Journal: American Journal of Cancer Research

Article Title: Alpelisib and radiotherapy treatment enhances Alisertib-mediated cervical cancer tumor killing

doi:

Figure Lengend Snippet: Alpelisib enhanced Alisertib-mediated toxicity in cervical cancer cells. A. Hela cells were treated with ALISERTIB at doses ranging from 40 uM to 40 nM in a 96 well plate. Cell viability was measured by the MTT assay at 3-days post-treatment. A dose dependent curve was generated using GraphPad Prism v9 to determine IC50 values. Data points are representative of the mean ± SEM (n = 3). B. Cells were treated with Alpelisib at increasing doses either in the presence or absence of Alisertib (concentration used: 0.5 Alisertib IC50 = 73.25 nM) before measuring cell viability at 3-days post-treatment by the MTT assay. Data is representative of one out of three independent experiments. Data points represent the mean ± SEM. C. Standard isobologram analysis of cell killing by the drug combinations. IC90 values of each drug are plotted on the axes; the solid line represents the additive effect, while the point represents concentrations of each drug resulting in 90% inhibition of growth. The point falls below the line indicating synergism between the drugs. Combination index (CI) at fractional response 0.9 (90% killing) is also shown. CI value is <1, indicative of a synergistic effect. Data is representative of three independent experiments. D. Proteins from cells collected at 24 h were immunoblotted for cleaved PARP. β-actin was used as a loading control. Individual blots shown are representative of three independent experiments. E. HeLa cells exposed to Alisertib, Alpelisib, or a combination of both were stained with annexin V-FITC and PI for FACS analysis after 48 h. Cell populations shown in the lower left quadrant represent living cells; lower right quadrant represents apoptotic cells, upper right quadrant represents necrotic cells and upper left quadrant represents pre-necrotic cells. Data is representative of 3 experiments. F. HeLa cells were treated either alone, with Alisertib (500 nM) or in combination with Alpelisib (20 uM). Images were captured by time-lapse microscopy over 72 h. Still images shown were captured at times indicated using the Hstudio 2.7.5™ live imaging. Data presented are representative of one out of three independent experiments. G. 10 individual cells were tracked from time-lapse microscopy videos using the Hstudio 2.7.5™ live imaging software and the time in and number of cell divisions were plotted on the graph over a 72 h period.

Article Snippet: The percentage of cells undergoing apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Kit (#K101) (BioVision, Milpitas, CA) as per manufacturer’s protocol.

Techniques: MTT Assay, Generated, Concentration Assay, Inhibition, Staining, Time-lapse Microscopy, Imaging, Software

Journal: Cell Death Discovery

Article Title: Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

doi: 10.1038/s41420-024-02225-7

Figure Lengend Snippet: a Western blotting analysis of endogenous BID cleavage induced by ITCs. HeLa cells were incubated with ITCs (20 µM) for 4 h in the absence or presence of Z-VAD-FMK (20 µM) or Z-IETD-FMK (20 µM). b Western blotting analysis of exogenous BID cleavage induced by ITCs. HeLa cells expressing GFP-tagged BID were incubated with ITCs (20 µM) for 4 h in the absence or presence of Z-VAD-FMK (20 µM) or Z-IETD-FMK (20 µM). Red asterisks indicate the cleaved proteins. c Confocal fluorescence imaging of BID. HeLa cells expressing GFP-tagged C -BID or full-length BID (FL-BID) and TOMM-mCherry were treated with ITCs (20 µM) as indicated for 4 h and imaged by confocal fluorescence microscopy. Scale bars = 10 µm. d Western blotting analysis of caspases after treatment with ITCs. HeLa cells were treated with ITCs (20 µM) as indicated for 4 h. Red asterisks indicate the cleaved proteins. e Caspase-3/7 activity measured by a caspase-3 substrate and flow cytometry. HeLa cells were treated with ITCs (20 µM) for 4 h. Data are shown as mean ± sd ( n = 4). f Fluorescence imaging of mitochondrial membrane potential using TMRE in HeLa cells treated with ITCs (20 µM) for 4 h. Scale bars = 20 µm. g Quantification of fluorescence imaging data shown in ( f ). Data are shown as mean ± sd ( n = 50 cells per condition). h Percentages of apoptotic cells measured by Annexin V staining and flow cytometry. HeLa cells were treated with ITCs (20 µM) for 4 h. Data are shown as mean ± sd ( n = 4). Statistical analyses in ( e , g , h ) were performed with one-way ANOVA test (* p < 0.05, **** p < 0.0001, and ns p > 0.05).

