Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: The Cnot genes maintain self-renewal by repressing early trophectoderm (TE) transcription factors. (A): Cnot1, Cnot2, and Cnot3 knockdown did not immediately affect known self-renewal factors and pathways. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) in M15 medium. Cells were collected 48 hours after transfection, and total Stat3, Smad1, b-Catenin as well as phospho-Stat3, phospho-Smad1, phosphor-b-Catenin, Oct4, and Nanog levels were determined by Western blot. Starved: control-transfected ESCs cultured in serum-free and LIF-free medium for additional 4 hours. (B): Comparing gene expression changes caused by perturbations of known self-renewal factors: Cnot1, 2, and 3 silencing induced similar changes to those of Oct4 or Sox2 silencing. Pearson's correlation coefficients were calculated between microarray datasets and depicted in a heatmap. The self-renewal factors were clustered by unsupervised hierarchical clustering based on the correlation coefficients. Microarray datasets used for this plot are listed in Supporting Information Table 2. (C): Cnot2 or Cnot3 overexpression cannot rescue Oct4 or Sox2 silencing-induced differentiation. Oct4GiP cells and Oct4GiP cells overexpressing Cnot2 (Cnot2-Rescue, same as in Fig. 1C) or Cnot3 (Cnot3-Rescue, same as in Fig. 1C) were transfected with control, Oct4 (Oct4-KD), or Sox2 (Sox2-KD) siRNAs, and the % differentiation was determined by the Oct4GiP reporter assay. (D): Cnot1, Cnot2, and Cnot3 knockdown induced TE differentiation in the presence of sustained Oct4 expression. ZHBTc4 cells that constitu-tively express Oct4 at the normal level from a Tet-Off promoter were transfected with control or Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), Cnot3-siRNA2 (Cnot3-KD), and the expression of TE markers Cdx2 and Gata3 was determined by qRT-PCR after 4 days. (E): Cdx2 deletion partially rescued Cnot1, Cnot2, and Cnot3 silencing-induced differentiation. Oct4GiP (WT) or dKO23-5 (Cdx2-/- ) cells were transfected with Control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD), and the expression of lineage markers was determined by qRT-PCR 96-hour after transfection. Abbreviations: ESC, embryonic stem cell; KD, Knockdown; WT, wild type.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Microarray, Over Expression, Reporter Assay, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Silencing Cnot1, Cnot2, or Cnot3 led to mouse embryonic stem cell (ESC) differentiation. (A): Silencing Cnot1, Cnot2, or Cnot3 resulted in ESC differentiation based on the Oct4GiP reporter assay. Oct4GiP ESCs were transfected with indicated siRNAs (two different siR NAs for each CCr4-Not complex gene) in M15 medium and cultured for 4 days. The percentage of differentiated cells (% differentiation) was determined by measuring the percentage of green fluorescent protein-negative cells by fluorescence-activated cell sorting (FACS) at the end of the culture. (B): Expression of siRNA-resistant Cnot2 or Cnot3 rescued the differentiation caused by Cnot2 or Cnot3 knockdown, respectively. Oct4GiP cells or Oct4GiP cells expressing siRNA-resistant Cnot2 (Cnot2-Rescue) or Cnot3 (Cnot3-Rescue) were transfected with Control, Cnot1-siRNA1, Cnot2-siRNA2, or Cnot3-siRNA2, and the percentage of differentiated cells was determined by the Oct4GiP reporter assays. Note that Cnot2-Rescue cells were not able to rescue the differentiation caused by Cnot1 or Cnot3 silencing, and Cnot3-Rescue cells were not able to rescue Cnot1 or Cnot2 silencing. ***, p < .001. (C): Silencing Cnot1, Cnot2, or Cnot3 resulted in morphological changes and loss of alkaline phosphatase (AP) staining in ESCs. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were stained with the AP staining kit and imaged 4 days after transfection. (D): Cnot1, Cnot2, or Cnot3 silencing led to downregulation of ESC marker and upregulation of differentiation markers. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 4 days after transfection. ESC marker: Oct4; differentiation markers: Cdx2, Eomes, Gata3, Hand1, and Krt8. (E): Cnot1, Cnot2, or Cnot3 silencing reduced cell proliferation or viability in 2i medium. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) and cul tured in 2i medium. Cell numbers were counted by FACS 4 days after transfection and normalized to control-transfected cells. (F): Cnot1, Cnot2, or Cnot3 silencing led to differentiation in 2i medium. Oct4GiP cells were transfected with indicated siRNAs and cultured in 2i medium. Cells were harvested for qRT-PCR analysis 4 days after transfection. (G): Expression of C-terminally HA-tagged Cnot2 (Cnot2-HA) in E14Tg2a cells. Expression of the exogenous Cnot2-HA was detected in Western blot with the HA-antibody, and Ran was used as a loading control. Expression of total (endogenous and exogenous) Cnot2 was determined by qPCR in wild-type E14Tg2a (E14) and Cnot2-HA expressing cells. The expression of the Cnot2-HA was estimated to be ∼2-fold of the endogenous Cnot2 on the mRNA level. (H): Identification of Cnot1 and Cnot3 in Cnot2-HA immunoprecipitation. HA-pull-down was carried out in E14Tg2a cells expressing Cnot2-HA. The presence of Cnot1, Cnot2-HA, and Cnot3 in the total lysate and pull-down sample (HA-beads) were detected by Western blot. Note that Oct4 was not detected in the pull down sample. As a negative control, protein-A beads were used in an independent pull-down. Abbreviations: HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Reporter Assay, Transfection, Cell Culture, Fluorescence, FACS, Expressing, Staining, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are required for human embryonic stem cell (ESC) self-renewal. (A): Cnot1, Cnot2, and Cnot3 were down-regulated during human ESC differentiation. H1 human ESCs were differentiated for 7 days using 100 ng/ml human recombinant BMP4. The expression levels of Cnot1, Cnot2, and Cnot3 as well as Oct4 and differentiation markers Cdx2 and Hand1 were determined by quantitative realtime PCR (qRT-PCR). (B): Silencing of Cnot1, Cnot2, or Cnot3 led to morphological changes of human ESCs. H1 cells were imaged 6 days after transfection. Phase-contrast images highlight the undifferentiated morphology of human ESCs in the lipids-only transfected cells (mock) versus the differentiated phenotype in the Cnot1, Cnot2, or Cnot3 siRNA transfected cells. (C): Silencing of the Cnot genes led to upregulation of the Cdx2 and Gata3 proteins. H1 cells were transfected with lipids-only (mock), Oct4, Cnot2, or Cnot3 siRNAs. Cells were fixed and stained for Cdx2 or Gata3 expression by immunofluorescence staining 6 days after transfection. (D): Silencing of the Cnot genes led to downregulation of the ESC marker and upregulation of the extraembryonic markers. H1 cells were harvested 6 days after transfection and marker expression was determined by qRT-PCR. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4′-6-diamidino-2-phenylindole.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: Perfluorocarbon Nanoemulsions with Fluorous Chlorin-Type Photosensitizers for Antitumor Photodynamic Therapy in Hypoxia

