Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: bioRxiv

Article Title: TXNIP mediates LAT1/SLC7A5 endocytosis to reduce amino acid uptake in cells entering quiescence

doi: 10.1101/2024.10.29.620655

Figure Lengend Snippet: (a) TXNIP KO and WT cells were grown in growth medium (+ serum) or serum starved for 24 h (-serum). Total cell lysates were analyzed by SDS-PAGE and WB with the indicated antibodies. (b) WB quantification of TXNIP (n=4, paired t-test), SLC7A5 (n=4, two-way ANOVA, Sidak’s multiple comparisons test) and SLC7A11 protein levels (n=4, two-way ANOVA, Sidak’s multiple comparisons test). Values were normalized to proliferating WT cells (+ serum). (c) IF of PFA fixed cells (WT, TXNIP KO and TXNIP KO reconstituted with TXNIP) stained for SLC3A2 (green) and DAPI (blue) were analyzed by confocal microscopy. The images show a single plane of a Z-stack. Regions of interest (ROI, white box) were magnified. Scale bar = 10 µm. (d) Quantification of SLC3A2 cell surface FACS of the indicated cells, normalized to proliferating WT cells (n=6, N>7000, two-way ANOVA, Sidak’s multiple comparisons test). (e) IF of PFA fixed TXNIP KO reconstituted with ALFA-TXNIP. The localization of ALFA-TXNIP (red) and SLC3A2 (green) was analyzed by confocal microscopy. The images show a single plane of a Z-stack. Regions of interest (ROI, white box) were magnified. Scale bar = 10 µm.

Article Snippet: The SLC7A5 inhibitor JPH203 (also KYT-0353, Selleckchem, #S8667), dissolved in DMSO, was used at a final concentration of 10 µM.

Techniques: SDS Page, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: TXNIP mediates LAT1/SLC7A5 endocytosis to reduce amino acid uptake in cells entering quiescence

doi: 10.1101/2024.10.29.620655

Figure Lengend Snippet: (a) Schematic representation of TXNIP protein harboring PPxY motifs. (b) Total cell lysates from WT cells or TXNIP KO reconstituted with ALFA-TXNIP PPxY331 mutants were analyzed by SDS-PAGE and WB with the indicated antibodies. The asterisk (*) labels a second band that was only detected using the monoclonal SLC7A5 antibody. (c, e) Indirect IF of PFA fixed cells (WT, TXNIP KO and TXNIP KO reconstituted with ALFA-TXNIP or ALFA-TXNIP PPXY331 ) was analyzed by confocal microscopy. The images show a single plane of a Z-stack (c) Cells were stained for SLC3A2 (green) and DAPI (blue). Scale bar = 10 µm. (d) Quantification of SLC3A2 cell surface FACS of the indicated cells, normalized to proliferating WT cells (n=5, N>7000 cells, two-way ANOVA, Sidak’s multiple comparisons test). (e) Cells were stained for ALFA-TXNIP (red) and SLC3A2 (green). Regions of interest (ROI, white box) were magnified. Scale bar = 10 µm . (f) SDS-PAGE and WB analysis with the indicated antibodies from non-denaturing ALFA-immunoprecipitations (IP) from HEK293T cells expressing ALFA-TXNIP or the mutated ALFA-TXNIP PPxY331 variant and the indicated YFP-tagged HECT-type ubiquitin ligase. (g) SDS-PAGE and WB analysis with the indicated antibodies from denaturing ALFA-SLC7A5 IP from SLC7A5 KO RPE1 reconstituted with ALFA-SLC7A5.

Article Snippet: The SLC7A5 inhibitor JPH203 (also KYT-0353, Selleckchem, #S8667), dissolved in DMSO, was used at a final concentration of 10 µM.