Article Snippet: Apoptotic cells were measured with the Annexin V-FITC apoptosis detection kit (Beyotime, C1062S).

Techniques: Western Blot, Incubation, Expressing, Fluorescence, Imaging, Microscopy, Activity Assay, Flow Cytometry, Membrane, Staining

Journal: Cell Death Discovery

Article Title: Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

doi: 10.1038/s41420-024-02225-7

Figure Lengend Snippet: a Co-immunoprecipitation analysis of the effects of PEITC and cysteine mutations on the interaction between N -BID and C -BID. HEK293T cells expressing the indicated proteins were treated with PEITC (20 µM) for 4 h and lysed for anti-GFP immunoprecipitation and Western blotting detection of co-immunoprecipitated HA-tagged N -BID. b Co-immunoprecipitation analysis of the effects of cysteine mutations on the interaction between N -BID and C -BID. HEK293T cells expressing the indicated proteins were lysed for anti-GFP immunoprecipitation and Western blotting detection of co-immunoprecipitated HA-tagged N -BID. c Co-immunoprecipitation analysis of the effects of cysteine mutations on the interaction between N -BID and FL-BID. HEK293T cells expressing the indicated proteins were lysed for anti-GFP immunoprecipitation and Western blotting detection of co-immunoprecipitated HA-tagged N -BID. d Co-immunoprecipitation analysis of the effects of cysteine mutations on the interaction between Bcl-xL and FL-BID. HEK293T cells expressing the indicated proteins were lysed for anti-FLAG immunoprecipitation and Western blotting detection of co-immunoprecipitated GFP-tagged FL-BID. e Western blotting analysis of cytochrome c release from mitochondria induced by BID proteins. Recombinant BID proteins were cleaved by caspase-8, treated with or without PEITC at indicated concentrations, incubated with purified mitochondria, and centrifuged. The pellet and supernatant were analyzed for cytochrome c release. f N -terminal cysteine mutations enhance the apoptotic activity of BID. HeLa cells were transfected to express the indicated proteins and assayed for apoptotic cells using Annexin V staining and flow cytometry. Data are shown as mean ± sd ( n = 3). Statistical analysis was performed with one-way ANOVA test (*** p < 0.001, **** p < 0.0001).

Article Snippet: Apoptotic cells were measured with the Annexin V-FITC apoptosis detection kit (Beyotime, C1062S).

Techniques: Immunoprecipitation, Expressing, Western Blot, Recombinant, Incubation, Purification, Activity Assay, Transfection, Staining, Flow Cytometry

Journal: Cell Death Discovery

Article Title: Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

doi: 10.1038/s41420-024-02225-7

Figure Lengend Snippet: PEITC covalently modifies the N -terminal cysteines of BID, disrupting the interaction between the N - and C -terminal fragments of BID. This releases the pro-apoptotic C -terminal fragment from the autoinhibitory noncovalent complex to induce cytochrome c release and promote apoptosis. Simultaneously, PEITC modification exposes the BH3 domain in the C -terminal region, enabling BID to bind with Bcl-xL to suppress its anti-apoptotic activity.

Article Snippet: Apoptotic cells were measured with the Annexin V-FITC apoptosis detection kit (Beyotime, C1062S).