doi: 10.3390/ijms24097995

Figure Lengend Snippet: FC-PFC-NE accumulation in HCT116 cells determined by flow cytometry. ( A ), Time course of cell-associated fluorescence after loading with 3b-PFC-NE. Percentages of positive (drug accumulated) cells are given in parentheses as mean ± SD. ( B ), 3a-c-PFC-NE uptake at 24 h. Shown are representative experiments ( n = 3).

Article Snippet: Human breast adenocarcinoma MCF-7 and colon adenocarcinoma HCT116 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Perfluorocarbon Nanoemulsions with Fluorous Chlorin-Type Photosensitizers for Antitumor Photodynamic Therapy in Hypoxia

doi: 10.3390/ijms24097995

Figure Lengend Snippet: Distribution of 3b and 3b-PFC-NE (10 μM chlorin each) in HCT116 cells. ( A ), Colocalization with LysoTracker Green, ( B ), Colocalization with MitoTracker Green. Cells were loaded with the indicated compounds for 24 h, washed, stained with intracellular dyes and analyzed by confocal laser scanning microscopy. Fluorescence signals: green—Lyso- or MitoTracker Green, red— 3b , blue—Hoechst 33342. Bar, 5 μm.

Article Snippet: Human breast adenocarcinoma MCF-7 and colon adenocarcinoma HCT116 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining, Confocal Laser Scanning Microscopy, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Perfluorocarbon Nanoemulsions with Fluorous Chlorin-Type Photosensitizers for Antitumor Photodynamic Therapy in Hypoxia

doi: 10.3390/ijms24097995

Figure Lengend Snippet: Photoinduced cytotoxicity (IC 50 , μM) in human cell lines.

Article Snippet: Human breast adenocarcinoma MCF-7 and colon adenocarcinoma HCT116 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Perfluorocarbon Nanoemulsions with Fluorous Chlorin-Type Photosensitizers for Antitumor Photodynamic Therapy in Hypoxia

doi: 10.3390/ijms24097995

Figure Lengend Snippet: Efficacy to trigger photonecrosis depends on oxygen content and PS formulation. The HCT116 cells were loaded with indicated compounds (each 10 µM chlorin) for 24 h. BODIPY™ 581/591 C11 (yellow—reduced form, magenta—oxidized form), Hoechst 33342 (blue, cyan) and PI (red) were added, and cells were illuminated with a light source (405 nm). Top panel : normoxia. ( A ), lipid peroxidation; ( B ), PI fluorescence. Bottom panel : PI fluorescence in hypoxic cells. ( C ), prolonged; ( D ), acute. Bar, 5 μm.

Article Snippet: Human breast adenocarcinoma MCF-7 and colon adenocarcinoma HCT116 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Formulation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

doi: 10.3390/ijms241713589

Figure Lengend Snippet: Activation of CRSTAL by ectopic GZMB expression. ( a ) Structure of the constructs of full-length GZMB containing the inactivation peptide GE as well as the mutated version lacking GE (GZMBΔGE); SP signal peptide. ( b ) CRSTAL-293T cells were transfected with pGZMB, pGZMBΔGE or a mock plasmid. CRSTAL fluorescence images and transmitted light (TL) images were acquired and analysed using the ImageXpress ® Pico instrument and Cell Reporter express software 2.9.

Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC ® CRL-3216TM, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS; 10270106, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL gentamicin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany), 2 mM GlutaMAXTM (35050061, Thermo Fisher Scientific) and 25 mM HEPES (15630080, Thermo Fisher Scientific).

Techniques: Activation Assay, Expressing, Construct, Transfection, Plasmid Preparation, Fluorescence, Software

Journal: International Journal of Molecular Sciences

Article Title: A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

doi: 10.3390/ijms241713589

Figure Lengend Snippet: General functionality test of CRSTAL. CRSTAL-293T-iGZMBΔGE were seeded and treated with doxycycline for 48 h. The cells were either ( a ) analysed via fluorescence microscopy or ( b ) lysed and analysed via Western blot.

Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC ® CRL-3216TM, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS; 10270106, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL gentamicin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany), 2 mM GlutaMAXTM (35050061, Thermo Fisher Scientific) and 25 mM HEPES (15630080, Thermo Fisher Scientific).

Techniques: Fluorescence, Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

doi: 10.3390/ijms241713589

Figure Lengend Snippet: Time course of CRSTAL fluorescence. ( a ) CRSTAL-293T-iGZMBΔGE cells were seeded and induced with 100 ng/mL doxycycline every 12 h for the last time points, then every 2 h for the time points 0–36 h. Fluorescence was measured by flow cytometry. ( b ) CRSTAL-293T-iGZMBΔGE cells were induced with 100 ng/mL doxycycline in steps of 2 h over 24 h. Cells were lysed at 24 h and analysed via Western blot.

Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC ® CRL-3216TM, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS; 10270106, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL gentamicin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany), 2 mM GlutaMAXTM (35050061, Thermo Fisher Scientific) and 25 mM HEPES (15630080, Thermo Fisher Scientific).

