed using a two-sided unpaired t-test with Welch’s correction, n = 4 biological replicates. g H69 tumour cell xenografts in Balb/cNj-Foxn1 nu /Gpt mice were randomized into four treatment groups when the tumours are palpable, including Vehicle, GSK2256098 monotherapy (75ppm, p.o per day), EP chemotherapy (carboplatin 60ppm + etoposide 12ppm, i.p. per week), and triple therapy (GSK2256098 + EP) (Left panel); Tumour volume (mm 3 ) was monitored for ~50 days. Data are presented as mean ± SEM. Statistical significance was determined using one-sided two-way ANOVA. Tumour growth curves were shown for n = 4 and 6 independent mice. Source data are provided in Source data file. Created in BioRender. Shen, S. (2026) https://BioRender.com/5p1ea8h .

Article Snippet: The following chemicals (stored at −20 °C or −80 °C) were used for experiments where indicated: Carboplatin (Selleckchem #S1215, stock 10 mM), Etoposide (Selleckchem #S1225, stock 50 mM in DMSO), Harringtonine (Abcam #ab141941, stock 100 μg ml − 1 in DMSO), Cycloheximide (MCE, #HY12320, stock 100 mg ml − 1 in DMSO), GSK-126 (MCE, #HY13470, stock 10 mM in DMSO), ifebemtinib (Selleckchem #E1114, stock 10 mM in DMSO), SCH772984 (Selleckchem #S7101, stock 10 mM in DMSO), GSK2256098 (Selleckchem #S8523, stock 10 mM in DMSO), Cobimetinib (Selleckchem #S8041, Stock 10 mM in DMSO), Rapamycin (Selleckchem #S1039, stock 10 mM in DMSO).

Techniques: Western Blot, Phospho-proteomics, Control, Knockdown, Gene Expression, Expressing, RNA Sequencing, CCK-8 Assay, Clonogenic Cell Survival Assay, Concentration Assay

Journal: Cancer Cell International

Article Title: The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging

doi: 10.1186/s12935-024-03361-x

Figure Lengend Snippet: DC-5163 suppresses aerobic glycolysis in breast cancer cells. MCF-7, MDA-MB-231, and BT549 cells were treated with 80 µM DC-5163 or DMSO for 24 h. ( A–C ) The glycolytic activity was evaluated by Seahorse XF Glycolytic Rate Assay, ECAR, and glycoPER were measured with the administration of mitochondrial inhibitors Rot/AA and glycolytic inhibitor 2-DG. D Basal and compensatory glycoPER values were determined based on measurements from before and after administration of Rot/AA, respectively. Relative glucose consumption E , lactate production F , and cellular ATP content G were measured and calculated after treatment. H The protein level of GLUT1 was measured by Western blot analysis. I Cellular uptake of 18 F-FDG was detected and calculated. Data are shown as mean ± SD ( n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. the DMSO group

Article Snippet: After attachment, cells were treated with 80 μM DC-5163 or DMSO for 24 h. The concentrations of glucose and lactate in the medium were measured by a glucose assay kit (Sigma, St. Louis, MO, USA) and a lactate assay kit (Solarbio, Beijing, China), respectively, following the manufacturer’s protocol.

Techniques: Activity Assay, Western Blot

Journal: Cancer Cell International

Article Title: The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging

doi: 10.1186/s12935-024-03361-x

Figure Lengend Snippet: DC-5163 inhibits breast cancer cell growth and induces cell cycle arrest. MCF-7, MDA-MB-231, and BT549 cells were treated with different concentrations of DC-5163 for the indicated times. A Cell viability was assessed using the CCK8 assay. B Colony formation assay. C Cell cycle distribution was detected by flow cytometry. D The protein levels of cyclin D1, CDK4, and TK1 were measured by Western blot analysis. E Cellular uptake of 18 F-FLT was detected and calculated. Data are shown as mean ± SD ( n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. the DMSO group

Article Snippet: After attachment, cells were treated with 80 μM DC-5163 or DMSO for 24 h. The concentrations of glucose and lactate in the medium were measured by a glucose assay kit (Sigma, St. Louis, MO, USA) and a lactate assay kit (Solarbio, Beijing, China), respectively, following the manufacturer’s protocol.

