Journal: Nature Communications

Article Title: BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects

doi: 10.1038/s41467-020-20679-y

Figure Lengend Snippet: a SH-SY5Y cells were incubated with cortisol for 12 h and mRNA expressions were analyzed by real-time PCR. n = 5. b , c Hippocampal neurons and SH-SY5Y cells were treated with corticosterone for 24 h and with cortisol for various time, respectively. Expression of NIX, PTEN-induced kinase 1 (PINK1), and BCL2 interacting protein 3 (BNIP3) were detected via western blot. n = 5. d , e Hippocampal neurons and SH-SY5Y cells were treated with corticosterone and cortisol, respectively, for 24 h. NIX (green), TOMM20 (red), and DAPI (nuclear counterstaining, blue) were visualized. Scale bars, 20 μm (magnification, ×1000). n = 5. f Nontargeting (NT) or PARK2 siRNA was transfected to SH-SY5Y cells for 24 prior to cortisol for 24 h. NIX was co-immunoprecipitated with parkin. Parkin levels in immunoprecipitated samples were quantified. n = 5. g , h Both hippocampal neurons and SH-SY5Y cells were transfected with mitochondria-targeted Keima (mt-Keima)-Red-Parkin for 24 h prior to corticosterone and cortisol for 24 h, respectively. Western blot was performed. n = 5. i – l Both hippocampal neurons and SH-SY5Y cells were pretreated with phorbol 12-myristate 13-acetate (PMA, 10 nM) for 30 min before corticosterone and cortisol for 24 h, respectively. i , j Hippocampal neurons and SH-SY5Y cells were transfected with mt-Keima at DIV 5 and 48 h prior to treatment, respectively. DAPI was used for nuclear counterstaining (blue). Ratio of red to sum of red and green was quantified. Scale bars, 20 μm (magnification, ×1000). n = 5. k , l TOMM20 levels were detected by western blot. n = 5. m SH-SY5Y cells were transfected with pcDNA3.1/c-eGFP or pcDNA3.1/NIX-c-eGFP vector for 24 h prior to cortisol for 24 h. TOMM20 levels were detected by western blot. n = 5. All blots and immunofluorescence images are representative. n = 5 from independent experiments with two technical replicates each. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging. Two-sided unpaired student’s t test: Figs. 3a, b, d, e. Two-sided one-way ANOVA: Fig. 3c. Two-sided two-way ANOVA: Figs. 3f–m. ** indicates p < 0.01 versus control. ## indicates p < 0.01 versus corticosterone in hippocampal neurons and cortisol in SH-SY5Y. Data are provided as a Source data file.

Article Snippet: The antibodies of LC3 (#NB100-2220, 1:3000 for western blot), PGC1α (#NBP1-04676, 1:3000 for western blot, 1:200 for immunostaining), NIX (#NBP1-88558, 1:3000 for western blot, 1:200 for immunostaining), and PINK1 (#BC100-494, 1:3000 for western blot) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Incubation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Immunoprecipitation, Plasmid Preparation, Immunofluorescence, Imaging, Control

Journal: Nature Communications

Article Title: BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects

doi: 10.1038/s41467-020-20679-y

Figure Lengend Snippet: a – f Mice were exposed to vehicle, corticosterone (10 mg/kg), corticosterone with phorbol 12-myristate 13-acetate (PMA pretreatment, 200 μg/kg), or PMA alone for 7 days. a Slide samples for IHC were immunostained with LAMP1 (green), TOMM20 (red), and DAPI (blue). Scale bars, 100 μm (magnification, ×200). n = 5. b The expressions of NIX, PTEN-induced kinase 1 (PINK1), and BCL2 interacting protein 3 (BNIP3) were detected with western blot where β-actin was used as a loading control. n = 5. c Slide samples for IHC were immunostained with synpatophysin (green), PSD95 (red), and DAPI (blue). Scale bars, 100 μm (magnification, ×200). n = 5. d Synaptophysin and PSD95 were detected by western blot. Loading control is β-actin. n = 5. e The mice were subjected to Y-maze test to evaluate spatial memory function. n = 6. f The mice were subjected to forced swim test to evaluate depression-like behavior. n = 5. g Vehicle or RU 486 (5 mg/kg) injected mice were presented with/without corticosterone (10 mg/kg) for 3 days. The expressions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and NIX were visualized via western blotting. Loading control is β-actin. n = 5. h The schematic model for mechanisms of inhibition in NIX-dependent mitophagy by glucocorticoid was shown. All blots and immunofluorescence images are representative. n = 5 or 6 from each animal with two technical replicates each in results of IHC and western blot. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided two-way ANOVA was conducted except Fig. 8b, data of which were analyzed by two-sided unpaired student’s t test. ** indicates p < 0.01 versus control and ## indicates p < 0.01 versus corticosterone, respectively. Data are provided as a Source data file.