Techniques: Fluorescence, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

doi: 10.3390/ijms241713589

Figure Lengend Snippet: Caspase-8-independent activation of CRSTAL. ( a ) Design of the inducible, self-cleaving variant of caspase-8 (iCasp8FT) based on the sequence of pro-caspase-8. ( b , c ) Expression of active caspase-8 or GZMB was induced in CRSTAL-293T-iCasp8FT or CRSTAL-293T-iGZMBΔGE via treatment with 1 µg/mL doxycycline in presence or absence of 100 µM Z-VAD-fmk. Cells were analysed 48 h post induction ( b ) via flow cytometry or ( c ) via Western blot. Statistics: Student’s t -test; **: p < 0.01; ***: p < 0.005; ****: p < 0.001; n.s.: not significant. Detailed flow cytometry information is depicted in .

Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC ® CRL-3216TM, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS; 10270106, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL gentamicin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany), 2 mM GlutaMAXTM (35050061, Thermo Fisher Scientific) and 25 mM HEPES (15630080, Thermo Fisher Scientific).

Techniques: Activation Assay, Variant Assay, Sequencing, Expressing, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity

doi: 10.3390/ijms241713589

Figure Lengend Snippet: CAR-T cell killing assay on CRSTAL-expressing target cells. CRSTAL-293T-CD19 cells were seeded and co-incubated with CD19FBBz-CTL or CAR-negative CTL at effector-to-target ratios of ( a , b ) 5:1 or © 3:1 for 48 h. Cells were stained and analysed ( a ) via flow cytometry, ( b ) analysed via ImageXpress ® Pico, or ( c ) via confocal laser scanning microscopy. Statistics: Student’s t -test; *: p <0.05; **: p < 0.01. Detailed flow cytometry information is depicted in .

Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC ® CRL-3216TM, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; 11500516, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS; 10270106, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL gentamicin (1405-41-0, Serva Electrophoresis, Heidelberg, Germany), 2 mM GlutaMAXTM (35050061, Thermo Fisher Scientific) and 25 mM HEPES (15630080, Thermo Fisher Scientific).

Techniques: Expressing, Incubation, Staining, Flow Cytometry, Confocal Laser Scanning Microscopy

Journal: PLoS Pathogens

Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

doi: 10.1371/journal.ppat.1005860

Figure Lengend Snippet: eGFP-PLK-expressing constructs alone (left panels) or a combination of eGFP or eGFP-PLK and Gag-mCherry encoding expression constructs (right panels) were transfected into 293T cells, as indicated above each panel of images. Forty-eight hours post-transfection, protein localization patterns were examined in fixed cells by confocal laser scanning microscopy (CLSM). Channels of the individual fluorescence micrographs are indicated on top, and the PLK variant used is indicated on the left. White arrowheads indicate fluorescent PLK foci presumed to be centrosomes. Data are representative of n = 5 independent experiments. (A) Localization patterns of eGFP-tagged PLK proteins (detected in eGFP-PLK channel) in mitotic cells transfected with the corresponding constructs. (B) Localization patterns of eGFP-tagged PLK and mCherry-tagged Gag proteins detected in corresponding channels (Gag variant used labeled either as wt-mCh or T225A-mCh) in mitotic cells. (C) Localization of eGFP and wt Gag-mCherry in mitotic cells. (D) Localization patterns of eGFP-tagged PLK proteins (detected in eGFP-PLK channel) in interphase cells transfected with the corresponding constructs. (E) Localization patterns of eGFP-tagged PLK and mCherry-tagged Gag proteins detected in corresponding channels (Gag variant used labeled either as wt-mCh or T225A-mCh) in interphase cells. Scale bar: 10 μm.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573) [ ], the human epithelium HeLa cell line (ATCC CCL-2) [ ], the human primary fibroblast MRC-5 cell line (ATCC CCL171), immortalized mouse primary embryonic fibroblasts (obtained from M. Trilling, Essen, Germany) and the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [ ] as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR driven EGFP reporter gene expression cassette [ ] were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Expressing, Construct, Transfection, Confocal Laser Scanning Microscopy, Fluorescence, Variant Assay, Labeling

Journal: PLoS Pathogens

Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

doi: 10.1371/journal.ppat.1005860

Figure Lengend Snippet: PFV virions were produced by transient transfection of the four-component PFV vector system, containing the pcoPG4 variants denoted above each of the blots, into 293T cells. Viral particles were pelleted from cell-free tissue culture supernatants by ultracentrifugation through 20% sucrose and equal amounts of particle lysates separated by SDS-PAGE were blotted to nitrocellulose membranes. The phosphorylation status of the particle-associated Gag variants was determined by the corresponding antibodies specific for different phosphorylated amino acid motives as indicated. Data are representative of n = 4 independent experiments. (A) Schematic illustration of PFV Gag protein organization with highlighted putative T-P motifs and surrounding amino acids recognized when phosphorylated at the threonine residue by either the α-pT-P (orange letters) or α-Gag S-pT-P (blue letters) phosphopeptide-specific antibody. Solid vertical arrow: primary Gag processing site; dashed vertical arrows: secondary Gag processing sites; (B) Detection of the phosphorylated Thr-Pro (pT-P) motives in PFV Gag wt, iSTP and pmSTP virus particles (α-pT-P antiserum) and comparison with the total Gag content in the particle lysates (α-Gag). (C) Comparison of the pT-P phosphorylation status in the wt and T225A PFV particles in the absence (-) or presence (+) of the Lambda Phosphatase (λP) pretreatment of viral proteins. (D) Comparison of the PFV Gag-associated S-T-P motif phosphorylation status (α-Gag S-pT-P; detected with the corresponding PFV Gag-specific antibody) in virus preparations containing either the wt or the T225A Gag variant in the absence (-) or presence (+) of the λP pretreatment.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573) [ ], the human epithelium HeLa cell line (ATCC CCL-2) [ ], the human primary fibroblast MRC-5 cell line (ATCC CCL171), immortalized mouse primary embryonic fibroblasts (obtained from M. Trilling, Essen, Germany) and the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [ ] as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR driven EGFP reporter gene expression cassette [ ] were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Produced, Transfection, Plasmid Preparation, SDS Page, Phospho-proteomics, Residue, Virus, Comparison, Variant Assay