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Western Blot

Journal: Cancer Cell International

Article Title: The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging

doi: 10.1186/s12935-024-03361-x

Figure Lengend Snippet: DC-5163 induces apoptosis in breast cancer cells. MCF-7, MDA-MB-231, and BT549 cells were treated with different concentrations of DC-5163 for 48 h. A The apoptosis rates were detected by flow cytometry. B–C The protein level of cleaved PARP was measured by Western blot analysis. D Cellular uptake of 18 F-ML-10 was detected and calculated. Data are shown as mean ± SD ( n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. the DMSO group

Article Snippet: After attachment, cells were treated with 80 μM DC-5163 or DMSO for 24 h. The concentrations of glucose and lactate in the medium were measured by a glucose assay kit (Sigma, St. Louis, MO, USA) and a lactate assay kit (Solarbio, Beijing, China), respectively, following the manufacturer’s protocol.

Techniques: Flow Cytometry, Western Blot

Journal: Cancer Cell International

Article Title: The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging

doi: 10.1186/s12935-024-03361-x

Figure Lengend Snippet: DC-5163 suppresses breast cancer in vivo with low cytotoxicity. The tumor-bearing mice were intraperitoneally injected with DC-5163 (80 mg/kg, q2d) or DMSO diluted with saline for 28 days. A The tumor volume growth curve was calculated to investigate tumorigenesis. B Photographs of tumor samples. C The weights of tumor samples. D Representative images of immunohistochemistry and TUNEL assay of the tumor samples (magnification, ×400). E Body weight of tumor-bearing mice. No significant difference in body weight was observed between the DC-5163 group and the control group. F HE staining images of vital organs (magnification, ×200). Data are shown as mean ± SD ( n = 6); * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. the DMSO group

Article Snippet: After attachment, cells were treated with 80 μM DC-5163 or DMSO for 24 h. The concentrations of glucose and lactate in the medium were measured by a glucose assay kit (Sigma, St. Louis, MO, USA) and a lactate assay kit (Solarbio, Beijing, China), respectively, following the manufacturer’s protocol.

Techniques: In Vivo, Injection, Saline, Immunohistochemistry, TUNEL Assay, Control, Staining

Journal: Cancer Cell International

Article Title: The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging

doi: 10.1186/s12935-024-03361-x

Figure Lengend Snippet: Monitoring of the efficacy of DC-5163 in vivo by molecular imaging. The tumor-bearing mice underwent micro-PET/CT scans before and after the treatment of DC-5163 or DMSO. A Representative images acquired by 18 F-FDG micro-PET/CT scanning. B Tumor uptake of 18 F-FDG was measured by VOI analysis in mice bearing MDA-MB-231 human breast cancer xenografts. C Representative images acquired by 18 F-FLT micro-PET/CT scanning. D Tumor uptake of 18 F-FLT was measured by VOI analysis in tumor-bearing mice. Data are presented as average %ID/g (mean ± SD, n = 6); ns, no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. the DMSO group

Article Snippet: After attachment, cells were treated with 80 μM DC-5163 or DMSO for 24 h. The concentrations of glucose and lactate in the medium were measured by a glucose assay kit (Sigma, St. Louis, MO, USA) and a lactate assay kit (Solarbio, Beijing, China), respectively, following the manufacturer’s protocol.

