Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV envelope protein (ZIKV-E, green) and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h post-infection (p.i.) with ZIKV. Scale bar, 50 μm. (B) Quantification of infection efficiency in four GSC and NPC lines 48 h p.i. with ZIKV. (C) Quantification of ZIKV+ cells in a panel of human GSCs and NPCs. (D) Kinetics of viral RNA copies p.i. with ZIKV by measuring viral RNA copies by qRT-PCR in NPC C4–7 and GSC3565. (E) ZIKV infection efficiency of GSCs and NPCs was measured by direct measurement of viral RNA copies. (F) Representative bright-field images 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. Scale bars, 50 μm. (G) Cell viability normalized to day 5 mock, as measured 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. (H) GSCs (GSC3565), differentiated GSCs, NPCs (NPC C4–7), and differentiated NPCs were assayed for cell viability 72 h p.i. with ZIKV. (I) Apoptosis of GSCs (387, 3565) and primary (NPC194, fetal human [fh] NPC) or iPSC-derived NPCs (WT83, C4–7) p.i. with ZIKV was measured by cleaved caspase-3 (CC3) staining. (J) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (K) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 72 h p.i. with ZIKV. Scale bars, 50 μm. (L) Quantification of the percentage of CC3+ cells in DAPI+ cells for GSCs and NPCs 72 h p.i. with ZIKV. (M) Cell viability of patient-derived cultures from GBM (387 and 3565), pontine glioma (3752 and 007), meningioma (CH-157MN, IOMM-LEE), ependymoma (EP1), and medulloblastoma cell lines (DAOY, D283, HDMB03, D341) 72 h after ZIKV infection. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. NS, no significance. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Quantitative RT-PCR, Derivative Assay, Staining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV-E (green), SOX2 (red), and DAPI (blue) of GSCs and forebrain-specific hiPSC-derived NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (B) Quantification of the percentage of SOX2+ cells in DAPI+ cells for GSCs and NPCs 48 h p.i. with ZIKV. (C) Representative immunostaining for ZIKV-E (green), SOX2 or AXL (red), and DAPI (blue) of GSCs (GSC3565) without transduction (shRNA) or transduced with control shRNA (shCONT), AXL shRNA (shAXL.2), or SOX2 shRNA (shSOX2.53) for 72 h and then 48 h with ZIKV infection. Scale bars, 100 μm. (D) Quantification of the percentage of ZIKV+ cells in DAPI+ cells in GSCs 1517 and 3565 under conditions for (C), with a range of ZIKV infection. (E) Viral copy number by qRT-PCR of GSCs (GSC3565 or GSC1517) or NPC C4–7 transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53) for 72 h and then either exposed to mock conditions or infected with ZIKV for another 72 h. All comparisons are versus shCONT. (F) Gene set enrichment (GSE) bubble plots showing pathways positively (top, r > 0.4) or negatively (bottom, r < −0.4) correlated with SOX2 expression in the TCGA GBM HG-U133A microarray dataset. Each circle represents an enriched pathway, with the border color indicating the false discovery rate (FDR)-corrected p value. (G) GSE graph showing the top pathway enrichments positively or negatively correlated with SOX2 as described in (F). (H) Correlation of mRNA levels of SOX2 with IFNAR1, IRF1, promyelocytic leukemia (PML), and IFITIM1 from the TCGA GBM HG-U133A microarray dataset. (I) Correlation between SOX2 with ISGs from the TCGA GBM HG-U133A microarray dataset. The size and color of the dots indicate the degree of correlation (p < 0.001). Blank cells indicate a non-significant correlation. (J) qPCR of ISGs (IFNAR-1, ISH20, IRF1, IFITM1, TLR3, and OAS2) in GSCs (GSC3565) transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53). Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. **p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Derivative Assay, Transduction, shRNA, Control, Infection, Quantitative RT-PCR, Expressing, Microarray

