Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Impaired AJC formation in Albatross knockdown cells. (A) Double staining for Albatross (red) and the undercoat proteins (green) for each AJC component: TJ, ZO-1; AJ, afadin; DS, desmoplakin. Top and bottom columns show projections of x-y planes and z sections, respectively. Albatross knockdown A549 (Albatross KD) cells lack accumulation of these proteins at the cell–cell borders except in regions where residual Albatross is present. (B) Cell–cell adhesive properties evaluated by a cell aggregation assay. In the differential interference contrast images, control cells show cell aggregation. With Albatross knockdown A549 (A1050 and A1160) cells, the aggregated cell population is reduced and free cells are increased. The percentages of single cells in total cells (mean ± SD) are: control, 36.1 ± 3.9; A1050, 52.4 ± 2.8; A1160 cells, 59.4 ± 10.2. n = 4 and P < 0.01. (C) Immunoelectron microscopy of A549 cells with anti-Albatross antibodies. Note that the cytoplasm in the vicinity of AJCs is labeled. TJ, AJ, and DS are indicated. Arrows indicate cell–cell contacts. (D) Quantitative data from C. (E) BC fraction and AJ fraction were immunostained for Albatross with the indicated AJC proteins, PKCζ or Par3. Note that Albatross is well colocalized with them. (F) Immunoblotting of fractions derived from mouse liver: homogenates (left), BC (middle), and AJ (right). Not only Albatross but also Par3 is enriched in line with the concentrations of the indicated AJC components. (G) Immunoprecipitation of A549 cells with anti-Albatross antibodies. Start and IP indicate starting lysates and immunoprecipitates with preimmune (Pre.) and anti-Albatross (αAlb.) antibodies, respectively. Note the Par3 precipitation with Albatross. Among AJC components, ZO-1 also coprecipitated. (H) Immunoprecipitation analysis with tagged Albatross and Par3. Start and IP indicate starting lysates and immunoprecipitates with anti-GFP antibodies, respectively. Left lanes show results for negative controls expressing GFP alone. Par3 was the most precipitated with GFP-Albatross among coexpressed myc-Par3, -Par6, and -PKCλ. Bars: (A) 10 μm; (B) 100 μm; (C) 0.1 μm; (E, BC) 13 μm; (E, AJ) 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Double Staining, Immuno-Electron Microscopy, Labeling, Western Blot, Derivative Assay, Immunoprecipitation, Expressing

Journal: The Journal of Cell Biology

Article Title: The keratin-binding protein Albatross regulates polarization of epithelial cells

doi: 10.1083/jcb.200803133

Figure Lengend Snippet: Functions of keratins and Albatross–Par3 complexes. (A–C) The amounts of Albatross protein and mRNA were analyzed in both keratin 8 and keratin 18 (K8/18)-introduced SW13 cells. As a control, an empty vector was transfected. As loading controls, α-tubulin and GAPDH were used. Two independent experiments were performed. (A) Immunoblotting. In transiently K8/18-introduced SW13 cells, the amount of Albatross protein is elevated, along with the amount of keratin 18. (B) With stable lines, the same results were obtained. (C) RT-PCR. In K8/18-introduced SW13 cells, the mRNA level of K18 is elevated, but not that of Albatross. β-actin is included as an internal control. (D) Double staining for K8/18 and the indicated proteins: Albatross, AJC components of ZO-1 and afadin, and Par3. (top) In control cells, K8/18 is absent and only limited amounts of Albatross are apparent at cell–cell junctions. In stably K8/18-introduced SW13 cells, Albatross is well localized in cell–cell junctions compared with control cells. (middle and bottom) ZO-1, afadin, and Par3 similarly accumulated at the cell–cell borders in stably K8/18-introduced SW13 cells. (E) Immunostaining of stably K8/18-introduced SW13 cells transfected with control or Albatross siRNA. Note that ZO-1, afadin, and Par3 are reduced at cell–cell borders with knockdown of Albatross. (F) A model for the regulation of AJC and lateral domains with the Albatross–Par3 complex and keratins. Albatross–Par3 complexes regulate the formation of AJC and maintain lateral membrane identity. However, Par3 without Albatross regulates apical structures. Keratins stabilize Albatross, promoting the formation of AJC. Knockdown effects are also indicated. Bars, 10 μm.