Techniques: In Vivo, Imaging, Micro-PET

Journal: Science Advances

Article Title: Targeting actin-bundling protein L-plastin as an anabolic therapy for bone loss

doi: 10.1126/sciadv.abb7135

Figure Lengend Snippet: ( A ) TRAP staining images and quantitative analyses of preosteoclasts on the trabecular bone and periosteal bone surface of the femoral diaphysis. Arrowhead, multiple nuclear osteoclasts; arrow, TRAP + cells. Scale bars, 50 μm. ( B ) Quantification of TRAP + cells/bone surface (N. TRAP + cells/B.Pm) and multiple nuclear osteoclast numbers/bone surface (N.Mu.Oc/B.Pm). n = 5 per group. ( C ) Quantification of preosteoclast/periosteal bone surface (N.POCs/BS). ( D ) Angiographic images of femora. Scale bar, 20 μm. ( E ) Quantitative analyses of vessel surface and vessel volume. n = 5 per group. ( F ) Representative images of immunostaining of endomucin (EMCN) (red), CD31 (green), and Emcn hi CD31 hi (yellow) cells on the trabecular bone and periosteal bone. Scale bar, 50 μm. ( G ) The percentage of CD31 hi Emcn hi area on the trabecular bone and periosteal bone. n = 5 per group. ( H ) Flow cytometry plots with the percentage of CD31 hi Emcn hi endothelial cells (ECs) in total bone marrow cells. n = 3 per group. ( I ) Immunostaining of TRAP (red) and PDGF-BB (green) on the trabecular bone and periosteal bone. Scale bar, 50 μm. ( J ) Quantification of PDGF-BB + and VEGF + cell numbers per respective bone surface. n = 6 per group. ( K ) Serum PDGF-BB and VEGF levels. n = 6 per group. * P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Samples were treated with 0.3% Triton X-100 for 20 min and then blocked with 5% donkey serum for 20 min. Rat anti-EMCN antibody (1:100; sc-65495, Santa Cruz Biotechnology) and rabbit anti-CD31 antibody (1:20; ab28364, Abcam) were incubated with the samples at room temperature for 2 hours.

Techniques: Staining, Immunostaining, Flow Cytometry

Journal: Science Advances

Article Title: Targeting actin-bundling protein L-plastin as an anabolic therapy for bone loss

doi: 10.1126/sciadv.abb7135

Figure Lengend Snippet: ( A ) Representative micro-CT images of femora of sham, OVX, and OVX + oroxylin A groups. Scale bars, 1 mm. ( B ) Quantitative micro-CT of BMD, BV/TV, and Tb.N of femora. n = 6 per group. ( C ) Quantitative micro-CT of the cortical thickness of femora. n = 6 per group. ( D and E ) Hematoxylin and eosin staining of femora with quantification of Tb area/tissue area × 100%. Scale bar, 500 μm. n = 5 per group. ( F ) Angiographic images of femora. Scale bar, 20 μm. n = 5 per group. ( G ) Quantitative analyses of vessel volume and vessel surface. n = 5 per group. ( H ) Representative images of immunostaining of EMCN (red), CD31 (green), and Emcn hi CD31 hi (yellow) cells on trabecular bone and periosteal bone. Scale bar, 50 μm. n = 5 per group. ( I ) The percentage of Emcn hi CD31 hi area on trabecular bone and periosteal bone. n = 5 per group. ( J ) Flow cytometry plots with the percentage of CD31 hi Emcn hi endothelial cells in total bone marrow cells. n = 3 per group. ( K to M ) Quantification of mineral apposition rate, bone formation rate per bone surface, mineralizing surface per bone surface in calcein double labeling. n = 5 per group. ( N ) Serum PDGF-BB and VEGF levels. n = 6 per group. ( O ) Serum CTX-1 and TRAcp5b levels. n = 6 per group. ( P ) Serum OCN levels. n = 6 per group. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Samples were treated with 0.3% Triton X-100 for 20 min and then blocked with 5% donkey serum for 20 min. Rat anti-EMCN antibody (1:100; sc-65495, Santa Cruz Biotechnology) and rabbit anti-CD31 antibody (1:20; ab28364, Abcam) were incubated with the samples at room temperature for 2 hours.

Techniques: Micro-CT, Staining, Immunostaining, Flow Cytometry, Labeling