Journal: Cytokine

Article Title: Exogenous Oncostatin M Induces Cardiac Dysfunction, Musculoskeletal Atrophy, and Fibrosis

doi: 10.1016/j.cyto.2022.155972

Figure Lengend Snippet: Primers.

Article Snippet: Application Primer Name Assay ID (Thermo Fisher Scientific) Amplicon length (bp) qRT-PCR Osm Mm01193966_m1 82 Osmr Mm01307326_m1 84 Bnip3 Mm01275600_g1 147 Bnip3-L Mm00786306_s1 141 Trim63 Mm01185221_m1 57 Fbxo32 Mm00499523_m1 73 Tbp Mm00446974_m1 105 Postn Mm01284919_m1 62 Vim Mm01333430_m1 62 Acta2 Mm00725412_s1 95 Open in a separate window Primers.

Techniques: Amplification

Journal: Cytokine

Article Title: Exogenous Oncostatin M Induces Cardiac Dysfunction, Musculoskeletal Atrophy, and Fibrosis

doi: 10.1016/j.cyto.2022.155972

Figure Lengend Snippet: A. qPCR results reveal Osm was not changed by qPCR in wildtype or Il6−/− quadriceps, but Osmr expression was elevated in WT and Il6−/− quadriceps. B. Atrophy genes Trim63, Bnip3, and Bnip3L were not changed but Fbxo32 was suppressed in Il6−/− quadriceps. Statistical Analysis: Two-Way ANOVA with Tukey’s Multiple Comparison Test.

Article Snippet: Application Primer Name Assay ID (Thermo Fisher Scientific) Amplicon length (bp) qRT-PCR Osm Mm01193966_m1 82 Osmr Mm01307326_m1 84 Bnip3 Mm01275600_g1 147 Bnip3-L Mm00786306_s1 141 Trim63 Mm01185221_m1 57 Fbxo32 Mm00499523_m1 73 Tbp Mm00446974_m1 105 Postn Mm01284919_m1 62 Vim Mm01333430_m1 62 Acta2 Mm00725412_s1 95 Open in a separate window Primers.

Techniques: Expressing, Comparison

Journal: Autophagy

Article Title: Autophagy regulates PVALB (parvalbumin) interneuron excitability and memory

doi: 10.1080/15548627.2025.2597463

Figure Lengend Snippet: Autophagy regulates the proteostasis of PVALB interneurons. (A) Representative CLEM images of the endoplasmic reticulum of control ( Pvalb-TdT) and Pvalb-atg5-TdT hippocampal PVALB interneurons. Scale bar: 5 μm. Insets show a higher magnification of the boxed areas. Inset scale bars: 1 µm. (B-C) Quantification of differences in cellular er volume (B) and diameter (C) in control and Pvalb-atg5-TdT PVALB cells. Statistical analysis was performed using unpaired, two-tailed Student’s t test (for er volume: **p = 0.0043, t = 5.849 and df = 4; for diameter: *p = 0.0189, t = 3.813 and df = 4). Bars represent mean values ± SEM. N = 3 animals per genotype. (D) Representative confocal images of TdT-positive PVALB cells (magenta) in the hippocampal CA1 area of control ( Pvalb-TdT ) and Pvalb-atg5-TdT animals, immunolabelled with antibodies against RTN3 (green) and stained with the nuclear dye Hoechst (blue). Scale bar: 20 µm. (E) Quantification of the mean RTN3 signal intensity in control versus Pvalb-atg5-TdT PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using unpaired, two-tailed Student’s t test (*p = 0.0172, t = 3.926, df = 4). Bars represent mean values ± SEM. N = 3 animals per genotype. (F) Representative confocal images of PVALB cells in the hippocampal CA1 area of control ( Pvalb-Cre ) and Pvalb-atg5 animals, immunolabelled with antibodies against PVALB (magenta) CALCOCO1 (green) and stained with the nuclear dye Hoechst (blue). Scale bar: 20 μm. (G) Quantification of the mean CALCOCO1 signal intensity in control versus Pvalb-atg5 PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using Mann-whitney U test (Mann-Whitney U = 2, n = 5 per group, *p = 0.0317). Bars represent mean values ± SEM. N = 5 animals per genotype. (H) representative CLEM images of mitochondria of control ( Pvalb-TdT) and Pvalb-atg5-TdT hippocampal PVALB interneurons. Arrowheads indicate differences in mitochondrial morphology between controls (left) and Pvalb-atg5-TdT (right) cells. Scale bars represent 5 μm. Insets show a higher magnification of the boxed areas. Inset scale bars: 1 µm. (I) Quantification of differences in cellular mitochondrial volume in control and Pvalb-atg5-TdT PVALB cells. Statistical analysis was performed using unpaired, two-tailed Student’s t test (*p = 0.0193, t = 3.790, df = 4). Bars represent mean values ± SEM. N = 3 animals per genotype. (J) Representative confocal images of TdT-positive PVALB cells (magenta) in the hippocampal CA1 area of control ( Pvalb-TdT ) and Pvalb-atg5-TdT animals, immunolabelled with antibodies against BNIP3 (green) and stained with the nuclear dye Hoechst (blue). Scale bar: 20 µm. (K) Quantification of the mean BNIP3 signal intensity in control versus Pvalb-atg5-TdT PVALB cells in hippocampal CA1. Results are expressed as a percentage of control values. Statistical analysis was performed using unpaired, two-tailed Student’s t test (**** p = < 0.0001, t = 15.76, df = 4). Bars represent mean values ± SEM. N = 3 animals per genotype. (L) representative confocal images of PVALB cells in the hippocampal CA1 area of control ( Pvalb-Cre ) and Pvalb-atg5 animals, immunolabelled with antibodies against PVALB (magenta), BNIP3L (green) and stained with the nuclear dye Hoechst (blue). Scale bar: 20 µm. (M) Quantification of the mean BNIP3L signal intensity in control versus Pvalb-atg5 PVALB cells. Results are expressed as a percentage of control values. Statistical analysis was performed using Mann-Whitney U test (Mann-Whitney U = 0, n = 5 per group, **p = 0.0079). Bars represent mean values ± SEM. N = 5 animals per genotype.