Journal: PLoS Pathogens

Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

doi: 10.1371/journal.ppat.1005860

Figure Lengend Snippet: (A) PFV virions were produced by transient transfection of 293T cells with the four-component PFV vector system, containing either the wt Gag or one of the denoted iSTP- and pmSTP Gag variants. Titers of harvested viruses were determined by flow cytometry analysis of infected HT1080 target cells three days post-infection. The mean values and standard deviation for each supernatant were calculated from samples of cells infected with serial virus dilutions as described in Material and Methods. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except mock) from independent experiments (n = 4–9) are shown. Differences between means of wt virus and the individual mutants were analyzed by Welch’s t test (**, p<0.01). Absolute titers of wt supernatants ranged between 1.2 x 10 6 and 1.2 x 10 7 eGFP ffu/ml. (B) Replication-competent PFV virions were produced by transient transfection of proviral expression vectors, containing either the wt Gag or one of the denoted iSTP- and pmSTP Gag variants into 293T cells. Viruses were harvested two days post-transfection and used to infect HT1080 PLNE target cells. Titers were determined by flow cytometry analysis one day post-infection. The values obtained using wt PFV Gag expression plasmids were arbitrarily set to 100%. Relative means and standard deviations normalized for Gag content (except mock) from independent experiments (n = 3–8) are shown. Differences between means of wt virus and the individual mutants were analyzed by Welch’s t test (**, p<0.01). Absolute titers of wt supernatants ranged between 1.7 x 10 4 and 7 x 10 4 eGFP ffu/ml. (C) Titers of iSTP- and pmSTP mutant PFV particles relative to wt over multiple rounds of target cell infection. Viruses were produced and harvested as described in panel B and Gag content normalized amounts of viral supernatants were used to infect HT1080 PLNE in serial dilutions. At different time points post-infection (as indicated on the x-axis) cells were harvested for flow cytometry analysis to determine viral titers. The values obtained using wt PFV supernatants at each time point were arbitrarily set to 100%. Relative means and standard deviations from two independent experiments are shown.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573) [ ], the human epithelium HeLa cell line (ATCC CCL-2) [ ], the human primary fibroblast MRC-5 cell line (ATCC CCL171), immortalized mouse primary embryonic fibroblasts (obtained from M. Trilling, Essen, Germany) and the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [ ] as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR driven EGFP reporter gene expression cassette [ ] were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Produced, Transfection, Plasmid Preparation, Flow Cytometry, Infection, Standard Deviation, Virus, Expressing, Mutagenesis

Journal: PLoS Pathogens

Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

doi: 10.1371/journal.ppat.1005860

Figure Lengend Snippet: Replication-competent PFV virions were produced by transient transfection of proviral expression constructs, containing either the wt Gag (wt) or one of the denoted iSTP- (S224A, T225A, P226A) or pmSTP (T225E) Gag variants into 293T cells. As controls, constructs containing wt Gag in combination with Pol with enzymatically inactive reverse transcriptase (iRT) or inactive integrase (iIN) domain, as well as constructs harboring translationally inactivated ORFs for Gag and Pol (ΔGag/Pol) or Env (ΔEnv) were used. The mock control (mock) included cells transfected with pUC19 alone. (A) Representative Western blot analysis of viral particles (virus) purified from 293T cell culture supernatant by ultracentrifugation through 20% sucrose and 293T cell lysates (cell). PFV proteins were detected using polyclonal antibodies specific for PFV Gag (α-Gag) or PFV Env LP (α-LP), a mixture of hybridoma supernatants specific for PFV Pol PR/RT and IN (α-PR/RT + α-IN), or a commercial monoclonal antibody specific for GAPDH (α-GAPDH). Serial dilutions of the wt samples (wt; lanes 6–9) were quantified to determine their relative protein contents compared to other samples. The identity of the individual proteins detected is indicated on the right. (B) Viral particle release was determined by quantitative Western blot analysis of viral particle lysates. Mean values and standard deviations (n = 3) are shown as relative values compared to the wt control and normalized for cellular expression levels. (C) Quantification of PFV vgRNA in virus producing cells (vgRNA cell) and released particles (vgRNA virus) and particle-associated vgDNA (vgDNA virus). Mean values and standard deviation (n = 3–8) are shown as relative values compared to the wt control. Cellular values were normalized to GAPDH levels, viral particle values were normalized for Gag content.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573) [ ], the human epithelium HeLa cell line (ATCC CCL-2) [ ], the human primary fibroblast MRC-5 cell line (ATCC CCL171), immortalized mouse primary embryonic fibroblasts (obtained from M. Trilling, Essen, Germany) and the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [ ] as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR driven EGFP reporter gene expression cassette [ ] were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Produced, Transfection, Expressing, Construct, Reverse Transcription, Control, Western Blot, Virus, Purification, Cell Culture, Standard Deviation

Journal: PLoS Pathogens

Article Title: Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

doi: 10.1371/journal.ppat.1005860

Figure Lengend Snippet: Replication-deficient PFV supernatants were harvested after transient transfection of the PFV four-component vector system, containing the eGFP-tagged Gag p68 wt (wt) or T225A (T225A) variants in combination with wt (wt) or fusion-incompetent Env (iFuse) into 293T cells. HT1080 cells were synchronously infected with undiluted (undil.) and ten-fold diluted (1:10) PFV supernatants and eGFP MFI values were measured by flow cytometry at indicated time points until 24 h post-transduction. Representative data shown are from one out of three independent experiments.

Article Snippet: The human embryonic kidney cell line 293T (ATCC CRL-1573) [ ], the human epithelium HeLa cell line (ATCC CCL-2) [ ], the human primary fibroblast MRC-5 cell line (ATCC CCL171), immortalized mouse primary embryonic fibroblasts (obtained from M. Trilling, Essen, Germany) and the human fibrosarcoma cell line HT1080 (ATCC CCL-121) [ ] as well as the clonal variant HT1080 PLNE thereof containing a PFV LTR driven EGFP reporter gene expression cassette [ ] were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics.