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-keratin 8 (Ks 8.7; Progen Pharmaceuticals), monoclonal mouse anti-keratin 18 (CY-90; Sigma-Aldrich), polyclonal mouse anti-pan keratin (Sigma-Aldrich), polyclonal guinea pig anti-K8/18 (Progen Pharmaceuticals), polyclonal guinea pig anti–desmoplakin 1 (Progen Pharmaceuticals), monoclonal mouse anti–desmoplakin 1 and 2 (Progen Pharmaceuticals), monoclonal mouse anti–ZO-1 (1; BD Biosciences), monoclonal rat anti–ZO-1 (BM173; Acris Antibodies, GmbH), monoclonal rat anti–E-cadherin (ECCD-2; EMD), monoclonal mouse anti-neurofilaments, monoclonal rat anti–platelet/endothelial cell adhesion molecule (anti-PECAM; CD31; BD Biosciences), monoclonal mouse anti–α-tubulin (B-5-1-2; Sigma-Aldrich), monoclonal mouse anti–claudin-2 (12H12; Invitrogen), monoclonal mouse anti–desmocollin-2/3 (7G6; Invitrogen), monoclonal mouse anti–desmoglein 2 (10G11; Progen Pharmaceuticals), monoclonal mouse anti–nectin-1 (CK8; Invitrogen), monoclonal mouse anti–β-catenin (14; BD Biosciences), polyclonal rabbit anti-ezrin (Millipore), rabbit anti-Par3 polyclonal antibody (provided by S. Ohno, Yokohama City University, Yokohama, Kanagawa, Japan; Millipore), monoclonal mouse anti-occludin (OC-3F10; Invitrogen), monoclonal rat anti–nectin-2 (502–57; HyCult Biotechnology), polyclonal rabbit anti-GFP (Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-PKCζ (Santa Cruz Biotechnology, Inc.), and polyclonal rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) conjugated to HRP (Abcam).

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Double Staining, Stable Transfection, Immunostaining

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: Redox Biology

Article Title: NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa -induced acute lung injury ☆

doi: 10.1016/j.redox.2022.102467

Figure Lengend Snippet: Effects of cigarette smoke on P. aeruginosa -triggered NLRP3 inflammasome activation. C57BL/6 adult male mice were exposed to RA, CS for 6 h or 2 weeks (a), or CS for 2 weeks (b–h). One hour after the last CS exposure, mice were given 1.0 × 10 5 CFU (determined based on the optical density of bacterial suspension) of PA103 (PA) in 50 μl saline or Ctrl (an equal volume of saline) by intratracheal instillation. The actual alive bacteria mice received were 1.8 × 10 5 CFU. 18 h after administration of PA, BAL fluid was collected for assessment of bacterial amount by counting colony forming units (CFU) (a). The data is presented as mean ± SD of Log10 (CFU/ml). 3–7 mice per experimental group were used. Lung homogenates from the experiment was prepared for assessment of NLRP3 inflammasome platform proteins (NLRP3, ASC, pro-caspase-1, and pro-IL-1β) and inflammasome activation (p20 caspase-1 and mature (cleaved) IL-1β) by immunoblot, using β-actin as a protein loading control (b). The relative levels of each protein of interest in each sample were determined by densitometry of their immunoblots normalized by immunoblots of respective protein loading controls. The densitometry values are presented as mean ± SD (c). 6–8 mice per experimental group were used. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. *p < 0.05. IL-1β levels in lung homogenates from this experiment were also assessed by ELISA (d). Data are presented as mean ± SD. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. **p < 0.01; ****p < 0.0001. The cytospun slides of BAL leukocytes (e) and cryosections of lung tissue (f, h) from these experiments were prepared for immunofluorescence microscopy to examine NLRP3 expression/localization (green) in either AM (CD68 + , red; e, f) or lung endothelial cells (von Willebrand factor (vWF+), red; h). DAPI was used to stain nuclei (blue). Panels e, f, h are representative images for each experimental group. Scale bar = 15 μm. The arrows in panels e–f indicate alveolar macrophages (M) and the arrows in panel h indicate lung endothelial cells (EC). The percentage of AM expressing NLRP3 over the total AM counted are presented as mean ± SD (g). Five slides at 40X and five slides at 100X per mouse lung tissue or BAL cytospun slides were used to count AM. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means. ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Caspase-1 inhibitor (Ac-YVAD-cmk) was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Microscopy, Expressing, Staining