Techniques: Modification, Activity Assay

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Scheme 1. Schematics of the synthesized injectable and TME-modulated MTX-ss-MBGN GO hydrogels activated a cascade of orchestrating residual tumor apoptosis, restoring chemotherapy sensitivity and promoting bone regeneration for postoperative tumor-associated bone defects closed-loop management.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: Synthesized

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Fig. 3. MTX-ss-MBGN nanoparticles induced G1/S cell cycle arrest, ROS generation, and tumor apoptosis in UMR-106 osteosarcoma cells. (A) The cell cycle distribution of UMR-106 cells after different treatments for 72 h. (B) Analyzing the proportion of cell phases. (C) The mitochondrial membrane potential of UMR-106 cells treated with different nanoparticles for 24 h in JC-1 staining by CLSM observation. (D) The mitochondrial membrane potential of UMR-106 cells treated with different nanoparticles for 24 h or 48 h in JC-1 staining by flow cytometry. (E) Cell apoptosis was detected with Annexin V–FITC/PI double-staining using flow cytometry. (F) Statistical analysis of the apoptosis rate in different groups. (G) ROS detected with relative DCF fluorescence intensity as measured by flow cytometry. (H) Statistical analysis of relative DCF fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: Membrane, Staining, Flow Cytometry, Double Staining, Fluorescence

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Fig. 5. MTX-ss-MBGN GO hydrogels restrained osteosarcoma recurrence and prolonged survival days in mice by orchestrating tumor apoptosis and cell cycle exit. (A) Schematic illustrating MTX-ss-MBGN GO hydrogels treatment in a mouse model of orthotopic post-operative tumor-associated bone defects with residual tumor tissue. (B) In vivo bioluminescence imaging of K7M2 osteosarcoma tumors after surgical removal of tumors at day 21. Images of day 20 were taken before surgery. (C) Tumor growth in different groups. (D) Bodyweight changes in different groups. (E) Survival days of the mice under different treatments. (F) Histology evaluation of residual tumors under different treatments.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: In Vivo, Imaging

Journal: Frontiers in Immunology

Article Title: Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota

doi: 10.3389/fimmu.2022.927880

Figure Lengend Snippet: Recent works on marine invertebrate caspases in immunological or toxicological contexts.

Article Snippet: HEK293T cells were incubated in the presence or absence of CCCP (carbonyl cyanide 3-chlorophenylhydrazone; 100 μM), a protonophore which uncouples oxidative phosphorylation in mitochondria, at 37°C for 30 min. Then, apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit (Solarbio, China).

Techniques: Expressing, Activity Assay, In Vitro, In Vivo, Knockdown, Virus, Inhibition

Journal: Frontiers in Immunology

Article Title: Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota

doi: 10.3389/fimmu.2022.927880

Figure Lengend Snippet: Formation of cellular ROS and associated mitochondrial stress in CdCl 2 -treated coelomocytes. (A) In vitro dose-dependent effects of CdCl 2 (at 3 h) on ROS formation levels in primary coelomocytes as detected by the fluorescent DCF. Scale bar: 50 μm. (B) Quantification of DCF fluorescence intensities as reported in (A) . (C) Effects of Hl-CASP9 overexpression on apoptosis in HEK293T cells, as analyzed by flow cytometry. HEK293T cells were treated with or without CCCP (100 μM). Comparisons are made between group with vector alone (HEK293T cells transfected with pcDNA3.1(+) blank plasmid) and group overexpressing Hl-CASP9 (HEK293T cells transfected with pcDNA3.1(+)/ Hl-CASP9 recombinant plasmid). (D) Comparison on average apoptosis rates in HEK293T cells treated with or without CCCP in different groups. (E) Changes in mitochondrial membrane potential (Δ ψ m) in CdCl 2 -treated primary coelomocytes with or without dsRNA (including dsGFP and dsCASP9) as assessed by JC-1 (2.5 μM) in flow cytometry. For comparison, coelomocytes were treated with or without CCCP (100 μM) or CdCl 2 (20 μM). (F) Changes of Δ ψ m in coelomocytes in response to CdCl 2 were quantified by JC-1 intensity ratio for λ ex 543 nm/488 nm. (G) In vitro effects of CdCl 2 (20 μM) on mitochondrial superoxide formation in primary coelomocytes with or without dsRNA (including dsGFP and dsCASP9), as visualized by MitoSOX (2.5 μM) in confocal imaging. Scale bar: 15 μm. (H) Quantification of MitoSOX fluorescence intensities in coelomocytes. Data were analyzed by using ImageJ.