Techniques: Transfection, Plasmid Preparation, Infection, Flow Cytometry, Transduction

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 expression is upregulated in both sepsis and THP-1-derived macrophages upon LPS + INF-γ stimulation. A PD-L1 expression in septic patients was analyzed using datasets GES134347 and GSE65682 retrieved from the GEO database. Statistical significance was determined by the Wilcoxon test. * p < 0.05. B THP-1-derived macrophages (THP-1-M) were co-stimulated with LPS (100 ng/ml) and IFN-γ (20 ng/ml) for 12–24 h, followed by quantification of PD-L1 mRNA levels via qRT-PCR ( n = 3 independent biological replicates). C PD-L1 protein expression in THP-1-M after LPS + IFN-γ stimulation for 6, 12, or 24 h was assessed by Western blotting. D Flow cytometry analysis of PD-L1 protein expression in THP-1-M treated with LPS + IFN-γ for 24 h. E Subcellular localization of PD-L1 in THP-1-M following 12 h LPS + IFN-γ stimulation was visualized by confocal laser scanning microscopy. The relative fluorescence intensity and Pearson’s R value were analyzed by ImageJ ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B was performed by one-way ANOVA, and that for E was determined by Student’s t-test. * p < 0.05

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Confocal Laser Scanning Microscopy, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 expression positively correlates with proinflammatory markers in M1-polarized macrophages. (A-C) PD-L1-overexpressing (PD-L1 HI ) and control (PD-L1 NC ) THP-1-M cells were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h followed by RNA sequencing. A Heatmap of inflammatory gene expression in PD-L1 HI vs. PD-L1 NC cells. B MA plot showing differential gene expression between PD-L1 HI and PD-L1 NC cells. C TPM expression values of IL-6, IL-27 and NOS2. D - E PD-L1 HI and PD-L1 NC cells were stimulated with LPS + IFN-γ for 24 h. ( n = 3 independent biological replicates) ( D ) qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). E ELISA for IL-6/IL-27 and Griess reagent assay for NO in supernatants ( n = 3 independent biological replicates). (F-G) PD-L1-knockout THP-1-M (PD-L1 KO /THP-1-M) and PD-L1 NC /THP-1-M were stimulated with LPS + IFN-γ for 24 h. F qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). G ELISA and Griess reagent detection of IL-6/IL-27 proteins and NO in supernatants ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C, D, E, F and G was performed by Student’s t-test. * p < 0.05

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Control, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Bioinformatics analysis integrating RNA sequencing data and GEO datasets demonstrated PD-L1-mediated significant activation of the NOD-like receptor signaling pathway. A PD-L1 NC /THP-1-M cells were co-stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h followed by RNA sequencing, with subsequent KEGG pathway analysis of activated signaling pathways. B - C PD-L1-centered single-gene GSEA across datasets GSE57065 , GSE28750 and GSE95233 . B Ridge plot displaying top 20 activated pathways. C GSEA enrichment plot illustrating NOD-like receptor signaling pathway activation. D Comparative PD-L1-specific GSEA of NOD-like receptor signaling pathway activation in PD-L1 HI /THP-1-M vs. PD-L1 NC /THP-1-M cells following 12 h LPS + IFN-γ co-stimulation with RNA sequencing verification

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: RNA Sequencing, Activation Assay, Protein-Protein interactions

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Multi-omics integration identified AIM2 as the critical mediator for PD-L1-regulated macrophage M1 polarization. A - D PD-L1 NC /THP-1-M cells were co-stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h for PD-L1-targeted IP-MS analysis. A Global proteomic landscape showing peptide/protein distribution and Venn intersections between PBS control vs. LPS + IFN-γ groups. B Triple-dataset intersection analysis integrating IP-MS, RNA-seq and GEO datasets ( GSE57065 , GSE28750 , GSE95233 ) for core candidate screening. C GO enrichment of 208 unique interacting proteins in LPS + IFN-γ group with inflammatory pathway emphasis. D Representative tandem MS/MS spectra identifying AIM2. E Co-IP validation of PD-L1-AIM2 interaction post 12 h LPS + IFN-γ co-stimulation in PD-L1 NC /THP-1-M cells ( F ) Differential AIM2 expression in PD-L1 HI /THP-1-M vs. PD-L1 NC /THP-1-M cells by MA plot (left) and TPM expression profile (right) following 12 h LPS + IFN-γ stimulation ( n = 3 independent biological replicates). G Temporal AIM2 regulation: qRT-PCR analysis at 6/12/24 h (left, n = 3 independent biological replicates) with Western blot confirmation at 24 h (right). H - J PD-L1-AIM2 structural docking studies. H AlphaFold-predicted full-length molecular models of PD-L1 and AIM2. I Top 5 scoring complexes from GRAMM docking server simulations. J High-resolution interface visualization of optimal complex with potential interaction sites. The data are presented as the mean ± SEM. Statistical analysis for F was performed by Student’s t-test, and that for G was determined by one-way ANOVA. * p < 0.05

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Biomarker Discovery, Protein-Protein interactions, Control, RNA Sequencing, Tandem Mass Spectroscopy, Co-Immunoprecipitation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 overexpression potentiates AIM2-mediated downstream effector (IL-18/IFN-γ) production and amplifies STAT1 activation cascade. A PD-L1 HI /THP-1-M vs. PD-L1 NC /THP-1-M cells were co-stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h. RNA-seq-derived TPM profiles of IL-1β (left) and IL-18 (middle), with MA plot visualization of IL-18 differential expression (right) ( n = 3 independent biological replicates). B Dual-modality verification of IL-18 regulation: qRT-PCR analysis (left) with paired ELISA quantification (right) post 12 h co-stimulation ( n = 3 independent biological replicates). C Temporal dynamics of IFN-γ regulation: mRNA/protein measurement by qRT-PCR (left) and ELISA (right) at 24 h endpoint ( n = 3 independent biological replicates). D Western blot detection of STAT1 phosphorylation status and basal expression following 24 h LPS + IFN-γ co-stimulation. E - G PD-L1-specific single-gene GSEA analysis across GEO datasets ( GSE57065 , GSE28750 , and GSE95233 ). E Identification of 111 molecules positively correlated with PD-L1 expression. F MCODE algorithm-based core module identification in Cytoscape (40 hub molecules). G GO enrichment analysis of the 40-molecule module revealing JAK-STAT pathway activation. The data are presented as the mean ± SEM. Statistical analysis for A, B and C was performed by Student’s t-test. * p < 0.05