Journal: Redox Biology

Article Title: NLRP3 inflammasome activation in cigarette smoke priming for Pseudomonas aeruginosa -induced acute lung injury ☆

doi: 10.1016/j.redox.2022.102467

Figure Lengend Snippet: Effects of pharmacological inhibition of caspase-1 on PA- and CS/PA-induced acute lung injury. C57BL/6 adult male mice were exposed to RA or CS for 2 weeks. One hour after the last CS exposure, mice were administered a caspase-1 inhibitor, Ac-YVAD-cmk (Ac–Y) at a dose of 10 mg/kg or vehicle intraperitoneally. One hour later, mice were given 1.0 × 10 5 CFU (determined based on the optical density of bacterial suspension) of PA103 (PA) in 50 μl saline or Ctrl (an equal volume of saline) by intratracheal instillation. The actual alive bacteria mice received were 3.5 × 10 5 CFU. 18 h after administration of PA, BAL fluid was collected for assessment of inflammasome activation by measuring mature IL-1β levels in cell-free BAL fluid by ELISA (a), BAL total protein levels (b), BAL inflammatory cells (c–f), BAL chemokine/cytokines (g–j), and BAL bacterial loads (k). Animal survival was recorded up to 36 h after administration of PA in a separate set of mice (l). 4–7 mice per experimental group were used. Data are presented as mean ± SD. Two-way ANOVA and Tukey's multiple comparison test was used to determine differences among means for studies other than survival study, in which Log-rank (Mantel-Cox) test was used for determining difference among groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS: not significant.

Article Snippet: Caspase-1 inhibitor (Ac-YVAD-cmk) was purchased from Cayman Chemicals (Ann Arbor, MI).

Techniques: Inhibition, Activation Assay, Enzyme-linked Immunosorbent 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: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

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: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

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: Experimental hematology & oncology

Article Title: Targeting NRF2 uncovered an intrinsic susceptibility of acute myeloid leukemia cells to ferroptosis.

doi: 10.1186/s40164-023-00411-4

Figure Lengend Snippet: Fig. 3 ML385 cooperates with FIN56/RSL3 to reduce the viability of AML cells. (a) MV4;11 cells were treated with the indicated concentrations of RSL3 (left panel) or FIN56 (right panel) for 24 h, and the expression of GPX4 was detected by western blotting. (b) AML cell lines and PBMCs from HIs were treated with the indicated concentrations of RSL3 for 24 h, and the cell viability was detected by the CCK8 assay. (c) MV4;11 (left panel) and Kasumi-1 cells (right panel) were treated with ML385 alone, RSL3 alone, or the combination of ML385 and RSL3 at the indicated concentrations for 24 h, and the cell viability was measured by CCK8 assay. CI values were calculated by the Chou-Talalay method. The dashed line designates a CI value of 1, and CI < 1 indicates a synergistic interaction between the two agents in the combination. (d) MV4;11 (left panel) and Kasumi-1 cells (right panel) were treated with ML385 alone, FIN56 alone, or the combination of ML385 and FIN56 at the indicated concentrations for 24 h, and the cell viability was measured by CCK8 assay. CI values were calculated by the Chou-Talalay method. The dashed line designates a CI value of 1, and CI < 1 indicates a synergistic interaction between the two agents in the combination. Data are expressed as the mean ± SD. n = 3 or more independent biological replicates, presented as individual points. P value < 0.05 was considered significant (a, one-way ANOVA with Bonferroni post hoc test; b, two-tailed unpaired Student’s t test).

Article Snippet: Ferrostatin-1, ML385, FIN56, and RSL3 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Expressing, Western Blot, CCK-8 Assay, Two Tailed Test

Journal: Experimental hematology & oncology

Article Title: Targeting NRF2 uncovered an intrinsic susceptibility of acute myeloid leukemia cells to ferroptosis.

