Journal: Radiotherapy and Oncology

Article Title: Regulation of O 2 consumption by the PI3K and mTOR pathways contributes to tumor hypoxia

doi: 10.1016/j.radonc.2014.02.007

Figure Lengend Snippet: Hypoxia is reduced in a 3D spheroid model and is predicted to decline in vivo . (A) EMT6 and FaDu spheroids were treated for 24 h with BEZ235 or BKM120, prior to fixation and sectioning. Sections were DAB-stained for pAKT (Ser473) expression. Representative images are shown for BEZ235 (50 nM) and BKM120 (5 μM). (B) The percentage of DAB-positive pixels in the viable region of the spheroid was reduced with all treatments. (C) Spheroids were treated with 50 nM BEZ235 or 5 μM BKM120 for 24 h, 48 h or 24 h followed by 24 h incubation in drug-free medium (24 h + wash). Hypoxia was assessed by staining central spheroid sections for EF5 (red) with Hoechst as a counterstain (blue). Data shown are representative of two independent experiments, n = 10 spheroids. (D) Mean EF5 fluorescence in spheroids treated for 24 h was reduced in both cell lines tested. (E) To assess whether hypoxia was recoverable, mean EF5 fluorescence in spheroids treated for 24 h was compared with that in spheroids treated for either 48 h or 24 h followed by the 24 h wash in drug-free medium. In cells treated with BEZ235, the 24 h wash period led to a return of the hypoxia signal. (F and G) Simulated distribution of oxygen partial pressure (mmHg) in a representative 1497 μm × 1482 μm vascular network derived from ex vivo samples where oxygen consumption was either 100% (F) or 55% (G) of base level. Highly oxygenated regions are in red, scaling down to poorly oxygenated regions in blue. (H) Average hypoxic fraction (defined as <10 mmHg) calculated across 10 example networks as in F as a function of varying oxygen consumption. All data points represent mean ± 95% Confidence Interval. One-way ANOVAs were performed with Bonferroni-corrected post t -tests. ∗ P < 0.01.

Article Snippet: To assess signaling inhibition in spheroids, sections were stained with anti-pAKT antibody using ImmPRESS™ reagent kit (MP-7401, VectorLabs) and DAB Peroxidase substrate kit (SK-4100, VectorLabs).

Techniques: In Vivo, Staining, Expressing, Incubation, Fluorescence, Derivative Assay, Ex Vivo

Journal: bioRxiv

Article Title: Distinct Th17 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis

doi: 10.1101/2023.03.10.532135

Figure Lengend Snippet: ( A ) t -SNE plot of the sample identity for ECs isolated from the OBs of PBS (gray) and GAS-infected (blue) mice. ( B ) Gene set enrichment analysis (GSEA) for genes differentially expressed in ECs following GAS infection. Bars indicate significance of enrichment (-log 2 FDR) and red dots indicate the number of genes significantly enriched in each gene set. The inflammatory signatures are upregulated, whereas blood-brain barrier (BBB) gene signatures are downregulated in ECs from GAS-infected mice. ( C ) Heat map of genes related to the endothelial response to systemic LPS in ECs from the OB of PBS and GAS-infected mice. ( D-F ) Expression changes for three BBB transcripts ( Itm2a , Itih5 and Mfsd2a ) in ECs by scRNAseq (left). Statistical comparisons of scRNAseq by Wilcoxon Rank Sum test (* p-adj < 0.05). Representative images (center) and quantification (right) of expression changes in the BBB transcripts by fluorescence in situ RNA hybridization (FISH) for the mRNA probes (red) combined with immunofluorescence staining for EC marker Glut1 ( D-E ; cyan) and immunofluorescence staining for Mfsd2a (red) and EC marker CD31 ( F ). Scale bars = 25 μm. Statistical comparisons were performed with unpaired t test with Welch’s correction (* p < 0.05; n = 7-9 per condition). Error bars represent mean with SEM. ( G ) On the left, correlation of log 2 fold changes in CNS EC genes identified from GAS versus PBS (x-axis) with those identified from either acute experimental autoimmune encephalomyelitis (EAE) versus Complete Freund Adjuvant (CFA) control (top panels, y-axis) and chronic EAE versus CFA (bottom paneled; y-axis). Correlations on the right display only BBB-associated genes (labeled in red). The correlation coefficient is displayed in the top left corner of each plot. Gray dotted lines mark the line of identity; black dashed line indicates the line of best fit. See also Figure S2 .