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Activation Assay, RNA Sequencing, Derivative Assay, Quantitative Proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Triple-verification strategy (gene silencing/antibody neutralization/site-directed mutagenesis) confirms regulatory hierarchy of PD-L1/AIM2/IL-18/STAT1 signaling axis in macrophage M1 polarization. A PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were pretreated with AIM2-targeting siRNA or scramble siRNA (scr siRNA) prior to 24 h LPS (100 ng/ml) + IFN-γ (20 ng/ml) co-stimulation, with subsequent Western blotting analysis of STAT1 phosphorylation. B - C Anti-IL-18 neutralizing antibody (1 µg/ml) and isotype control (1 µg/ml) were pretreated for 24 h followed by LPS + IFN-γ 24 h co-stimulation. B qRT-PCR quantification of IFN-γ mRNA ( n = 3 independent biological replicates). C STAT1 phosphorylation profiling by Western blotting. D Truncation mutant validation: LPS + IFN-γ-stimulated (24 h) PD-L1 Δ35–90 cells analyzed for STAT1 activation vs. full length type. E Following AIM2 silencing in PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells, qRT-PCR quantification of IL-6, IL-27 and NOS2 mRNA after 24 h LPS + IFN-γ stimulation ( n = 3 independent biological replicates). F Post IL-18 neutralization, mRNA levels of IL-6, IL-27 and NOS2 were assessed by qRT-PCR under LPS + IFN-γ 24 h stimulation ( n = 3 independent biological replicates). G STAT1-silenced cells were analyzed for IL-6/IL-27/NOS2 transcriptional changes post 24 h LPS + IFN-γ exposure ( n = 3 independent biological replicates). H Truncated PD-L1 Δ35–90 mutants were evaluated for IL-6, IL-27 and NOS2 mRNA expression following LPS + IFN-γ 24 h challenge ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B, E, F, G and H was performed by Student’s t-test. * p < 0.05

Article Snippet: The human monocytic wild-type THP-1 cell line (PD-L1 WT ) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Neutralization, Mutagenesis, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Biomarker Discovery, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: PLGA nanoparticles co-delivering MDR1 and BCL2 siRNA for overcoming resistance of paclitaxel and cisplatin in recurrent or advanced ovarian cancer

doi: 10.1038/s41598-018-25930-7

Figure Lengend Snippet: Efficient cellular internalization of siRNA@PLGA NPs on MDR ovarian cancer cells. ( A ) Flow cytometric histogram showing cellular internalization of the fluorescent siRNA@PLGA NPs on SKOV3-TR and A2780-CP20 cancer cells respectively. The resistance cell lines were treated with fluorescent Cy5.5-siRNA@PLGA NPs, and 24 h later, sorted. A prefixed gate region was allowed only for Cy5.5 fluorescence. The untreated cells are indicated as a control. ( B ) Z-stack confocal microscopic images of SKOV3-TR and A2780-CP20 cancer cells treated with Cy5.5-labeled siRNA@PLGA NPs. Cy5.5-siRNA and DAPI dyes are represented by red and blue, respectively. Scale bar: 10 μm.

Article Snippet: For flow cytometry analysis, each cell lines treated with the fluorescent Cy5.5-siRNA-loaded PLGA NPs were examined by Guava EasyCyte flow cytometer (Merck Millipore) equipped with 640 nm laser.

Techniques: Fluorescence, Labeling

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wogonin attenuates the pathogenicity of Streptococcus pneumoniae by double‐target inhibition of Pneumolysin and Sortase A

doi: 10.1111/jcmm.17684

Figure Lengend Snippet: Toxic side effects of wogonin on host cells. A549 (A) and J774 cells (B) were cocultured with different concentrations of wogonin, and LDH release into the supernatant was quantified. Each column represents replicates ( n = 3), and error bars represent standard errors.

Article Snippet: A549 cells (human lung epithelial cells, purchased from ATCC, USA) were cultured in complete medium consisting of DMEM supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 after inoculation in 24‐well plates with 5 × 10 4 cells per well for an overnight culture.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Wogonin attenuates the pathogenicity of Streptococcus pneumoniae by double‐target inhibition of Pneumolysin and Sortase A

doi: 10.1111/jcmm.17684

Figure Lengend Snippet: Wogonin inhibits S. pneumoniae adhesion and colonization and neutralizes PLY‐mediated injury in A549 cells. (A,B) Wogonin (0, 16, 32, 64 or 128 μg/mL) was added to the S. pneumoniae D39 culture system, and the numbers of S. pneumoniae D39 and S. pneumoniae E1 strains adhering to A549 cells were measured at 3 and 6 h. (C) PLY‐treated A549 cells. Green and red fluorescence read‐outs were imaged using a confocal laser scanning microscope. (D–G) Wogonin was added at concentrations of 8, 16, 32 and 64 μg/mL and incubated with PLY‐treated A549 cells. (H) LDH release by A549 cells in the presence of various concentrations of wogonin. Each column represents replicates ( n = 3), and error bars represent standard errors. *Represents p < 0.05 and **indicates p < 0.01.

Article Snippet: A549 cells (human lung epithelial cells, purchased from ATCC, USA) were cultured in complete medium consisting of DMEM supplemented with 10% fetal bovine serum at 37°C with 5% CO 2 after inoculation in 24‐well plates with 5 × 10 4 cells per well for an overnight culture.