Techniques: SDS Page, Confocal Microscopy, Staining, Expressing, Variant Assay

Journal: bioRxiv

Article Title: TXNIP mediates LAT1/SLC7A5 endocytosis to reduce amino acid uptake in cells entering quiescence

doi: 10.1101/2024.10.29.620655

Figure Lengend Snippet: (a) WT and TXNIP KO cells were grown in growth medium (+ serum), serum starved for 24 h (-serum) or treated with 10 µM JPH203. Cells were incubated with [ 3 H]-leucine for 15 min, washed and lysed. Cell lysates were analyzed by scintillation counting. Cpm values were normalized to total protein content (n=5, two-way ANOVA, Tukey’s multiple comparisons test). (b) WT, SLC7A5 KO and TXNIP KO cells were grown in growth medium (+ serum) or serum starved for 24 h (-serum). Mass spectrometry analysis of free AAs, normalized to total protein content (nmol AA / µg protein, n=5). (c) Total cell lysates from WT and TXNIP KO cells before or after 24h serum starvation, were analyzed by SDS-PAGE and WB with the indicated antibodies (d-f) WB quantification of pS6K T389 , normalized to total S6K. WB quantification of pS6 S240/244 , normalized to total S6 and WB quantification of pEIF2α S51 , normalized to total EIF2α protein levels (n=5, two-way ANOVA). (g) Cells were incubated with [ 35 S]-methionine, [ 35 S]-cysteine for 2 minutes, before CHX (10µg/ml) was added, cells were lysed and analyzed by SDS-PAGE and autoradiography follow by densitometric analysis (n=3, paired t-test).

Article Snippet: The SLC7A5 inhibitor JPH203 (also KYT-0353, Selleckchem, #S8667), dissolved in DMSO, was used at a final concentration of 10 µM.

Techniques: Incubation, Mass Spectrometry, SDS Page, Autoradiography

Journal: bioRxiv

Article Title: TXNIP mediates LAT1/SLC7A5 endocytosis to reduce amino acid uptake in cells entering quiescence

doi: 10.1101/2024.10.29.620655

Figure Lengend Snippet: (a) WT, TXNIP KO and TXNIP KO cells reconstituted with TXNIP were harvested, permeabilized and fixed. The DNA content was analyzed by PI staining to assess the cell cycle profile by FACS (n=3, N>4000 cells). (b) Increase in confluency of the indicated cells, after refeeding serum starved cells, was measured by continuous live cell microcopy in an Incucyte incubator for 48 h (n=6). (c) The proliferation of the indicated cells was determined using CASY cell counting during the indicated time. The relative increase in cell number is normalized to 0 h and show as fold increase (n>3). (d) Model of TXNIP mediated downregulation of SLC7A5 during entry into quiescence.

Article Snippet: The SLC7A5 inhibitor JPH203 (also KYT-0353, Selleckchem, #S8667), dissolved in DMSO, was used at a final concentration of 10 µM.

Techniques: Staining, Cell Counting

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 reverses the ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) by tripartite motif-containing protein 21 (TRIM21) and ameliorates the phenotype of polycystic ovary syndrome (PCOS) mice. (A) Western blot (WB) analysis of neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) and CPT1A treated with MLN4924, with or without TRIM21 knockdown in KGN cell. (B and C) Coimmunoprecipitation (Co-IP) and WB analysis of TRIM21 neddylation in KGN cells treated with or without MLN4924. (D) Co-IP and WB analysis of exogenous K48 ubiquitination of CPT1A in KGN cells overexpressing UBE2M, with or without NEDD8 knockdown in the presence of MG132 (10 μM, 4 h). (E and F) Evaluation of the mitochondrial oxidative phosphorylation (OXPHOS) function by oxygen consumption rate (OCR) in KGN cells, including basal respiration, maximum respiration, ATP generation, and coupling efficiency ( n = 6). (G and H) Evaluation of fatty acid oxidation (FAO)-dependent mitochondrial function by OCR in KGN cells, including basal respiration and maximum respiration ( n = 6). (I) Activity of mitochondrial complex V in KGN cells ( n = 6). (J and K) Testosterone and luteinizing hormone (LH) levels of serum ( n = 15). (L) Anogenital distance in adult female mice ( n = 15). (M) Insulin tolerance test (ITT) test in adult female mice after 4 h of fasting ( n = 5). (N) Number of pups per birth ( n = 10). (O and P) WB and reverse transcription polymerase chain reaction (RT-PCR) analysis of TRIM21 and CPT1A in ovarian granulosa cells (GCs). (Q) Immunohistochemical staining of TRIM21 in mouse ovarian tissues (scale bars, 100 μm). Data are expressed as means ± standard error of the mean (SEM), and each symbol represents a biologically independent mouse. Significance was calculated by 1-way analysis of variance (ANOVA) multiple comparison test. Blood glucose analysis between groups (M) was determined by 2-way ANOVA and multiple comparison test. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. IB, immunoblot.