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Immunostaining of the subventricular zone (SVZ) of mice 72 h following ZIKV infection ZIKV-E (green), SOX2 (red, top panels), and integrin αvβ5 (red, bottom panels). Scale bars, 50 μm. (B) Higher magnification of images from (A), demonstrating ZIKV infection of SOX2+ (top panels) and integrin αvβ5+ cells. Scale bars, 10 μm. (C) Survival of ZIKV-infected NSG mice from (A) was plotted by the Kaplan-Meier method. (D) ZIKV-infected brains from the mice in (A) were collected upon death, and histology was assessed by H&E staining. Scale bars, 20 μm. (E) Survival of NSG mice following implantation of GSCs treated with isotype control, P1F6 antibody, ZIKV, combined P1F6 and ZIKV, combined CRISPR knockout (KO) of integrin β5 (sgRNA1 sgRNA2) with ZIKV inoculation, analyzed by log rank test; p < 0.01. (F) H&E staining of tumor-bearing brains from (E). Scale bars, 50 μm. (G) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection (10e3 FFU). Slices then underwent immunofluorescence staining for ZIKV-E (green), integrin αvβ5 (red), and DAPI (blue). Scale bars, 10 μm. (H) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection. Slices then underwent a viral RNA copy assay by qRT-PCR. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Staining, Control, CRISPR, Knock-Out, Incubation, Blocking Assay, Immunofluorescence, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b β3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of β3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, β3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. * p < 0.05; *** p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Staining, Western Blot, Expressing, Microarray, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Identification of the POU3F2 binding site on the NTF3 promoter. a Transcription factor response elements predicted by the Transcription Element Search System for the nucleotide sequence of the NTF3 promoter region (− 1823 to + 243). The transcription start site is indicated as + 1. b Comparison of NTF3 promoter sequence conservation between different species. c Biotin-labeled oligonucleotides containing the intact or mutated POU3F2 binding site were hybridized with total lysates prepared from NT2D1 cells. The POU3F2-DNA complexes were precipitated by streptavidin agarose beads. POU3F2 was analyzed by Western blot analyses. The input of nuclear extracts was used as loading control. Three independent experiments were performed. d Chromatin was prepared from NT2D1 cells treated with induction medium for 0, 2, and 6 h. Cell lysates were mixed with antibodies against POU3F2 or IgG and then precipitated. The precipitates were analyzed by PCR for the presence of the NTF3 promoter sequence. The DNA purified from the sonicated chromatin was directly analyzed by PCR using the ChIP primer, which was used as an input control (Input). e The values of the ChIP DNA were normalized to that of the NT2D1 cells at 0 h (as a control). Values of fold-change over the control are presented as mean ± SEM of three independent experiments for d . * p < 0.05 compared with the control

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Sequencing, Comparison, Labeling, Western Blot, Control, Purification, Sonication

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 on NTF3 promoter activity. a Schematic representation of NTF3-luciferase chimeric constructs. The negative numbers refer to the numbers of bases upstream of the transcription start (+ 1) site of the NTF3 gene. b NT2D1 cells were transiently transfected with the pGL3 basic vector or NTF3 promoter constructs of different lengths. The luciferase activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. c NT2D1 cells were transfected with the pGL3 basic vector, pNTF3-1902, and pNTF3-1902 POU3F2 mut. Approximately 24 h later, cells were treated with neuronal induction medium. The transcriptional activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. Values are presented as mean ± SEM of three independent experiments for b and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 silencing on neuronal differentiation and NTF3 expression in NT2D1 cells. a POU3F2 expression in POU3F2-knockdown (shPOU3F2) and control (shLuc) NT2D1 cells was determined by Western blot analyses after neuronal induction for 6 h. GAPDH was used as a loading control. The values represent the relative expression compared with that of the non-induced shLuc cells (to which a value of 1 was assigned). b NTF3 mRNA expression of the cells described in a was analyzed by real-time PCR. mRNA levels were calculated relative to that of the non-induced shLuc cells. * p < 0.05; *** p < 0.001. c Neuronal morphology of shLuc and shPOU3F2 cells that were treated with neuronal induction medium for 24 h or left untreated (non). d Quantification of cell numbers of shLuc and shPOU3F2 described in c . All the percentages of the shLuc and shNTF3 cells were compared to that of the non-induction shLuc cells (to which a value of 100% was assigned). e β3-tubulin staining was performed on shLuc and shPOU3F2 cells, which were treated with neuronal induction medium for 0, 6, or 24 h or left untreated, after which neuronal cells were detected. Values represent the mean ± SEM of three independent experiments for b and d