Techniques: Fluorescence, Laser-Scanning Microscopy, Incubation

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) Confocal laser scanning microscopy (CLSM) analyses of CXCR4 (green fluorescence) and PC-PLC (red fluorescence) localization on plasma membrane of unfixed cells. Scale bars, 18μm. B) Western blot (WB) of proteins isolated from U87MG cells by immunoprecipitation with anti-CXCR4 Ab (IP-α-CXCR4). Top panels show IP-α-CXCR4 preparations blotted with anti-PC-PLC Ab compared to control (CTRL IgG) (left) and total cell lysate (right). *IgG heavy chains. The bottom panels represent an α-CXCR4-IP preparation blotted with anti-CXCR4 Ab compared with CTRL IgG (left) and total lysate (right). C) CLSM analyses of CXCR4 (green fluorescence) and PC-PLC localization (red fluorescence) on plasma membrane of unfixed U87MG cells following the respective treatments (indicated on the left of the panel rows). Images represent untreated cells (CTRL) and cells exposed to either D609 or Plerixafor at different time-points (24h, 48h, 72h). Scale bars, 16 μm.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Confocal Laser Scanning Microscopy, Fluorescence, Clinical Proteomics, Membrane, Western Blot, Isolation, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: Representative WB of CXCR4 and PC-PLC detection in U87MG cells processed after 24h, 48h and 72h of treatment with either D609 or Plerixafor. β-actin was used as loading control. The histograms represent the mean value ± SD of relative fold change of CXCR4 (on the left) and PC-PLC (on the right) optical density (OD) normalized to β-actin, obtained by densitometric analyses of WB bands (Image J software). CTRL values normalized to 1 at each time point; n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA *** P = 0.0008 (CXCR4 expression), ***P = 0.0008 (PC-PLC expression).

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Control, Software, Expressing

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) U87MG cell proliferation after 24h, 48h, 72h of treatments with D609 or Plerixafor evaluated by cell counting using the Trypan blue excluding assay. Curves represent mean values ± SD of U87MG cell counts at different time-points (n = eight independent experiments). ● CTRL; ■ (dark grey square) D6099; and ▲ (light grey triangle) Plerixafor. Statistical analyses were performed with two-tailed unpaired t Student’s test. CTRL versus D609: 24h * P = 0.05; 48h *** P = 0.0001; 72h ** P = 0.0005. B) U87MG cell invasion evaluated by transwell chamber assay using Matrigel, in response to D609 or Plerixafor 24h treatment. The histograms represent the percentage of the area occupied by U87MG cells. CTRL values = 100%. Mean values ± SD (n = 6). Statistical analyses were performed with one-way ANOVA. ***P = 0.0001. C) Representative WB of p-EGFR and total EGFR expression in U87MG cells either untreated (CTRL), or treated with D609 or with Plerixafor for 24h, 48h and 72h. β-actin was used as loading control. The histograms represent the mean values of relative fold changes of p-EGFR and EGFR optical density normalized to β-actin, obtained with densitometric analyses of the WB protein bands (Image J software). CTRL values = 1. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA * P = 0.0333 (p-EGFR); * P = 0.0141 (EGFR). D) Representative WB of p-AKT and AKT expression in U87MG control cells (CTRL) and in cells treated with either D609 or Plerixafor for 24h, 48h and 72h. β-actin was used as loading control. Histogram represents the mean of relative fold change of p-AKT and AKT optical density normalized to β-actin, obtained with densitometric analyses of WB protein bands (Image J software). CTRL values = 1. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with one-way ANOVA * P = 0.0230 (p-AKT); * P = 0.0165 (AKT).

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Cell Counting, Two Tailed Test, Transwell Chamber Assay, Expressing, Control, Software

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: The histograms represent the means ± SD of percentage values obtained from quantitative 1 HMRS analysis of each metabolite/percentage of total metabolites content in U87MG untreated (CTRL), treated with D609 or Plerixafor at 24h (A), 48h (B), 72h (C) of treatment. Metabolites reported are the compounds that mainly contribute to the U87MG metabolic profile: Gly, glycine; GPC+PCho, glycerophosphocholine+phosphocholine; Cho, choline; tCr, total creatine (creatine+phosphocreatine); Glu, glutamate; Ala, alanine; Lac, Lactate. Total metabolites = 100%. Means ± SD of n = 3 independent experiments. Statistical analyses were performed with two-tailed unpaired t Student’s test. CTRL versus D609: 24h GPC+PCho **P = 0.0096, Lac *P = 0.0170; 72h GPC+PCho *P = 0.0286, Lac *P = 0.047.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Two Tailed Test

Journal: PLoS ONE

Article Title: Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells

doi: 10.1371/journal.pone.0176108

Figure Lengend Snippet: A) Histogram represents mean values of percentage of intracellular Lac concentration in U87MG obtained colorimetric assay after 24h, 48h, 72h of treatments. Mean value ± SD of n = 3 independent experiments. Statistical analyses were performed using one-way ANOVA. ***P = 0.0003. B) Histogram represents mean ± SD of percentage values (n = 3 independent experiments) of LDH intracellular activity measured by colorimetric assay at different treatment time-points (24h, 48h, 72h). Statistical analyses were performed using one-way ANOVA. ***P = 0.0006; unpaired two-tailed Student-t test CTRL 72h VS D609 72h P = 0.004. C) Histogram represents mean ± SD of percentage values of Lac extracellular concentration measured by a colorimetric assay on U87MG-conditioned medium in response to treatments (24h-48h-72h) (n = 3 independent experiments). All values were normalized for μg of proteins. CTRL values = 100%.

Article Snippet: The human glioblastoma multiforme (GBM) cell line U87MG, purchased from American Type Culture and Collection (ATCC, Manassas, VA, USA) was cultured in MEM at 5% CO2 and 95% humidified atmosphere air at 37°C.

Techniques: Concentration Assay, Colorimetric Assay, Activity Assay, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: IR783-Stabilized Nanodrugs Enhance Anticancer Immune Response by Synergizing Oxidation Therapy and Epigenetic Modulation.

doi: 10.1002/advs.202415684

Figure Lengend Snippet: Figure 2. Preparation and characterization of IR/Lap/CUDC NPs. A) Size distribution and transmission electron microscopy (TEM) image of IR/Lap/CUDC NPs (Scale bar: 200 nm). B) Zeta potential of IR/Lap/CUDC NPs. C) UV–vis absorption spectra of free 𝛽-lap, CUDC101, IR783, and IR/Lap/CUDC NPs. D) Stability test of IR/Lap/CUDC NPs under physiological conditions for 48 h. E) Size distribution changes of IR/Lap/CUDC NPs after H2O2 incubation. F) Fluorescent spectra of IR/Lap/CUDC NPs with or without H2O2 incubation. G) UV–vis absorption spectra of IR/Lap/CUDC NPs with or without H2O2 incubation. H) Cell viability of 4T1 cells treated with gradient concentrations of free 𝛽-lap and CUDC101. I) Synergy score of free 𝛽-lap and CUDC101 in 4T1 cells at gradient concentrations calculated by the Highest Single Agent (HSA) model in SynergyFinder. "NPs" shown in (E), (F), and (G) refers to IR/Lap/CUDC NPs.