Article Snippet: Following TRIzol RNA extraction, polyA libraries were prepared with the TruSeq Stranded mRNA kit (Illumina, San Diego, CA).

Techniques: Isolation, Infection, Expressing, Fluorescence, In Situ, Hybridization, Immunofluorescence, Staining, Marker, Adjuvant, Control, Labeling

Journal: International Journal of Nanomedicine

Article Title: A Novel Airway-Organoid Model Based on a Nano-Self-Assembling Peptide: Construction and Application in Adenovirus Infection Studies

doi: 10.2147/IJN.S413743

Figure Lengend Snippet: Growth and proliferation activity of airway organoids after adenovirus infection. ( A ). Fluorescence microscopy observation of airway organoids at 3 dpi, 6 dpi and 9 dpi. Green fluorescent cells were observed at 3 dpi, and the number of fluorescent cells and the fluorescence intensity increased gradually from the edge of the gel, peaking at 9 dpi ( B ). Inverted microscope observation of airway organoids at 3 dpi, 6 dpi and 9 dpi. With the extension of infection time, the cells in airway organoids became round and scattered. Adenovirus (Adenovirus-EGFP, green fluorescence) ( C ). PI staining observation of airway organoids at 9 dpi. The red fluorescence signal was strong, there were more dead cells, and the lesions of cells were visible under the bright field view ( D ). The proliferative activity curves of airway organoids after adenovirus infection were obtained by MTS. The cell activity of airway organoids in the infected group was significantly lower than that in the uninfected group at 12 to 20 days after culture (4 dpi to 12 dpi). Each data point represents the mean of at least three independent experiments ( E ). The viable cell proliferation curves of airway organoids after adenovirus infection were calculated from DNA quantification. The cell proliferation of airway organoids in the infected group was significantly lower than that in the uninfected group at 12 to 20 days after culture (4 dpi to 12 dpi). Each data point represents the mean of at least three independent experiments; PI (red, indicating dead cells) and DAPI (blue, indicating nuclei). The dotted line indicates the onset of infection;**Compared with the uninfected group, P < 0.01.

Article Snippet: The DNA density (μg/mL) was determined by fluorometric quantification of the DNA content using a DNA fluorescence assay kit according to the manufacturer’s instructions (Invitrogen, molecular probes).

Techniques: Activity Assay, Infection, Fluorescence, Microscopy, Inverted Microscopy, Staining

Journal: PLoS ONE

Article Title: Expression of a Finger Millet Transcription Factor, EcNAC1, in Tobacco Confers Abiotic Stress-Tolerance

doi: 10.1371/journal.pone.0040397

Figure Lengend Snippet: (A) Adapting a gradual stress imposition protocol, 25-day-old finger millet seedlings were subjected to water-deficit stress following gravimetric approach. (B) NaCl treatment was given to seedlings 3-days after germination. Accumulation of EcNAC1 transcripts was determined by qRT-PCR with 2 µg of total RNA. The Actin gene was used as normalizer.

Article Snippet: To study the expression pattern of EcNAC1 under water-deficit stress and salinity, quantitative RT-PCR (qRT-PCR; Opticon 2, MJ research, USA) was performed using the fluorescent dye SYBR-Green (DyNAmo SYBR-Green qRT-PCR kit, Finnzymes, Finland) for 30 cycles following the manufacturer’s protocol.

Techniques: Quantitative RT-PCR

Journal: Viruses

Article Title: Infectious Spleen and Kidney Necrosis Virus Triggers Ferroptosis in CPB Cells to Enhance Virus Replication.