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Knockdown, Control, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of NTF3 silencing and NTF3 recombinant protein treatment on the viability and neuronal differentiation of NT2D1 cells. a NTF3 mRNA levels in NTF3-knockdown (shNTF3) and control (shLuc) NT2D1 cells, which were treated with neuronal induction medium for 0, 24, or 48 h or left untreated (Non), were determined by real-time PCR. mRNA levels were calculated as the relative expression compared with the untreated shLuc cells. *** p < 0.001. b Phase contrast microscopy images of untreated shLuc and shNTF3 cells and those cells 24 h after neuronal induction with concomitant treatment of rNTF3 (5, 20 ng/ml) or vehicle. c Quantification of neuron number of shLuc and shNTF3 cells as described in b . All the percentages of neurons differentiated from shLuc and shNTF3 cells were compared to that of neurons differentiated from the vehicle-treated shLuc cells (to which a value of 100% was assigned). * p < 0.05; ** p < 0.01. d A suggested model of the POU3F2/NTF3 pathway that mediates the process of neuron differentiation. Values are presented as mean ± SEM of at least three independent experiments for a and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Recombinant, Knockdown, Control, Real-time Polymerase Chain Reaction, Expressing, Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Infection Causes a Significant Increase in the Protein HSp27, Accompanied by Large Spatial Accumulation of Hsp27 with the Bacteria in a Temporal Manner in Primary GECs. ( A ) Representative confocal microscopy images of P. g -infected human primary GECs at an MOI 100, at 6 h and 24 h after infection. GECs were then stained for P. g (rabbit anti-P. g; Alexa 488; green) or HSp27 (mouse anti-HSp27; Alexa 568; red). GECs were then imaged via the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( Ai ) Imaris Software was used to create a zoomed image of infected GECs and was used to calculate the amount of co-localization between P. g and HSp27. HSp27 was found to readily colocalize with P. g , having an average Pearson correlation coefficient of 0.87 via the Imaris software. Scale bar is 30 µm for 63x and Zoomed Magnification. (B ) P. g was added at MOI 100 to GECs, which were incubated 6 or 12h. Cell lysates were then analyzed via western blot. ( Bi ) Quantitative ImageJ analyses of western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as significant via Two-Tailed Student T-test. *p<0.05.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Bacteria, Confocal Microscopy, Staining, Software, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Integrity of P. gingivalis (P. g) -Specific Autophagosomes is Highly Dependent on HSp27 Presence. Human primary GECs were treated with HSP27siRNA (100nM) for 48 h prior to incubation with P. g ( MOI 100) for 6 h. Autophagosomes were then isolated and analyzed via Confocal Microscopy. ( A ) Schematic autophagosomal isolation method of infected GECs. ( B ) Confocal microscopy images of autophagosomes (ThiolTracker Violet; blue) were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( C ) Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal microscopy images of autophagosomes were obtained via Super Resolution Zeiss Airyscan LSM 880 at 20x. ( Ci ) Quantitative ImageJ analysis of Confocal microscopy results was then performed. Data is represented as Mean±SD, where n=25 and p<0.05 was considered as statistically significant (Student two-tailed T-test). **p<.005