doi: 10.1186/s40164-023-00411-4

Figure Lengend Snippet: Fig. 4 ML385 cooperates with FIN56/RSL3 to induce the cell death of AML cells. (a) MV4;11 (upper panel) and Kasumi-1 cells (lower panel) were treated with 0.5 µM RSL3, 5 µM ML385, or the combination of RSL3 and ML385 for 24 h, and the percentage of Annexin V + cells was determined by flow cytom etry. (b) MV4;11 (upper panel) and Kasumi-1 cells (lower panel) were treated with 10 µM FIN56, 10 µM ML385, or the combination of FIN56 and ML385 for 48 h, and the percentage of Annexin V + cells was determined by flow cytometry. (c) MV4;11 cells were co-treated with RSL3 and ML385 (upper panel) or with FIN56 and ML385 (lower panel) at the indicated concentration for 24 h, and the cell cycle was determined by flow cytometry. (d) BMMCs from newly diagnosed AML patients were treated with 2 µM RSL3, 10 µM ML385, or a combination of RSL3 and ML385 for 48 h (upper panel), as well as with 10 µM FIN56, 10 µM ML385, or a combination of FIN56 and ML385 for 48 h (lower panel), and cell viability was analyzed using the CCK8 assay. (e) BMMCs from newly diagnosed AML patients were treated with 2 µM RSL3, 10 µM ML385, or a combination of RSL3 and ML385 for 48 h (upper panel). In addition, cells were treated with 10 µM FIN56, 10 µM ML385, or a combination of FIN56 and ML385 for 48 h (lower panel). The percentage of Annexin V + cells was then detected using flow cytometry. Data are expressed as the mean ± SD. n = 3 or more independent biological replicates, presented as individual points. P value < 0.05 was considered significant (a-e, one-way ANOVA with Bonferroni post hoc test).

Article Snippet: Ferrostatin-1, ML385, FIN56, and RSL3 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Flow Cytometry, Concentration Assay, CCK-8 Assay

Journal: Experimental hematology & oncology

Article Title: Targeting NRF2 uncovered an intrinsic susceptibility of acute myeloid leukemia cells to ferroptosis.

doi: 10.1186/s40164-023-00411-4

Figure Lengend Snippet: Fig. 5 ML385 cooperates with FIN56/RSL3 to promote the ferroptosis of AML cells. (a) MV4;11 and Kasumi-1 cells were treated with 5 µM ML385, 0.5 µM RSL3, or a combination of ML385 and RSL3 for 24 h (upper panel). In addition, cells were treated with 10 µM FIN56, 10 µM ML385, or a combination of FIN56 and ML385 for 48 h (lower panel), and the levels of lipid reactive oxygen species (ROS) were determined by flow cytometry using the C11-BODIPY 581/591 probe. (b) Histogram showing the levels of lipid ROS. (c) MV4;11 cells were treated with 0.5 µM RSL3, 5 µM ML385, or a combination of RSL3 and ML385. To visualize the effect of these compounds on cell death, cells were stained with propidium iodide (PI) and images were obtained using a fluo rescence microscope. (d) MV4;11 cells were treated with 5 µM ML385, 0.5 µM RSL3, 10 µM ferroptosis inhibitor ferrostatin-1 (Fer-1), or the combination of ML385 and RSL3 with Fer-1 for 24 h, the percentage of Annexin V + cells was determined by flow cytometry. Data are expressed as the mean ± SD. n = 3 or more independent biological replicates, presented as individual points. P value < 0.05 was considered significant (b, d, one-way ANOVA with Bonferroni post hoc test).

Article Snippet: Ferrostatin-1, ML385, FIN56, and RSL3 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Flow Cytometry, Staining, Microscopy

Journal: Experimental hematology & oncology

Article Title: Targeting NRF2 uncovered an intrinsic susceptibility of acute myeloid leukemia cells to ferroptosis.

doi: 10.1186/s40164-023-00411-4

Figure Lengend Snippet: Fig. 6 NRF2/GPX4 expression contributes to ferroptosis resistance. (a) MV4;11 and Kasumi-1 cells were co-treated with 0.5 µM RSL3 and 5 µM ML385 for 24 h, and the expression levels of NRF2 and GPX4 were detected by western blotting (upper panel). Similarly, MV4;11 and Kasumi-1 cells were co-treated with 10 µM FIN56 and 10 µM ML385 for 48 h, and the expression levels of NRF2 and GPX4 were detected by western blotting (lower panel). (b) Western blots were quantified and are presented in the form of a histogram. (c) MV4;11 cells transfected with shNRF2 were treated with 10 µM FIN56 or 0.5 µM RSL3 for 48 h, and the percentage of Annexin V + cells was determined by flow cytometry. (d) MV4;11 cells transfected with shNRF2 were treated with 10 µM FIN56 or 0.5 µM RSL3 for 24 h, and the lipid ROS levels were determined by flow cytometry using C11-BODIPY 581/591 probe. (e) MV4;11 cells trans fected with LV-NC or LV-NRF2 vector were treated with the indicated concentrations of RSL3 for 24 h, and the cell viability was analyzed by the CCK8 assay. (f) The expression levels of GPX4 and NRF2 in MV4;11 cells transfected with small interfering RNAs (siRNAs) targeting GPX4 (siGPX4) were determined by western blotting. (g) MV4;11 cells transfected with siGPX4 were treated with 10 µM FIN56 or 0.5 µM RSL3 for 24 h, and the percentage of Annexin V + cells was analyzed by flow cytometry. Data are expressed as the mean ± SD. n = 3 or more independent biological replicates, presented as individual points. P value < 0.05 was considered significant (b-d, f-g, one-way ANOVA with Dunnett’s post hoc test; e, two-tailed unpaired Student’s t test).