Article Snippet: HEK293T cells were incubated in the presence or absence of CCCP (carbonyl cyanide 3-chlorophenylhydrazone; 100 μM), a protonophore which uncouples oxidative phosphorylation in mitochondria, at 37°C for 30 min. Then, apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit (Solarbio, China).

Techniques: In Vitro, Fluorescence, Over Expression, Flow Cytometry, Plasmid Preparation, Transfection, Recombinant, Comparison, Membrane, Imaging

Journal: Frontiers in Immunology

Article Title: Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota

doi: 10.3389/fimmu.2022.927880

Figure Lengend Snippet: Coelomocyte apoptosis analysis by flow cytometry following Hl-CASP9 knock-down in vivo. (A) Coelomocytes treated with the indicated stimulants including H 2 O 2 (2 μM), NOC-18 (1 μM) or CdCl 2 (20 μM). Effects are compared among the “-dsRNA” group (sea cucumber injected of RNase-free saline solution), “+dsGFP” group (sea cucumber injected of dsGFP) and “+dsCASP9” group (sea cucumber injected of dsCASP9). (B) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to H 2 O 2 (2 μM) for 24 h in different groups. (C) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to NOC-18 (1 μM) for 24 h in different groups. (D) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to CdCl 2 (20 μM) for 24 h in different groups.

Article Snippet: HEK293T cells were incubated in the presence or absence of CCCP (carbonyl cyanide 3-chlorophenylhydrazone; 100 μM), a protonophore which uncouples oxidative phosphorylation in mitochondria, at 37°C for 30 min. Then, apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit (Solarbio, China).

Techniques: Flow Cytometry, Knockdown, In Vivo, Injection, Saline

Journal: Frontiers in Immunology

Article Title: Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota

doi: 10.3389/fimmu.2022.927880

Figure Lengend Snippet: Conceptualization of Cd-induced mitochondria-dependent apoptosis in sea cucumber coelomocytes.

Article Snippet: HEK293T cells were incubated in the presence or absence of CCCP (carbonyl cyanide 3-chlorophenylhydrazone; 100 μM), a protonophore which uncouples oxidative phosphorylation in mitochondria, at 37°C for 30 min. Then, apoptosis was detected by an Annexin V-FITC/PI cell apoptosis detection kit (Solarbio, China).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pharmacological inhibition of ABCC3 slows tumour progression in animal models of pancreatic cancer

doi: 10.1186/s13046-019-1308-7

Figure Lengend Snippet: Pharmacological inhibition of ABCC3 reduces cell proliferation through STAT3 and HIF1α dysregulation and induction of apoptosis. a Representative Western blot images show the effects of pharmacological inhibition of ABCC3 with MCI-715 on the expression of pSTAT3 Y705 and HIF1α in three PDAC cell lines (AsPC1, HPAFII, CFPAC-1). Cells were treated with MCI-715 at the concentration of 10 μM and collected after 24 h (CFPAC-1) or 48 h (AsPC1, HPAFII). The quantitative analysis of n = 3 separate experiments is presented in Additional file : Figure S2; b The effects of the treatment of AsPC1, HPAFII and CFPAC-1 cell lines with 10 μM MCI-715 on the Caspase 3/7 activity (72 h post treatment) measured with Caspase 3/7 fluorigenic probe; c Representative Western blotting images and quantitative analysis of cleaved caspase 3 expression following treatment of indicated PDAC cell lines with 10 μM MCI-715. All results are presented as mean ± SEM of 3 independent experiments. The quantitative analysis was performed with the use of ImageJ and Image Lab software, unpaired Student’s t-test was performed for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The following day cells were treated with 10 μM MCI-715 and incubated with Incucyte Caspase 3/7 fluorogenic apoptosis detection reagent (1:1000) (Essen Bioscience) according to manufacturer’s instruction and monitored for up to 72 h using IncuCyte Life Cell Analysis Imaging System (Sartorius).