Article Snippet: The antibodies of LC3 (#NB100-2220, 1:3000 for western blot), PGC1α (#NBP1-04676, 1:3000 for western blot, 1:200 for immunostaining), NIX (#NBP1-88558, 1:3000 for western blot, 1:200 for immunostaining), and PINK1 (#BC100-494, 1:3000 for western blot) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control, Injection, Inhibition, Immunofluorescence, Imaging

Journal: Current Issues in Molecular Biology

Article Title: Combination Treatment of Carboxyl Esterase 2-Overexpressing hTERT-Immortalized Human Adipose Stem Cells Enhances the Inhibition of Tumor Growth by Irinotecan in PC3, a Castration-Resistant Prostate Cancer Model

doi: 10.3390/cimb47110902

Figure Lengend Snippet: Expression of chemoattractant factors and receptors in hTERT-ADSC.CE2 cells. Real-time PCR analysis comparing unmodified hTERT-ADSC vs. hTERT-ADSC.CE2. The bar graphs show mRNA levels (relative to the ADSC control, set as 1) for key chemoattractant ligands—VEGF, SCF, and SDF-1—and their receptors—VEGFR-2, VEGFR-3, c-Kit, and CXCR4—in ADSC.CE2 cells. The expression of all these factors was higher in hTERT-ADSC.CE2 than in parental ADSCs (* p < 0.05 for each). These factors are known to mediate stem cell migration to tumors. Elevated levels in ADSC.CE2 suggest an enhanced readiness to respond to tumor-derived signals. (VEGFR-1 was also tested but showed only baseline expression, not shown.) Error bars represent SE (n = 3). Abbreviations: ADSC = parental hTERT-ADSC; ADSC.CE2 = CE2-transduced hTERT-ADSC. Y-axis: Normalized band intensities relative to β-actin.

Article Snippet: After blocking, the membranes (Merck KGaA, Darmstadt, Germany) were probed with antibodies against Nanog, Oct4, Sox2, BAX, cleaved Caspase-3, BCL-2, and β-actin (Santa Cruz Bio-technology; 1:3000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Migration, Derivative Assay

Journal: Current Issues in Molecular Biology

Article Title: Combination Treatment of Carboxyl Esterase 2-Overexpressing hTERT-Immortalized Human Adipose Stem Cells Enhances the Inhibition of Tumor Growth by Irinotecan in PC3, a Castration-Resistant Prostate Cancer Model

doi: 10.3390/cimb47110902

Figure Lengend Snippet: Phenotypic characterization of hTERT-ADSC.CE2 cells. ( A ): Flow cytometry for surface markers. Histograms show that hTERT-ADSC.CE2 cells (shaded curves) are positive for mesenchymal stem cell markers CD29, CD90, and CD105 and negative for hematopoietic markers CD34 and CD45, identical to parental ADSCs. The overlay of ADSC vs. ADSC.CE2 was nearly superimposable for each marker (isotype control indicated by dotted line). Y-axis: Cell count. ( B ): Western blot analysis of stemness markers Oct4, Nanog, and Sox2 in parental and CE2-modified ADSCs. Both cell lines express these transcription factors, indicating maintenance of stem cell characteristics. ( C ): Densitometric quantification of band intensities from ( B ) (normalized to β-actin). There were significant differences in Oct4/Nanog/Sox2 levels between hTERT-ADSC and hTERT-ADSC.CE2, confirming that the introduction of hTERT and CE2 did not diminish the cells’ stemness. Overall, hTERT-ADSC.CE2 retains a typical MSC immunophenotype and pluripotency marker expression profile, validating it as a bona fide mesenchymal stem cell line. Y-axis: Normalized band intensities relative to β-actin. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking, the membranes (Merck KGaA, Darmstadt, Germany) were probed with antibodies against Nanog, Oct4, Sox2, BAX, cleaved Caspase-3, BCL-2, and β-actin (Santa Cruz Bio-technology; 1:3000).