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Isolation, Confocal Microscopy, Infection, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Magnetic-Labelling Process Does Not Impact P. gingivalis (P. g) Infection in GECs. P. g was labeled with lipobiotin (5 μM) and were then incubated in MagCellect Streptavidin Ferrofluid. Human Primary GECs were then infected for 24 h. Representative confocal microscopy images of P. g- infected GECs at an MOI 100, were taken at 24 h after infection using via Zeiss LSM 880 (63x). P. g (rabbit anti-P . gingivalis ; Alexa 488; green) was detected in the GECs. Actin (red) was stained utilizing Rho-Phallodin.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Incubation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Δ ndk-P. gingivalis (P. g) Undergoes Canonical Degradative Autophagosomal Trafficking in Human Primary GECs. Primary GECs were infected with WT P. gingivalis (P. g) versus Δ ndk - P. g at MOI 100 for 3, 12, or 24h. ( A ) TEM analysis used Immunogold labeling for P. g was performed on WT P. g versus Δ ndk - P. g at 3 or 24h post-infection. Images were acquired at 40000x magnification utilizing a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc.) affixed to a Veleta camera with iTEM. ( B ) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Co-localization analysis of P. g and LAMP-1 was additionally carried out using the Zeiss LSM 880 Confocal Software, determining that the WT P. g had a Pearsons value of .137 while the Δ ndk - P. g had a Pearsons value of .704.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Infection, Labeling, Staining, Software

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Depletion of HSp27 Abrogates the Inhibition of Oxidative Stress and Severely Impacts the Intracellular Survival of P. gingivalis (P. g) Studied in Human Primary Organotypic Cultures of Gingiva. To create the organotypic culture systems, human primary GECs and Fibroblasts Cells (FBCs) were co-cultured together upon a collagen raft. Select rafts were then treated with HSP27 siRNA (100nM) for 48 h. Select rafts were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h. P. g was added at MOI 100 to rafts, which were incubated for 24 h. Rafts were then collected and sectioned so that immunofluorescence could be performed. ( A ) Representative images of H&E stained raft culture systems at 20x magnification, which clearly mimic the oral gingival crevice. Scale bar is 50 µm. E: Multilayer Undifferentiated Epithelial Cells, C: Collagen Matrix; F: Fibroblasts. ( B ) Rafts were stained for P. g (rabbit anti- P. g; Alexa 488; green) or HpS27 (mouse anti-HSp27; Alexa 568; red). Rafts were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 20x and 63x. Scale bar is 50 um for 20x and 20 um for 63x and Zoomed View. The range of z-stacks was kept consistent. HSp27 was once again found to readily co-localize with P.g with a Pearson coefficient of .83 as determined via the Imaris Software. ( Bi ) A Zoomed (4x) version of the 63x magnification was created using the Imaris Software, highlighting the intracellular nature of individual P. g. ( C ) GECs were additionally treated with HSp27 siRNA (100nM) for 48 h. Select GECs were then treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 h. If any extracellular bacteria were present, they were killed by gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment for 1 h. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular level of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via One-Way Anova Test. ** p<0.005.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: Inhibition, Cell Culture, Incubation, Immunofluorescence, Staining, Confocal Microscopy, Software, Bacteria, Synthesized

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Isolated P. gingivalis specific autophagosomes were also fixed in 10% NBF for 1 h, and were stained for 1 h at RT with anti- P. gingivalis ATCC 33277 rabbit antibody (1:1000), followed by incubation in anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen, A-11008; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) A provisional network was generated from integration of two microarray data sets. Node color represents increases (red), no significant changes (yellow), and decreases (green) in gene expression in murine prostate tissue after cholesterol alteration as ascertained by cDNA microarray. Changes in RNA expression levels of the corresponding nodes in LNCaP cells are shown as colored node boundaries (donut shape) and the color represents increases (red), no significant change (yellow), and decreases (green) in gene expression under CDM conditions compared to control. Arrows indicate direct activation, T-shaped lines direct repression, dashed arrows indirect activation, and lines physical interaction. ( B ) Gene expression under Normo and Hyper conditions ( in vivo ). To verify in vivo microarray data obtained from SCID experiments, mRNA levels of the indicated genes were determined. GAPDH expression was used to normalize gene expression. Error bars represent SD (n = 3). ( C ) Gene expression under Control and Cholesterol-depleted conditions ( in vitro ). LNCaP cells were incubated in CDM for 0, 3 or 16 h, and mRNA levels of the indicated genes were measured by RT-PCR analysis to validate cDNA microarray data. Error bars represent SD (n = 3). * p <0.05 (Student’s t-test).