Techniques: Inhibition, Western Blot, Expressing, Concentration Assay, Activity Assay, Software

Journal: STAR Protocols

Article Title: In vitro modeling of myocardial ischemia/reperfusion injury with murine or human 3D cardiac spheroids

doi: 10.1016/j.xpro.2022.101751

Figure Lengend Snippet: 3D cell death analyses of confocal images of an I/R CS using Imaris software 3D rendering analyses of confocal images of an I/R CS stained with ethidium homodimer (yellow), and with antibodies against CD31 for endothelial cells (blue), cTNT for cardiomyocytes (red) and vimentim for fibroblasts (green). (A–D) (A) shows overlay of (B) highlighting all the dead cardiomyocytes, (C) highlighting dead endothelial cells and (D) highlighting dead fibroblasts, respectively. Scale Bar: 100 μM. Reused from ( Sharma et al., 2022 ).

Article Snippet: Figure 3 3D cell death analyses of confocal images of an I/R CS using Imaris software 3D rendering analyses of confocal images of an I/R CS stained with ethidium homodimer (yellow), and with antibodies against CD31 for endothelial cells (blue), cTNT for cardiomyocytes (red) and vimentim for fibroblasts (green). (A–D) (A) shows overlay of (B) highlighting all the dead cardiomyocytes, (C) highlighting dead endothelial cells and (D) highlighting dead fibroblasts, respectively.

Techniques: Software, Staining

Journal: STAR Protocols

Article Title: In vitro modeling of myocardial ischemia/reperfusion injury with murine or human 3D cardiac spheroids

doi: 10.1016/j.xpro.2022.101751

Figure Lengend Snippet:

Article Snippet: Figure 3 3D cell death analyses of confocal images of an I/R CS using Imaris software 3D rendering analyses of confocal images of an I/R CS stained with ethidium homodimer (yellow), and with antibodies against CD31 for endothelial cells (blue), cTNT for cardiomyocytes (red) and vimentim for fibroblasts (green). (A–D) (A) shows overlay of (B) highlighting all the dead cardiomyocytes, (C) highlighting dead endothelial cells and (D) highlighting dead fibroblasts, respectively.

Techniques: Isolation, Recombinant, Saline, Plasmid Preparation, Software, Microscopy, Imaging