Article Snippet: Cells were exposed to 100 nM DHT for 72 h. To investigate specific cellular processes, cells were also subjected to various inhibitors: 100 nM MLN4924 (Selleck, cat# S7109) for 24 h to inhibit neddylation; 5 μM MG132 (Selleck, cat# S2619) for 24 h to inhibit proteasomal degradation; 500 nM CHX (MCE, cat# HY-12320) for 4 h to inhibit protein synthesis; and 50 μM etomoxir (MCE, cat# HY-50202) for 24 h to inhibit FAO.

Techniques: Ubiquitin Proteomics, Western Blot, Knockdown, Co-Immunoprecipitation Assay, Phospho-proteomics, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Comparison

Journal: Research

Article Title: Inhibiting TRIM21 Neddylation Rejuvenates Oocyte Quality in PCOS by Regulating Ubiquitination of CPT1A

doi: 10.34133/research.1223

Figure Lengend Snippet: MLN4924 improves the quality of oocytes of polycystic ovary syndrome (PCOS) mice. (A) Hematoxylin and eosin (H&E) staining of ovaries and the number of follicles in ovary including primordial follicles (*), growing follicles (#), and atretic follicles (arrows; scale bars, 100 μm). (B) Number of oocytes after superovulation ( n = 5 mice; scale bars, 100 μm). (C) Representative images and percentage of first polar body extrusion in mouse oocytes, fertilization rate, and blastocyst maturation rate ( n = 5 mice; scale bars, 100 μm). (D) Transmission electron microscopy (TEM) analysis of mitochondrial morphology of mouse ovarian granulosa cells. Green arrows indicate normal mitochondria, red arrows indicate abnormal mitochondria, and # indicates lipid droplets ( n = 5 mice; scale bar, 500 nm). (E) Representative images of spindle morphology and chromosome alignment in oocytes at the metaphase II stage by confocal microscopy ( n = 5 mice; scale bar, 10 μm). The percentage of aberrant spindles and misaligned chromosomes were quantified in oocytes at metaphase II from control ( n = 23), PCOS ( n = 19), and MLN4924 ( n = 21) mice. (F) Representative images for JC-1 staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected (G) Representative images for tetramethylrhodamine ethyl ester perchlorate (TMRE) staining of mouse oocytes ( n = 15; scale bar, 50 μm) and relative fluorescence intensities. Three fields of view of each mouse were selected. (H) Representative images for lipid content in oocytes ( n = 15; scale bar, 50 μm) and ratio of the fluorescence intensities. Three fields of view of each mouse were selected. (I) Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA levels of follicle development-related genes in mouse cumulus–oocyte complexes (COCs) ( n = 5). Data are expressed as means ± SD, and each symbol represents a biologically independent mouse. (J) Schematic diagram of the pathway by which tripartite motif-containing protein 21 (TRIM21) regulates ubiquitination of carnitine palmitoyltransferase 1A (CPT1A) leading to abnormal fatty acid oxidation in ovarian granulosa cells. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were exposed to 100 nM DHT for 72 h. To investigate specific cellular processes, cells were also subjected to various inhibitors: 100 nM MLN4924 (Selleck, cat# S7109) for 24 h to inhibit neddylation; 5 μM MG132 (Selleck, cat# S2619) for 24 h to inhibit proteasomal degradation; 500 nM CHX (MCE, cat# HY-12320) for 4 h to inhibit protein synthesis; and 50 μM etomoxir (MCE, cat# HY-50202) for 24 h to inhibit FAO.