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Generated, Microarray, Gene Expression, RNA Expression, Control, Activation Assay, In Vivo, Expressing, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) RT-PCR analysis in vivo . ATF3 levels are reduced in all prostatic lobes from Hyper mice, compared to those from the Normo group (AP = anterior prostate; VP = ventral prostate; DLP = dorsal prostate). ( B ) Immunoblot analysis. Immunoblot of PrEC lysates showed induction of ATF3 protein by CDM (left panel) and by β-cyclodextrin (right panel). MG132, a proteasome inhibitor, also increased ATF3 expression. ( C ) Immunofluorescence analysis. Induction of ATF3 protein by CDM in LNCaP cells as shown by IF. LNCaP cells were treated with CDM for 18 h, stained with anti-ATF3 antibody and nuclei were counterstained with DAPI (left panel: ATF3; middle panel: DAPI; right panel: overlay). ( D ) RT-PCR analysis. ATF3 mRNA levels in LNCaP cells treated with CDM were normalized to levels of GAPDH. RT-PCR analysis shows induction of ATF3 mRNA levels by CDM. ( E–F ) Promoter reporter analysis. A full-length ATF3 promoter was cloned into a luciferase reporter vector and transfected into LNCaP (D) or PrEC (E). Cells were then incubated in Control and CDM medium. ATF3 promoter activity was plotted as arbitrary units (± SD) after normalization with total protein concentration.

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Control, Activity Assay, Protein Concentration

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 1. Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, human samples of normal urothelium (N ¼ 10) and urothelial carcinoma of the bladder (N ¼ 8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma (UCC) compared with the normal urothelium. B, upregulation of these genes was validated by 2-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N ¼ 3) and normal urothelium (N ¼ 3). Ten of 13 genes (asterisked) showed statistical significance (t test; P < 0.05) for differential expression in urothelial carcinoma compared with normal urothelium.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 2. MLN8237 (MLN) induces cell-cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 showed specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, PI staining with flow cytometry analysis was conducted to assess cell-cycle changes. T24, UM-UC- 3, and RT4 cells were treated with 10 nmol/L to 1 mmol/L MLN8237 for 48 hours. All 3 cell lines showed dramatic cell-cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also showed a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Cytometry

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 3. Cellular phenotypes of T24 and RT4 cells differ after MLN8237 (MLN) treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells show a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic Aurora A expression, whereas RT4 cells lacked cytoplasmic Aurora A expression. E, real-time imaging of T24 and RT4 cells treated with MLN8237 was conducted over 48 hours. T24 cells exhibited dramatic increases in cell size as a result of repeated cell-cycle progressions without separation of daughter cells. RT4 cells seemed to become arrested after one failed mitotic attempt, preventing repeated cell-cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Microscopy, Expressing, Imaging

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 4. MLN8237 (MLN) induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 hours. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 and 45 nmol/L, respectively) and lowest potency in RT4cells (IC50 of 120 nmol/L). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 hours after initiation of treatment. C, annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 hours after initiation of MLN8237 treatment. D, clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nmol/L MLN8237.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Cytometry, Inhibition

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 6. Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 hours (left), or sequentially, with one drug for 48 hours, followed by washout and the other drug for 48 hours (middle and right). MTS assay was used to quantify the effect on cell viability of these combination treatments. MLN8237 showed synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right), and antagonistic effects when dosed simultaneously (left).