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: (A) Diagram showing OL morphological changes and cell death during in vitro differentiation. (B-E’) Representative micrographs of control OLs (B-C’) and TFEB cKO OLs (D-E’) that have differentiated for 2 days (B and D) and 4 days (C, C’, E, and E’) in culture. A subset of control OLs undergo programmed cell death at day 4 (C and C’), as evidenced by their fragmented cellular processes (red arrows in C) and the presence of cleaved Caspase-3 immunofluorescent signals (red arrows in C’). In contrast, TFEB cKO OLs display reduced cell death (compare E to C, and E’ to C’; quantified in F and G). (F and G) Quantification of live cell ratio (F) and cleaved Caspase-3+ cell ratio (G) in TFEB cKO and littermate control OLs. n≥6 biological replicates. (H) Representative live-imaging micrographs of littermate control (top panels) and TFEB cKO OLs (bottom panels) throughout OL differentiation (left three columns), labeled by Annexin V (red). Note that a subset of control OLs became Annexin V+ at 72 hr post differentiation, a stage when the OPCs have just differentiated into the pre-OL stage but not yet become fully mature. (I) Quantification of Annexin V+ cell area throughout OL in vitro differentiation. n=4 independent experiments with 2–12 replicates per experiment. (J-K’) Representative micrographs of OLs transfected with plasmids expressing GFP alone (J and K) or GFP-TFEB (J’ and K’) at day 1 (J and J’) and day 7 (K and K’) after transfection followed with differentiation. (L) Quantification of the ratio of GFP+ cells to the total cells following the transfection with the plasmid expressing GFP or GFP-TFEB. n ≥6 biological replicates for each condition. Error bars represent SEM. Scale bars: 100 μm in (E’) for (B)-(E’); 100 μm in (H); and 100 μm in (K’) for (J)-(K’).

Article Snippet: The “differentiation medium” was supplemented with Annexin V Red Reagent for Apoptosis (Essen Bioscience, 1:200, Cat#4641).

Techniques: In Vitro, Imaging, Labeling, Transfection, Expressing, Plasmid Preparation

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: (A) Representative live imaging micrographs of wildtype (WT; left) and PUMA−/− OLs (right) labeled by CaleinAM (green; live cells) and Annexin V (red; dead cells) 7 days after in vitro differentiation. Note that a subset of WT OLs died (yellow arrows in the left panel) whereas most PUMA−/− OLs survived (right panel). (B) Quantification of the ratio of the total live cell area (CalceinAM+) to the total cell area (CalceinAM+ and Annexin V+) in WT and PUMA−/− OLs. (C) Quantification of the total Annexin V+ cell area throughout OL differentiation. n=3 independent experiments with more than 5 replicates per experiment. (D-D” and F-F”) Representative confocal micrographs of the cerebellar ML (D-D”) and upper cortical layers (F-F”) from P14 WT (D and F), PUMA−/− (D’ and F’), and CNP-Cre; BaxF/F; Bak−/− mice (D” and F”), showing that PUMA−/− and CNP-Cre; BaxF/F; Bak−/− mutant mice exhibit ectopic MBP immunofluorescence in the cerebellar ML (D’ and D”, respectively) and cortical layer I (F’ and F”, respectively; white vertical bars demarcate layer I). (H-H”) A representative P7 sagittal brain section showing that MBP immunofluorescent signals are primarily located in the corpus callosum and deep cortical layers in WT (H; see also Figure 3A). In contrast, PUMA−/− (H’) and CNP-Cre; BaxF/F; Bak−/− mice (H”) display widespread MBP fluorescent signals precociously in many brain regions including upper cortical layers, fornix, internal capsule, and the thalamus. (E, G, and I) Quantification of the averaged MBP fluorescent intensity in P14 cerebellar ML (E), P14 cortical layer I (G), and P7 forebrain (I) from WT (black circles), PUMA−/− (red circles), and CNP-Cre; BaxF/F; Bak−/− mice (blue circles). (J) Performance of littermate control (TFEBF/F, n=9) and TFEB cKO mice (CNP-Cre; TFEBF/F, n=9) in an open-field test, showing the total distance traveled and averaged velocity through the entire 10-minute session. Left two panels show the representative movement paths of a littermate control and a TFEB cKO mouse through the entire session, respectively. (K) Performance of littermate control (TFEBF/F, n=7) and TFEB cKO (CNP-Cre; TFEBF/F, n=5) on an accelerating rotarod (4–40rpm). The averaged latency to fall off for individual trials (left) and the averaged latency to fall off for a given day (right) are plotted. *P<0.05, two-way ANOVA followed by Bonferroni’s multiple comparisons test. (L) The TFEB-PUMA-Bax/Bak axis controls the location and timing of CNS myelination. TFEB is highly expressed by pre-OLs and it facilitates programmed cell death of a subset of pre-OLs during development. TFEB induces PUMA mRNA expression, which encodes a pro-apoptotic factor that triggers Bax/Bak-dependent programmed cell death. The continuous expression of TFEB in myelinating OLs acts as a brake on OL maturation and myelination. Error bars represent SEM. Scale bars: 100 μm in (A); 100 μm in (F”) for (D)-(F”); and 1 mm in (H”) for (H)-(H”).