doi: 10.3390/v17050713

Figure Lengend Snippet: Figure 3. ISKNV infection induces ferroptosis in CPB cells. (A) Transmission electron microscopy of CPB cells treated with DMSO (72 h), erastin (10 µmol/L, 72 h), and ISKNV (100 MOI, 24 h, 48 h, 72 h). Scale bars = 1 µm. (B) Analysis of Fe2+ levels in CPB cells after treatment with ISKNV (100 MOI) using the fluorescent probe FerroOrange by laser scanning confocal microscopy. Scale bars = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity of (B) using Image J. (D) Analysis of intracellular ROS levels using DCFH-DA staining, and laser scanning confocal microscopy of CPB cells treated with ISKNV (100 MOI) for 24–72 h. Scale bars = 10 µm. (E) Quantitative analysis of the mean fluorescence intensity of (D) using Image J. (F–H) Detection of Fe2+, ROS, and MDA levels in cell lysates treated with ISKNV (100 MOI) for 24–72 h by microplate reader. * p < 0.05, ** p < 0.01, and *** p < 0.001, with p > 0.05 considered not significant (ns).

Article Snippet: The Fe2+ content of the cells was detected by laser scanning confocal microscopy and a microplate reader using the fluorescent probe FerroOrange (Elabscience, E-BC-F101, Wuhan, China).

Techniques: Infection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Fluorescence, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Mitochondrial deoxyguanosine kinase is required for female fertility in mice

doi: 10.3724/abbs.2024003

Figure Lengend Snippet: DGUOK deficiency results in smaller ovaries and fewer ovulations (A) Representative images of ovary morphology between WT and Dguok–/– mice. Scale bar: 1 mm. (B,C) The number of developing follicles (B) and corpus hemorrhaglcum (C) per section was counted in WT and Dguok–/– mice; n=3. *P<0.05 and **P<0.01. (D) Representative images of HE staining of the ovarian structure. Ovaries from 6-week-old WT and Dguok–/– mice were embedded, sectioned, and stained with hematoxylin and eosin (H&E). Developing follicles were indicated by yellow circle. Scale bar: 200 μm. (E) Representative images of H&E staining of ovarian structures in 6-week-old WT and Dguok–/– mice stimulated by PMSG and HCG. Corpus hemorrhagicum (green circle) from WT and anovulatorydominant follicles from Dguok–/– mice (red circle) are indicated. Scale bar: 200 μm. (F) The BrdU+ cell number of developing follicles was counted; n=3, ns (not significant) indicates P>0.05. (G) Statistical analysis of the number of apoptotic and necrotic granulosa cells in WT and Dguok–/– mice; n=3, *** P<0.001. (H) Representative immunofluorescence images of WT and Dguok–/– ovarian sections stained with BrdU-FITC antibody; n=3. Scale bar: 20 μm. (I) Representative images of Annexin-V/PI staining indicating granulosa cell apoptosis and necrosis in WT and Dguok–/– mice; n=3. Scale bar: 20 μm.

Article Snippet: An Annexin V-EGFP cell apoptosis detection kit (Beyotime) was used to measure the level of oocyte or granulosa cell apoptosis.

Techniques: Staining, Immunofluorescence

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Mitochondrial deoxyguanosine kinase is required for female fertility in mice

doi: 10.3724/abbs.2024003

Figure Lengend Snippet: DGUOK deficiency significantly increases ROS levels and apoptosis of oocytes (A) Annexin-V staining in oocytes (maintained in vitro for 6 h and 36 h) from WT and Dguok–/– mice. Scale bar: 50 μm. (B) Representative image of DCFH-DA signals in oocytes from WT and Dguok –/– mice. Scale bar: 50 μm. (C,D) Representative images and quantification analysis of ROS fluorescence intensity in oocytes from WT and Dguok–/– mice, n=3. Scale bar: 10 μm. ***P<0.001.

Article Snippet: An Annexin V-EGFP cell apoptosis detection kit (Beyotime) was used to measure the level of oocyte or granulosa cell apoptosis.

Techniques: Staining, In Vitro, Fluorescence

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 2 ｜Cellular ATP levels and F1F0-ATPase activity in isolated mitochondria in a in SH-SY5Y cell model of Parkinson’s disease. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector + MPP+: LV-Flag-vector- transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag + MPP+: LV-CHCHD2-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Detection of ATP levels in control or MPP+-treated SH-SY5Y cells stably transfected with empty vector or constructs encoding WT or T61I-mutant CHCHD2. (B) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in control or MPP+-treated cells. (C) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in AVV-transfected mice with or without MPTP treatment. CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); Flag-vector: AVV-Flag (CMV- betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ATP: Adenosine triphosphate; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Activity Assay, Isolation, Plasmid Preparation, Transfection, Cell Culture, Control, Stable Transfection, Construct, Mutagenesis, Saline