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Phosphorylation of Trop-2. A, FLAG-tagged Trop-2 was immunoprecipitated (IP) from lysates of HCT116/M (M) and HCT116/WT (WT: #1) cells and subjected to SDS-PAGE, followed by immunoblotting (IB). B, FLAG-tagged Trop-2 immunoprecipitated from a lysate of HCT116/WT (#1) cells with anti-FLAG magnetic beads was treated with or without N-glycanase and then subjected to SDS-PAGE, followed by immunoblotting. C, after N-glycanase treatment, FLAG-tagged Trop-2 obtained from a lysate of HCT116/WT (#1) cells as described above was subjected to SDS-PAGE using a Phos-tag–containing gel (Phos-tag SDS-PAGE), followed by immunoblotting. The top and bottom bands represent phosphorylated and unphosphorylated FLAG-tagged Trop-2, respectively. D, schematic model of WT and mutated FLAG-tagged Trop-2. ED, ectodomain; TM, transmembrane domain; CD, cytoplasmic domain. E, after N-glycanase treatment, FLAG-tagged Trop-2 immunoprecipitated from lysates of HCT116/WT (#1 and #2), HCT116/S303A (S303A: #1 and #2), and HCT116/S322A (S322A: #1 and #2) cells as described above was subjected to Phos-tag (top) and ordinary (bottom) SDS-PAGE, followed by immunoblotting. The arrowhead indicates phosphorylated FLAG-tagged Trop-2. F, intensities of the bands in the Phos-tag gel panel in E were measured, and then the ratio of phosphorylated to total FLAG-tagged Trop-2 was calculated (means ± S.E. (error bars), n = 4).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Western Blot, Magnetic Beads

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Interaction of Trop-2 with claudin-7. A, lysates (left) of HCT116/M (M) and HCT116/WT (WT: #1) cells and the immunoprecipitates (IP) (right) obtained from the lysates with anti-FLAG magnetic beads were subjected to SDS-PAGE, followed by immunoblotting (IB), and detection of claudin-7, occludin, E-cadherin, and FLAG-tagged Trop-2. β-Actin served as a loading control. B and C, HCT116 cells were stably transfected with both a Tet-ON regulator plasmid and a response plasmid–inserted FLAG-tagged Trop-2 gene. Lysates of the cells treated with or without 10 μg/ml doxycycline (Dox) for 1–6 days were subjected to SDS-PAGE, followed by immunoblotting. β-Actin served as a loading control. The intensity of the claudin-7 and β-actin bands in B was measured, and the ratio of claudin-7 to β-actin in each Dox-treated cell is indicated, with that in Dox-nontreated cells being taken as 1 (means ± S.E. (error bars), n = 3; *, p < 0.05; **, p < 0.01). D and E, immunoprecipitates from the lysates of HCT116/WT (#1), HCT116/S322A (SA: #1), and HCT116/S322E (SE: #1) cells with anti-claudin-7 antibodies were subjected to SDS-PAGE, followed by immunoblotting. Histograms show the intensity of the FLAG-tagged Trop-2 bands, each density being normalized as to that of claudin-7, and the value for HCT116/WT (#1) cells was taken as 1 (means ± S.E., n = 3; *, p < 0.05; **, p < 0.01). F, after treatment of HCT116/WT (#1) cells with PMA or 4α-PMA for 2 h, their lysates (left) and claudin-7 coimmunoprecipitated with anti-FLAG magnetic beads (right) were subjected to SDS-PAGE, followed by immunoblotting, and detection with anti-claudin-7 and anti-FLAG antibodies. β-Actin served as a loading control.

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Magnetic Beads, SDS Page, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Expression and distribution of Trop-2 and claudin-7 in various types of Trop-2–expressing cells and in cancer tissues. A, levels of claudin-7 mRNA in HCT116/M (M), HCT116/WT (WT: #1), HCT116/S322A (SA: #1), and HCT116/S322E (SE: #1) cells were determined by DNA microarray analysis, and its level in HCT116/M cells was taken as 1. B, expression of claudin-7 protein in the four cell types described above was analyzed by SDS-PAGE, followed by immunoblotting, and that in HCT116/M cells was normalized as to β-actin, and the value for HCT116/M cells was taken as 1. β-Actin served as a loading control (means ± S.E. (error bars), n = 4; *, p < 0.05; **, p < 0.01). C, distribution of claudin-7 (green) and FLAG-tagged Trop-2 (magenta) in HCT116/M, HCT116/WT (#1), HCT116/S322A (#1), and HCT116/S322E (#1) cells was observed immunochemically. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. D, distribution of claudin-7 and Trop-2 was observed immunohistochemically in colorectal cancer tissues. Nuclei were stained with hematoxylin. Scale bars, 100 μm.