Article Snippet: The “differentiation medium” was supplemented with Annexin V Red Reagent for Apoptosis (Essen Bioscience, 1:200, Cat#4641).

Techniques: Imaging, Labeling, In Vitro, Mutagenesis, Immunofluorescence, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leishmania donovani Induces Autophagy in Human Blood-Derived Neutrophils.

doi: 10.4049/jimmunol.1801053

Figure Lengend Snippet: FIGURE 1. Leishmania infection induces autophagy in hPMNs. (A) Immunofluorescence imaging of LC3-II punctae in hPMNs during L. donovani infection (Inf Ld), serum starvation (Starv), and in the presence of 3-MA. Scale bar, 5 mm. (B) Western blots of cell extracts of above groups showing the levels of LC3-II at 5 h. b-Actin housekeeping gene expression was used as a loading control. Respective densitometric analysis of LC3-I/b-actin and LC3-II/b-actin has been shown as a bar graph. The densitometric analysis was done based upon three independent experiments. **p , 0.01. Underlined labeling, comparison of LC3-I level between groups; nonunderlined labeling, comparison between LC3-II level between groups. (C) Time kinetics of changes in infection-induced autophagy as detected by LC3-II punctae. Note the highest concentration at 12 h. Arrows indicates position of Leishmania inside the neutrophils. Scale bar, 5 mm. (D) Western blots of treated cell extracts showing p62 levels from 0 to 12 h postinfection along with LC3-II expression levels in the absence of autophagy inhibition by Bafilomycin A1 (Baf2). (E) Western blots of treated cell extracts showing p62 levels from 0 to 12 h postinfection in the presence of the autophagy inhibitor Bafilomycin A1 (Baf+). b-Actin was used as a loading control. (F) Blots showing phosphorylation of ATG14 (S29) at 2 and 3 h postinfection. Expression levels of ATG14 are given below. (G) Phosphorylation of AMPK-a (T172) and Beclin-1 (S93) at different time points are shown on Western blots. Respective expression levels of the proteins are given below each phosphorylation blot. b-Actin expression was used as a loading control. Results are based upon at least three independent experiments with blood samples of three healthy human volunteers.

Article Snippet: Dilutions of Abs used for Western blot analysis were as follows: LC3-II polyclonal rabbit Ab (1:2000; Novus Biologicals and CST); phospho-autophagy–related protein 14 (pATG14) (S29) rabbit mAb (1:1000); ATG 14 rabbit mAb (D1A1N) (1:1000); phospho-p42/44 MAPK (T202/Y204) (pERK1/2) rabbit mAb (D13.14.4E) (1:1000); p42/44 MAPK (ERK1/2) rabbit mAb (137F5) (1:1000); phospho-Akt (S473) (pAkt) rabbit mAb (D9E) (1:1000); pan Akt polyclonal Ab (C67E7) (1:1000) rabbit mAb; phospho-59 AMP-activated protein kinase (pAMPK-a) (T172) rabbit mAb (40H9) (1:1000); AMPK-a rabbit polyclonal Ab (23A3) (1:1000); phospho–Beclin-1 (pBeclin-1) (S93) rabbit mAb (D9A5G) (1:1000) (CST); p62 mouse Ab (1:1000) (BD Biosciences); Beclin-1 mouse Ab (1:1000) (BD Biosciences); b-actin mouse mAb (AC-15) (1:10,000) (Sigma-Aldrich); CD47 (1:500); and phospho-SAPK/JNK (pJNK) (T183/ Y185) (Thermo Fisher Scientific).