Techniques: Flow Cytometry, Marker, Control, Cell Counting, Western Blot, Modification, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Combination Treatment of Carboxyl Esterase 2-Overexpressing hTERT-Immortalized Human Adipose Stem Cells Enhances the Inhibition of Tumor Growth by Irinotecan in PC3, a Castration-Resistant Prostate Cancer Model

doi: 10.3390/cimb47110902

Figure Lengend Snippet: In vitro therapeutic effects of the ADSC.CE2 + CPT-11 system. ( A ): Suicide effect on ADSCs: Viability of hTERT-ADSC.CE2 vs. hTERT-ADSC after 72 h exposure to increasing CPT-11 concentrations. Data are mean % of untreated control viability ± SE (n = 4). At CPT-11 ≥ 1 µM, ADSC.CE2 viability drops significantly below ADSC viability ( p < 0.05), indicating CE2-mediated conversion of CPT-11 to SN-38 kills the CE2-expressing cells. ( B ): Selective cytotoxicity in co-culture: PC3 prostate cancer cells were co-cultured with either ADSC or ADSC.CE2 (1:0.05 ratio) and treated with CPT-11 (5 µM, 72 h). Cell viability is expressed relative to the untreated PC3 control. Co-culture with ADSC.CE2 led to ~40% viability of PC3, significantly lower than PC3 + CPT-11 alone (~80%) or PC3 + ADSC + CPT-11 (~85%) ( p < 0.05). This demonstrates a strong bystander killing effect on cancer cells due to SN-38 release by ADSC.CE2. ( C ): Apoptosis induction: Western blot images of co-cultured PC3 cells (after 72 h CPT-11) show increased BAX and cleaved caspase-3 and decreased BCL-2 in the PC3 cells when ADSC.CE2 were present (right lane) compared to control conditions (PC3 + CPT-11 without ADSC.CE2, left lane). ( D ): Densitometry of apoptosis protein levels (each normalized to β-actin, and expressed relative to control = 1). The ADSC.CE2 + CPT-11 co-culture caused ~2-fold increase in BAX and cleaved caspase-3 and ~50% reduction in BCL-2, confirming activation of apoptosis in tumor cells ( p < 0.05). PC3: PC3 alone, CPT-11: PC3 + CPT-11, ADSC.CE2: PC3 + CPT + 11 + ADSC.CE2. Y-axis: Normalized band intensities relative to β-actin. Together, these results highlight that the enzyme/prodrug therapy not only kills cancer cells but does so by triggering apoptotic pathways. (Error bars = SE, n = 3 experiments.) ** p < 0.01, *** p < 0.001, ## p < 0.01.

Article Snippet: After blocking, the membranes (Merck KGaA, Darmstadt, Germany) were probed with antibodies against Nanog, Oct4, Sox2, BAX, cleaved Caspase-3, BCL-2, and β-actin (Santa Cruz Bio-technology; 1:3000).

Techniques: In Vitro, Control, Expressing, Co-Culture Assay, Cell Culture, Western Blot, Activation Assay

Journal: Journal of Translational Medicine

Article Title: Multiomic analysis of monocyte-derived alveolar macrophages in idiopathic pulmonary fibrosis

doi: 10.1186/s12967-024-05398-y

Figure Lengend Snippet: Phenotypic Mo_AMs Subpopulations and Cellular Interaction Between Fibroblasts and Mo_AMs. UMAP plots identify the cells with the higher levels of FVC%pred (Scissor+) and the cells with lower levels of FVC%pred (Scissor−; A ). Barplots show the constitution of Mo_AMs in Scissor + cells and Scissor − cells ( B ). Volcano plots of differentially expressed genes in Scissor + cells versus Scissor − cells in Mo_AMs ( C–F ). The ligand-receptor analysis with CellChat ( G ). The expression of CXCR4 and CXCL12 ( H ) and the hierarchical plot ( I ) of the CXCL12/CXCR4 signalling pathway

Article Snippet: After blocking with 5% BSA for 1 h, the membranes were subjected to overnight incubation at 4°C with primary antibodies targeting CXCL12 (Rabbit, #17042-1-AP, 1:3000, proteintech), CXCR4 (Mouse, #60042-1-Ig,1:2000, proteintech), p-MEK (Rabbit,#28930-1-AP,1:2000, proteintech), MEK (Rabbit, #11049-1-AP, 1:10000, proteintech), p-ERK (Rabbit, #28733-1-AP, 1:5000, proteintech), ERK (Rabbit, #51068-1-AP,1:10000, proteintech), α-SMA (Rabbit, #67735-1-Ig, proteintech), and collagen type 1α (Mouse,1:10000,#67288-1-Ig, proteintech), followed by a 1 h incubation with secondary antibodies (Rabbit, #SA00001-2, proteintech; Mouse,# SA00001-2, proteintech).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Multiomic analysis of monocyte-derived alveolar macrophages in idiopathic pulmonary fibrosis