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Microarray, SDS Page, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Phosphorylation of Trop-2 by PKCα and PKCδ. A, expression of PKC and PKD isoforms in HCT116/WT (WT: #1) cells was examined by DNA microarray analysis. B and C, HCT116/WT (#1) cells were treated with or without a PKC or PKD inhibitor for 1 h and subsequently with PMA or 4α-PMA for 2 h. Each cell lysate was subjected to SDS-PAGE, followed by immunoblotting. The intensities of the bands in B were determined. Histograms show the inhibitory effect of each inhibitor, the levels of phosphorylated Trop-2 being represented as the ratio of phosphorylated to total FLAG-tagged Trop-2 (p-Trop-2/FLAG), and the ratio obtained for 4α-PMA–treated cells was taken as 1 (means ± S.E. (error bars), n = 3; **, p < 0.01; NS, not significant). D and E, HCT116/WT (#1) cells were transiently transfected with PKCα siRNA (#1), PKCδ siRNA (#1), both PKCα and PKCδ siRNAs (#1), or a control siRNA and then treated with PMA or 4α-PMA for 2 h. A lysate of each cell type was subjected to SDS-PAGE, followed by immunoblotting. β-Actin served as a loading control. The intensities of the bands in D were determined, and the relative value (p-Trop-2/FLAG) was calculated as described in B and C. Histograms show the p-Trop-2/FLAG ratio in PMA-treated cells and that in 4α-PMA–treated cells (mean ± S.E., n = 3; **, p < 0.01; NS, not significant).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Expressing, Microarray, SDS Page, Western Blot, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility

doi: 10.1074/jbc.RA119.008084

Figure Lengend Snippet: Effects of Trop-2 phosphorylation on motility of WT and mutated Trop-2–expressing HCT116 cells. A and B, migration of HCT116/M (M, n = 7), HCT116/WT (WT: #1, n = 4), HCT116/S322A (SA: #1, n = 4), and HCT116/S322E (SE: #1, n = 3) cells was evaluated by means of Transwell assays. A representative photograph of each migrated cell type is shown in A. Scale bars, 100 μm. The number of migrated cells per field was determined and is shown as a histogram in B (means ± S.E. (error bars); **, p < 0.01). C, proliferation of HCT116/M (n = 7), HCT116/WT (#1: n = 4), HCT116/S322A (#1: n = 4), and HCT116/S322E (#1: n = 3) cells was analyzed using MTT assays (means ± S.E.). D and E, migration of HCT116/WT (#1) cells treated with or without PKC inhibitors was measured as described in A and B. A representative photograph of each migrated cell is shown in D. Scale bars, 100 μm. The number of migrated cells per field was determined as described in A and B and is shown as a histogram in E (means ± S.E., n = 3; **, p < 0.01; NS, not significant). F, Proliferation of cells was examined using MTT assays as described in C (means ± S.E., n = 3). G and H, migration of HCT116/WT (#1) cells treated with both PKCα and PKCδ siRNAs (#1) or a control siRNA and then with PMA or 4α-PMA was analyzed by using Transwells. A representative photograph of each migrated cell type is shown in G. Scale bars, 100 μm. The number of migrated cells per field was determined as described in A and B and is shown as a histogram in H (means ± S.E., n = 3; **, p < 0.01; NS, not significant). I, proliferation of cells was analyzed using MTT assays as described in C (means ± S.E., n = 3).

Article Snippet: Cell culture and transfection of plasmids A human colorectal cancer cell line, HCT116 cells, and a human pancreatic cancer cell line, PANC-1 cells, were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Phospho-proteomics, Expressing, Migration, Control

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Cell reports

Article Title: Pulsatile MEK Inhibition Improves Anti-tumor Immunity and T Cell Function in Murine Kras Mutant Lung Cancer

doi: 10.1016/j.celrep.2019.03.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse; LLC , ATCC , ATCC® CRL-1642.

Techniques: Flow Cytometry, Purification, In Vitro, Activation Assay, In Vivo, Control, Virus, Plasmid Preparation, Recombinant, Staining, Luminex, Multiplex Assay, Cell Viability Assay, Gene Expression, Transgenic Assay, Software, Imaging, Microarray