Techniques: Staining, Transmission Assay, Electron Microscopy, Confocal Microscopy, Control, Fluorescence, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: BMC pharmacology & toxicology

Article Title: Narciclasine induces autophagy-mediated apoptosis in gastric cancer cells through the Akt/mTOR signaling pathway.

doi: 10.1186/s40360-021-00537-3

Figure Lengend Snippet: Fig. 4 Narciclasine promotes autophagy of gastric cancer cells. A-C, Laser confocal scanning microscopy was used to observe the formation of autolysosomes in gastric cancer cells treated with narciclasine (0.5 μM) for 24 h. D-F, Western blotting was used to detect the effect of narciclasine combined with autophagy inhibitor 3-MA (5 mM) and CQ (2.5 μM) on LC3-II and p62 protein after treatment of gastric cancer cells for 24 h. Data are shown as mean ± SD. NCS: narciclasine; 3-MA: 3-methyladenine; CQ: chloroquine

Article Snippet: The human gastric cancer cell lines BGC-823, MGC-803, GES-1, MKN28 and SGC-7901 were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China); Roswell Park Memorial Institute 1640 (RPMI 1640), fetal bovine serum (FBS), penicillin and streptomycin from Gibco Life Technologies (NY, US); chloroquine (CQ) and 3-methyladenine (3-MA) from Selleck Chemicals (Texas, US); insulin from Sigma-Aldrich (CA, US); and AktiVIII from Macklin Inc. (Beijing, China).

Techniques: Confocal Laser Scanning Microscopy, Western Blot

Journal: Cell death & disease

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns.

doi: 10.1038/cddis.2014.450

Figure Lengend Snippet: Figure 7 Calpain activity is a prerequisite for the release of nucleosomes and DAMPs in dying cells. HeLa cells were incubated in amino acid-depleted medium (HBSS) or staurosporine-containing medium (Stau) (1 μg/ml) for 24 or 8 h, respectively, in the presence of solvent control, BAPTA-AM (50 μM) or A23187 (1 μg/ml). Released DNA was quantified with PicoGreen (a). Amount of intracellular free Ca2+ was measured in cells treated with solvent control or staurosporine at the indicated time periods by eFluor 514 (b). Cells were treated with staurosporine for 8 h in the presence of control solvent, ALLN (20 μM), or PD150606 (20 μM), and released DNA or protein was detected by PicoGreen staining (c) or western blot for histones and DAMPs (d), respectively. Genomic DNA was separated in agarose gel electrophoresis from staurosporine-treated cells with or without PD150606 (e). The cells treated with staurosporine and PD150606 for 8 h, were stained for lamin A/C and histone H1, and examined by confocal microscopy; the arrows indicate nuclear fragmentations (f). Cells treated with control solvent, zVAD-fmk (20 μM), or PD150606 with or without staurosporine for 8 h were measured for viabilities with Calcein assay (g). Cells treated with staurosporine were measured for calpain activity, caspases 3 and 7 activity, or released DNAs at the indicated time periods (h). HeLa cells transfected with non- targeting shRNA (NT), calpastatin shRNA, calpain S1 shRNA, or overexpressing calpain 1, calpain 2, or calpastatin were incubated with staurosporine for 8 h and the released DNAs were measured by PicoGreen staining (i). Cells expressing calpain 1 or calpain 2 tagged with Flag at the N-terminus and HA at the C-terminus were treated with staurosporine and western-blotted with anti-Flag antibody or anti-HA antibody (j). HeLa cells expressing halide-sensitive YFP were treated with staurosporine and/or PD150606 for 4 h, and their chloride ion currents were measured by fluorometry by detecting quenching of YFP fluorescence (k). Data performed in triplicate are presented as mean± S.D. (a–c and g–i)

Article Snippet: Cl-amidine was purchased from Cayman Chemical (Ann Arbor, MI, USA). zVAD-fmk, zDEVD-fmk, Necrostatin-1, BAPTA-AM, diphenyliodonium, PD150606, ALLN and A23187 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Incubation, Solvent, Control, Staining, Western Blot, Agarose Gel Electrophoresis, Confocal Microscopy, Transfection, shRNA, Expressing, Fluorescence