Techniques: Infection, Imaging, Western Blot, Gene Expression, Control, Labeling, Comparison, Concentration Assay, Expressing, Inhibition, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leishmania donovani Induces Autophagy in Human Blood-Derived Neutrophils.

doi: 10.4049/jimmunol.1801053

Figure Lengend Snippet: FIGURE 2. Both canonical and noncanonical forms of autophagy is triggered in response to Leishmania infection. (A) Western blots showing LC3-II levels at different hours with or without ULK1/2 inhibition using MRT68921 as ULK1/2 inhibitor. Bar graphs represent densitometric analysis of the blots. *p # 0.05, **p , 0.01, ***p , 0.001. NS, p . 0.05. (B) Photomicrographs showing concentration of LC3-II punctae at different hours postinfection with (T) or without (UT) ULK inhibition. Scale bar, 5 mm. (C) LC3-II punctae formation after starvation with or without ULK1/2 inhibition. Scale bar, 5 mm. (D) Western blots of treated cell extracts showing levels of LC3-II, pATG14, and pBeclin-1 under ULK1/2 inhibition and starvation in the presence of Bafilomycin A1. b-Actin is the loading control in all Western blots. (E) Colocalization of Rubicon–Beclin-1 after 1 h postinfection showing highest colocalization at 1 h. Scale bar, 5 mm. (F) Concentration of LC3-II punctae in infected cells treated with or without VPS34 inhibitor Vps34-IN1 (Vps34inh). Scale bar, 5 mm. (G) LC3-II expression levels shown in cell extracts from infected cells treated with or without VPS34 inhibitor Vps34-IN1. b-Actin is the loading control in all Western blots. (H) Rubicon–Beclin-1 colocalization in the presence or absence of VPS34 inhibitor Vps34-IN1. Scale bar, 5 mm. Results are based upon at least three independent experiments with three different blood donors. P.C., Pearson coefficient.

Article Snippet: Dilutions of Abs used for Western blot analysis were as follows: LC3-II polyclonal rabbit Ab (1:2000; Novus Biologicals and CST); phospho-autophagy–related protein 14 (pATG14) (S29) rabbit mAb (1:1000); ATG 14 rabbit mAb (D1A1N) (1:1000); phospho-p42/44 MAPK (T202/Y204) (pERK1/2) rabbit mAb (D13.14.4E) (1:1000); p42/44 MAPK (ERK1/2) rabbit mAb (137F5) (1:1000); phospho-Akt (S473) (pAkt) rabbit mAb (D9E) (1:1000); pan Akt polyclonal Ab (C67E7) (1:1000) rabbit mAb; phospho-59 AMP-activated protein kinase (pAMPK-a) (T172) rabbit mAb (40H9) (1:1000); AMPK-a rabbit polyclonal Ab (23A3) (1:1000); phospho–Beclin-1 (pBeclin-1) (S93) rabbit mAb (D9A5G) (1:1000) (CST); p62 mouse Ab (1:1000) (BD Biosciences); Beclin-1 mouse Ab (1:1000) (BD Biosciences); b-actin mouse mAb (AC-15) (1:10,000) (Sigma-Aldrich); CD47 (1:500); and phospho-SAPK/JNK (pJNK) (T183/ Y185) (Thermo Fisher Scientific).

Techniques: Infection, Western Blot, Inhibition, Concentration Assay, Control, Expressing

Journal: Cell reports

Article Title: A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors

doi: 10.1016/j.celrep.2021.109953

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-DR5 (Human) , Cell Signaling Technology , RRID:AB_10692107.

Techniques: Virus, Recombinant, Plasmid Preparation, Proliferation Assay, Modification, Protease Inhibitor, Transfection, Generated, Expressing, Knock-Out, Mutagenesis, Software, Imaging, Chromatography