Article Snippet: Cell Culture: The human triple-negative breast cancer cell line (MDAMB-231), mouse macrophage-like cell line (RAW264.7), and mouse breast cancer cell line (4T1) were purchased from ATCC.

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Incubation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: IR783-Stabilized Nanodrugs Enhance Anticancer Immune Response by Synergizing Oxidation Therapy and Epigenetic Modulation.

doi: 10.1002/advs.202415684

Figure Lengend Snippet: Figure 3. Cytotoxicity and ROS generation ability of IR/Lap/CUDC NPs. A) Cell viability of 4T1 cells treated with different formulations. B) Lactate dehydrogenase (LDH) release level of 4T1 cells after different treatments. C) Cellular uptake level of IR/Lap/CUDC NPs in 4T1 cells with different endocytosis inhibitors. D) Live and dead staining of 4T1 cells treated with different formulations (Scale bar: 200 μm). E) Apoptosis level of 4T1 cells treated with different formulations. F) Quantitative analysis of the apoptosis assay. G) Flowcytometric analysis of ROS generation in 4T1 cells. H) Representative confocal laser scanning microscopy (CLSM) images of ROS in 4T1 cells after different treatments (Scale bar: 50 μm; Grid side length in Z-stack: 30 μm). Data were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns (not significant).

Article Snippet: Cell Culture: The human triple-negative breast cancer cell line (MDAMB-231), mouse macrophage-like cell line (RAW264.7), and mouse breast cancer cell line (4T1) were purchased from ATCC.

Techniques: Staining, Apoptosis Assay, Confocal Laser Scanning Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: IR783-Stabilized Nanodrugs Enhance Anticancer Immune Response by Synergizing Oxidation Therapy and Epigenetic Modulation.

doi: 10.1002/advs.202415684

Figure Lengend Snippet: Figure 4. NQO1-dependent ROS generation and HDAC inhibition caused by IR/Lap/CUDC NPs. A) Flowcytometric analysis of ROS generation in 4T1 cells after different treatments with or without dicoumarol (DIC). B) Cell viability of 4T1 cells after different treatments with or without DIC. C) Schematic illustration of NQO1-dependent ROS generation and subsequent IR783 oxidation. D) Intracellular IR783 fluorescence in 4T1 cells treated with IR/Lap/CUDC NPs with or without DIC. E) Western blot analysis of acetylated 𝛼-tubulin, acetylated histone H3, and GAPDH expression levels in 4T1 cells after different treatments. F) Representative CLSM images of 𝛾-H2AX in 4T1 cells after different treatments (Scale bar: 50 μm). G) Heatmap of specific DEGs between normal 4T1 cells and IR/Lap/CUDC NPs-treated 4T1 cells. H) Volcano plot of total DEGs between normal 4T1 cells and IR/Lap/CUDC NPs-treated 4T1 cells. Data were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns (not significant).

Article Snippet: Cell Culture: The human triple-negative breast cancer cell line (MDAMB-231), mouse macrophage-like cell line (RAW264.7), and mouse breast cancer cell line (4T1) were purchased from ATCC.

Techniques: Inhibition, Western Blot, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: IR783-Stabilized Nanodrugs Enhance Anticancer Immune Response by Synergizing Oxidation Therapy and Epigenetic Modulation.

doi: 10.1002/advs.202415684

Figure Lengend Snippet: Figure 5. Immunogenic cell death and pro-inflammatory effects of IR/Lap/CUDC NPs. A) Representative CLSM images of HMGB1 in 4T1 cells after different treatments (Scale bar: 50 μm). B) Representative CLSM images of CRT in 4T1 cells after different treatments (Scale bar: 50 μm). C) Quantitative analysis of HMGB1 confocal imaging assay. D) Quantitative analysis of CRT confocal imaging assay. E) Relative intracellular ATP level of 4T1 cells treated with different formulations. F) Flowcytometric analysis of bone marrow-derived dendritic cells (BMDCs) maturation after incubation with the supernatant of 4T1 cells treated with different formulations. G) Quantitative analysis of CD80+CD86+ BMDCs maturation level. H) mRNA expression of TNF-𝛼in RAW264.7 cells after different treatments. I) Quantitative analysis of CD80 expression level in RAW264.7 cells. J) Flowcytometric analysis of CD80 expression level in RAW264.7 cells after different treatments. Data were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns (not significant).

Article Snippet: Cell Culture: The human triple-negative breast cancer cell line (MDAMB-231), mouse macrophage-like cell line (RAW264.7), and mouse breast cancer cell line (4T1) were purchased from ATCC.

Techniques: Imaging, Derivative Assay, Incubation, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: IR783-Stabilized Nanodrugs Enhance Anticancer Immune Response by Synergizing Oxidation Therapy and Epigenetic Modulation.

doi: 10.1002/advs.202415684

Figure Lengend Snippet: Figure 6. Biodistribution and antitumor growth effect of IR/Lap/CUDC NPs. A) Fluorescence imaging and biodistribution of free IR783 and IR/Lap/CUDC NPs in the 4T1 tumor-bearing BALB/c mouse model (n = 3). B) Ex vivo fluorescence image of tumors and main organs collected from the mice treated with free IR783 or IR/Lap/CUDC NPs after 24 h. C) Fluorescence intensity within the tumor area at different time points after the intravenous injection of IR783 or IR/Lap/CUDC NPs. D) Quantitative analysis of the fluorescence intensity in tumor tissues and main organs collected from the treated mice. E) Therapeutic schedule using IR/Lap/CUDC NPs for oxidation therapy and epigenetic modulation against 4T1 subcutaneous tumors. F) Tumor volume

Article Snippet: Cell Culture: The human triple-negative breast cancer cell line (MDAMB-231), mouse macrophage-like cell line (RAW264.7), and mouse breast cancer cell line (4T1) were purchased from ATCC.

Techniques: Fluorescence, Imaging, Ex Vivo, Injection