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 3 ｜CHCHD2 T61I mutation promotes mitochondrial permeability transition pore (mPTP) opening in SH-SY5Y cells. CHCHD2-Flag: LV-CHCHD2-Flag-transfected SH-SY5Y cells; CHCHD2-T61I: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells; Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells. (A) Calcein-AM and CoCl2 were administered to live cells expressing WT or T61I-mutant CHCHD2, and fluorescence intensity was detected by flow cytometry. (B) Effect of WT or the T61I-mutant CHCHD2 on mPTP opening as determined by detecting mitochondrial membrane potential using JC-1 fluorescent probes (white squares) in control or MPP+-treated SH-SY5Y cells. The fluorescence intensity of intracellular JC-1 aggregates (red) in the MPP+ + CHCHD2-Flag group was greater than that in the MPP+ + Flag-vector group. The CHCHD2-T61I group showed an increased amount of JC-1 monomers (green). Scale bars: 10 μm. (C) The mean fluorescence intensity of JC-1 aggregates or monomers in control or MPP+-treated SH-SY5Y cells expressing WT or T61I-mutant CHCHD2. Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Calcein-AM: Calcein acetoxymethyl ester; MPP+: 1-methyl-4-phenylpyridinium; mPTP: mitochondrial permeability transition pore; WT: wild type.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Permeability, Transfection, Plasmid Preparation, Expressing, Fluorescence, Flow Cytometry, Membrane, Control

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 5 ｜CHCHD2 T61I mutation aggravates movement deficits and nigral DA neuron function in a mouse model of PD. (A) Experimental design. AAV-CHCHD2, AAV-CHCHD2 T61I, or AAV-Vector was stereotaxically injected into the substantia nigra pars compacta, and 4 weeks later mice were injected intraperitoneally with normal saline or MPTP for 5 weeks. One day after the last MPTP/normal saline injection, behavioral tests were performed, and then the mice were sacrificed. Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector+MPTP: AVV-Flag– transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag+MPTP: AVV-CHCHD2-Flag–infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I+MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (B) The grasping test was used to examine grip strength. (C) The pole-climbing test was used to examine bradykinesia. (D) The rotarod test was used to examine motor coordination. (E) Representative immunohistochemical staining of TH-positive neurons in the substantia nigra (SN). There were fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 800 μm. (F) Quantification of the TH-positive cells shown in E. (G) Representative images of double-immunofluorescent staining for CHCHD2-Flag (green, Alexa Fluor 488) and TH (red, Alexa Fluor 555) (white squares) in the SN. There were significantly fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 50 μm. (H) Quantification of TH mean fluorescence in the SN as shown in G. (I) Representative immunoblot for TH in the SN. (J) TH expression in the SN. Results are expressed as the mean ± SD (n ≥ 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; AAV: adeno-associated virus; DA: dopaminergic; DAPI: 4′,6-diamidino-2-phenylindole; MPTP: 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine; TH: tyrosine hydroxylase; SN: substantia nigra.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Plasmid Preparation, Injection, Saline, Transfection, Infection, Immunohistochemical staining, Staining, Fluorescence, Western Blot, Expressing, Virus

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 8 ｜CHCHD2 modulates OSCP expression in MPP+-/MPTP-induced PD models. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector+MPP+: LV-Flag-vector–transfected SH- SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag+MPP+: LV-CHCHD2-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Representative immunoblot of OSCP, CHCHD2, and ACTB in MPP+-treated SH-SY5Y cells. (B) Quantification of relative OSCP expression shown in A. (C, D) Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag- infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (C) Representative immunoblot of OSCP, CHCHD2, and ACTB in control or MPTP-treated mice. (D) Quantification of relative OSCP expression shown in C. Data normalized to SH-SY5Y cells overexpressing Flag-vector or C57BL/6J mice transfected with AAV-Flag are expressed as the mean ± SD (n = 3 independent experiments for cells, n = 6 for animals). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine; OSCP: oligomycin sensitivity conferral protein.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Western Blot, Saline, Infection, Control