doi: 10.1186/s12967-024-05398-y

Figure Lengend Snippet: High Expression of CXCR4 in Activated Fibroblasts and High Expression of CXCL12 in Profibrotic Macrophages. Establishment of the fibrosis model and expression validation of α -SMA and collagen type I expression by quantitative polymerase chain reaction (qPCR) and immunofluorescence staining ( A, B, D ; scale bars = 20 μm). Inducing M2 macrophage formation and validation of CD206 expression via flow cytometry ( C, E ). Validation of CCL2, IL10 and CCL18 in M2 macrophages by ELISA ( F ). RNA and protein expression of CXCL12 in unstimulated HELF cells and stimulated HELF cells through qPCR and Western blot ( G ). RNA and protein expression of CXCR4 in unstimulated THP1 cells and stimulated THP1 cells through qPCR and Western blot ( H )

Article Snippet: After blocking with 5% BSA for 1 h, the membranes were subjected to overnight incubation at 4°C with primary antibodies targeting CXCL12 (Rabbit, #17042-1-AP, 1:3000, proteintech), CXCR4 (Mouse, #60042-1-Ig,1:2000, proteintech), p-MEK (Rabbit,#28930-1-AP,1:2000, proteintech), MEK (Rabbit, #11049-1-AP, 1:10000, proteintech), p-ERK (Rabbit, #28733-1-AP, 1:5000, proteintech), ERK (Rabbit, #51068-1-AP,1:10000, proteintech), α-SMA (Rabbit, #67735-1-Ig, proteintech), and collagen type 1α (Mouse,1:10000,#67288-1-Ig, proteintech), followed by a 1 h incubation with secondary antibodies (Rabbit, #SA00001-2, proteintech; Mouse,# SA00001-2, proteintech).

Techniques: Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Translational Medicine

Article Title: Multiomic analysis of monocyte-derived alveolar macrophages in idiopathic pulmonary fibrosis

doi: 10.1186/s12967-024-05398-y

Figure Lengend Snippet: The CXCL12/CXCR4 Pathway Can Efficiently Stimulate the Chemotaxis of M2 Macrophages, Promoting the Activation of Fibroblasts. The expression of CD206 measured by M2 macrophages with the stimulation of AMD3100 (CXCR4 antagonist) and CXCR4 siRNA for 24 h ( A ). The efficacy of siRNA CXCR4 was verified by WB ( B ). Bar graph represents changes of protein expression on CXCR4 ( B ). M2 macrophages were cultured in conditioned media from the fibrosis models with or without AMD3100 and CXCR4 siRNA. M2 macrophages were seeded into the upper chambers of transwell. The conditioned media from in vitro fibrosis models was added into the lower chambers of transwell ( C ). Western blot showing expression of MEK, P-MEK, ERK, and P-ERK in M2 macrophages at different time points (0 h, 4 h, 8 h) and different concentrations (10 ng/ml and 50 ng/ml) of CXCL12 stimulation ( D ). Bar graph represents changes of protein expression on pERK and pMEK ( D ).Western blot of α-SMA and collagen type I and immunofluorescence of α-SMA in activated HELF cells cocultured with M2 macrophages and then stimulated with agonist (NUCC-390) and inhibitors of the ERK pathway (U0126 and PD98059), CXCR4 inhibitor (AMD3100), and antifibrotic agents (pirfenidone; E, F ; scale bars = 20 μm). Bar graphs represent changes of protein expression on α-SMA and Collagen I ( E ). M2 macrophages were seeded into the upper chambers of transwell. The fibrosis models were added into the lower chambers of transwell

Article Snippet: After blocking with 5% BSA for 1 h, the membranes were subjected to overnight incubation at 4°C with primary antibodies targeting CXCL12 (Rabbit, #17042-1-AP, 1:3000, proteintech), CXCR4 (Mouse, #60042-1-Ig,1:2000, proteintech), p-MEK (Rabbit,#28930-1-AP,1:2000, proteintech), MEK (Rabbit, #11049-1-AP, 1:10000, proteintech), p-ERK (Rabbit, #28733-1-AP, 1:5000, proteintech), ERK (Rabbit, #51068-1-AP,1:10000, proteintech), α-SMA (Rabbit, #67735-1-Ig, proteintech), and collagen type 1α (Mouse,1:10000,#67288-1-Ig, proteintech), followed by a 1 h incubation with secondary antibodies (Rabbit, #SA00001-2, proteintech; Mouse,# SA00001-2, proteintech).

Techniques: Chemotaxis Assay, Activation Assay, Expressing, Cell Culture, In Vitro, Western Blot, Immunofluorescence