Article Snippet: Ferrostatin-1, ML385, FIN56, and RSL3 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Expressing, Western Blot, Transfection, Flow Cytometry, Plasmid Preparation, CCK-8 Assay, Two Tailed Test

Journal: Cell death & disease

Article Title: LUBAC-mediated M1 Ub regulates necroptosis by segregating the cellular distribution of active MLKL.

doi: 10.1038/s41419-024-06447-6

Figure Lengend Snippet: Fig. 4 LUBAC and M1 poly-Ub segregate the subcellular distribution of activated MLKL. A Fractionation of phosphorylated and total MLKL in the micelle-poor (aqueous) and micelle-rich (detergent) fractions upon phase separation using Triton X-114 lysis buffer in untreated (UT) and HOIPIN-8 (30 µM) pre-treated HT-29 cells upon treatment with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 4 h. GAPDH was used as loading control for soluble proteins and CD9 as loading control for membrane proteins. Representative blots of at least two independent experiments are shown. B Representative fluorescence microscopy demonstrating the cellular distribution of phosphorylated MLKL (green) in untreated (UT) and HOIPIN-8 (30 µM) pre-treated HT-29 cells and after treatment with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 3 h. Nuclei were stained with DAPI (blue). Arrows indicate cytoplasmic clusters of phosphorylated MLKL. Representative images of at least two independent experiments are shown. Scale bars 50 µm. C Western blot analysis of TBZ-induced release of MLKL in exosomes, isolated from the supernatants of control, HOIPIN-8 (30 µM) or NSA (10 µM)-pre-treated HT-29 cells after treatment with TBZ (10 ng/mL TNFα, 1 µM BV6, 20 µM zVAD.fmk) for 3 h. Exosome isolation was confirmed with antibodies against ALIX, CD9 and Vinculin. Asterisk marks unspecific band. Expression of phosphorylated and total MLKL was determined by Western blotting of cell lysates from the same experiment. Vinculin was used as loading control. Representative blots of at least two independent experiments are shown. D Western blot analysis of lysosomal degradation of phosphorylated MLKL upon TBZ (10 ng/mL TNFα, 0.1 µM BV6, 20 µM zVAD.fmk) treatment of control, HOIPIN-8 (30 µM) or NSA (10 µM)-pre-treated HT-29 cells. HT-29 cells were treated with TBZ for 3 h and either harvested or the medium was exchanged with fresh medium with or without BafA1 (100 nM) after a washing step with PBS. Washed-out cells were harvested 12 h or 18 h after medium exchange. Vinculin was used as loading control. Representative blots of at least two independent experiments are shown. See also Figure S4.

Article Snippet: BV6 was kindly provided by Genentech Inc. (San Francisco, CA, USA), recombinant human TNFα was purchased from Biochrom Ltd. (Waterbeach, UK), Birinapant from Selleckchem (Houston, TX, USA), the pancaspase inhibitors zVAD.fmk and Emricasan from Bachem AG (Bubendorf, Switzerland) and Selleckchem, Nec-1s, GSK’872 and NSA from Merck KGaA (Darmstadt, Germany), HOIPIN-8 from Axon Medchem LLC (Reston, VA, USA), Gliotoxin from Tocris Biosciences (Bristol, UK), Doxycycline hydrochloride from Merck KGgA (Darmstadt, Germany) and BafA1 from Selleckchem.

Techniques: Fractionation, Lysis, Control, Membrane, Microscopy, Staining, Western Blot, Isolation, Expressing