Article Snippet: BNIP3L , Rabbit , Proteintech , 12,986–1-AP , 1:100 (IHC).

Techniques: Control, Two Tailed Test, Staining, MANN-WHITNEY

Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 4 BNIP3 mRNA and protein levels are increased upon compound treatment. (a) Fold change in gene expression in PANC-1 cells treated for 3 days with 2.5 mM gossypol alone or the combination of 2.5 mM gossypol and 2.5 mM BRD4770. The 10 gene transcripts that showed the greatest fold induction between the two conditions are shown. The total RNA was extracted, and the 84 key genes involved in autophagy were profiled by a 384-well format human autophagy pathway-focused expression real-time PCR array. Six housekeeping genes were used as internal controls for data normalization (see the Materials and Methods section). Data for BRD4770 treatment were generated by gene expression profiling (see the Materials and Methods section). (b) Protein levels of BNIP3, BNIP3L, beclin1, and BCL-xL were assessed in PANC-1 cells treated for 3 days with the indicated concentrations of BRD4770. BNIP3 was observed as a 30-kDa monomer and a 60-kDa dimer. Tubulin was used as an internal loading control

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Generated, Control

Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 5 BNIP3 overexpression induces LC3-II expression and autophagosome formation in the presence of gossypol. (a) PANC-1 cells were transiently transfected with BNIP3 and BNIP3L, either individually or in combination. The control cells were transfected with an empty vector. After 24 h, the cells were treated with 2.5 mM gossypol for another 24 h. Tubulin served as an internal loading control. (b) Fluorescence microscopy of mCherry-eGFP-LC3 HeLa cells treated as described in panel (a). Scale bar ¼ 20 mm

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Over Expression, Expressing, Transfection, Control, Plasmid Preparation, Fluorescence, Microscopy

Journal: Cell death & disease

Article Title: Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

doi: 10.1038/cddis.2013.191

Figure Lengend Snippet: Figure 6 Knockdown of G9a and GLP expression induces BNIP3 expression and cell death. PANC-1 cells were transiently transfected with siRNA against G9a and GLP, both individually and in combination. Scrambled siRNA was used as the negative control. (a) Quantification of the level of knockdown for G9a and GLP by real-time PCR. GAPDH and actin were used to normalize the data. (b) Cellular ATP levels were measured after 3-day treatment with gossypol and either 1.25 mM BRD4770 or siRNA knockdown of G9a and GLP. Data represent the mean and standard error of six independent replicates for each combination. (c) Western blot analysis to examine the protein levels of LC3, BNIP3, and BNIP3L 3 days after transfection. Tubulin served as an internal loading control

Article Snippet: Plasmids encoding human BNIP3 (RC205087) and BNIP3L (RC203315) were obtained from Origene (Rockville, MD, USA). siRNAs (25 nM) and plasmid DNA (0.4 mM) were transfected into PANC-1, mCherry-eGFP-LC3 HeLa, and PC-3 cells using Lipofectamine 2000 reagent (Invitrogen).

Techniques: Knockdown, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, 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 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 – e Nontargeting (NT) or GR siRNA was transfected to hippocampal neurons and SH-SY5Y cells for 24 h prior to corticosterone and cortisol for 12 h, respectively. a , b Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression was detected in western blot where β-actin was used as a loading control in both cell types. n = 5. c Colocalization of PGC1α (red) and DAPI (blue) in hippocampal neurons was visualized with SRRF imaging system. Scale bars, 20 μm (magnification, ×1000). n = 5. d Colocalization of PGC1α (green) and DAPI (blue) in SH-SY5Y was visualized with SRRF imaging system. Scale bars, 20 μm (magnification, ×1000). n = 5. e PGC1α protein expressions in subcellular fraction samples were detected by western blotting. Lamin A/C and α-tubulin were used as a nuclear and cytosolic loading control, respectively. n = 5. f , g SH-SY5Y cells were transfected with pcDNA3.1/c-eGFP or pcDNA3.1/PPARGC1A-c-eGFP vector for 24 h prior to cortisol treatment for 24 h. f NIX expression was detected in western blot where β-actin was used as a loading control. n = 5. g TOMM20 levels were detected by western blot. Loading control for western blot is β-actin. 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. Two-sided two-way ANOVA was conducted. ** indicates p < 0.01 versus control. # , ## indicates p < 0.05, p < 0.01 versus corticosterone in hippocampal neurons and cortisol in SH-SY5Y, 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: Transfection, Expressing, Western Blot, Control, Imaging, Plasmid Preparation, Immunofluorescence

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