Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Dose- and time-dependent effect of BAY and medium refreshment on cell viability. ( a ) Dose-dependent effect of BAY ( N =2, n =4) on the viability of G361 and SK-MEL-28 cells (at 72 h). A Boltzmann equation was used to determine the IC 50 ( x 0 ) value: y =( A 2 +( A 1 − A 2 )/(1+exp(( x − x 0 )/d x ))). ( b ) G361 and SK-MEL-28 melanoma cells were treated with 10 nM BAY ( N =3, n =6) and their viability was measured at different time points. A sigmoidal (logistic) equation was used to determine the T 1/2 ( x 0 ) value: y =([( A 1 − A 2 )/(1+( x/x 0 )]+ A 2 ). Statistics: In panel b, significant differences with the SK-MEL-28 cell line are indicated by * P <0.05 and *** P <0.001

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques:

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of TRAP1 knockdown/overexpression on the BAY-induced reduction in cell viability. ( a ) Detection of photoinduced mitochondrial membrane potential (Δ ψ ) depolarizations (‘Δ ψ -flickering') in a typical TMRM-stained G361 cells (see Results for details). ( b ) Effect of BAY (2 min), CsA (2 h), empty vector and TRAP1-OE on Δ ψ -flickering ( N =3, n ≥20). ( c ) Effect of BAY on reactive oxygen species (ROS) levels (at 24 h; N=3, n =3) in cells transfected with the empty or TRAP1-OE vector. ( d ) Effect of vehicle ( N =5, n =15), BAY ( N =5, n =15) and CsA ( N =3, n =6) on cell death (G361: at 48 h; SK-MEL-28: at 72 h). ( e ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siTRAP1, empty vector and TRAP1-OE. Statistics: Significant differences relative to the vehicle condition (‘Veh' in panel b) and between treatments (a, b, c, d in panels c and d ) are indicated by * P <0.05, ** P <0.01 and *** P <0.001. In panel ( e ), significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Over Expression, Membrane, Staining, Plasmid Preparation, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of ATG5 knockdown on the BAY-induced stimulation of autophagy, reactive oxygen species (ROS) increase and reduction in cell viability. ( a ) Effect of BAY in the absence and presence of BafA1, TOC and ATG5 knockdown on the number of green puncta in G361 and SK-MEL-28 cells (at 24 h; N =3, n =30). ( b ) Effect of BAY on ROS levels (at 24 h; N =3, n =9) in cells transfected with an empty or GPX4 overexpression vector (GPX-OE). ( c ) Effect of BAY on cellular MG fluorescence (G361: at 24 h; SK-MEL-28: at 24 h; N =3, n =9) in cells transfected with siCTRL, siATG5-no. 1 and siATG5-no. 2. ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siATG5-no. 1 and siATG5-no. 2. Statistics: Significant differences relative to the marked conditions are indicated by * P <0.05, ** P <0.01 and *** P <0.001. NS indicates nonsignificant. In panel ( d ), significant differences with the (siCTRL+10 nM BAY condition) are marked by ‘&'. In panel ( b ) significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Transfection, Over Expression, Plasmid Preparation, Fluorescence

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of PINK1 knockdown on the BAY-induced stimulation of mitophagy, Δ ψ depolarization, reactive oxygen species (ROS) levels and reduction in cell viability. ( a ) Effect of BAY treatment, siPINK1-no. 1 and siPINK1-no. 2 on the number of green puncta colocalizing with mitochondria in G361 and SK-MEL-28 cells (at 24 h; N =3, n ≥14). ( b ) Similar to panel a, but now for the effect on mitochondrial membrane potential (i.e. the JC-1 red/green ratio signal; N =3, n =6). ( c ) Similar to panel a, but now for the effect on cellular ROS levels ( N =3, n =6). ( d ) Similar to panel a, but now for the effect on cellular MG fluorescence intensity (G361: at 24 h; SK-MEL-28: at 24 h; N =3, n =9). ( e ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siPINK1-no. 1 and siPINK1-no. 2. Statistics: Significant differences relative to the indicated conditions are marked by * P <0.05, ** P <0.01 and *** P <0.001. In panel c statistical analysis was performed using a one-sample t-test against a value of 100. In panel d, NS indicates nonsignificant and significant differences with the (siCTRL+10 nM BAY condition) are marked by ‘&'. In panel ( e ) significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Membrane, Fluorescence, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of Drp1 knockdown on mitochondrial morphology and the BAY-induced reduction in cell viability. ( a ) Typical examples visualizing mitochondria morphology in MG-stained cells treated with siCTRL, siDrp1-no. 1 and siDrp1-no. 2 in the absence and presence of 10 nM BAY (G361: at 16 h; SK-MEL-28: at 24 h). ( b ) Quantification of mitochondrial morphology in multiple cells ( N =2; numerals indicate the number of cells analyzed) for the conditions in panel ( a ). ( c ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siDrp1-no. 1 and siDrp1-no. 2. Statistics: In panel ( c ), statistically significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of z-VAD-FMK, Nec-1, Nec-1s, Fer-1 and knockdown of RIPK1 and MLKL on the BAY-induced reduction in cell viability. ( a ) Effect of vehicle ( N =5, n =15), BAY ( N =5, n =15), the pancaspase inhibitor z-VAD-FMK ( N =3, n =9), Nec-1 ( N =3, n =9) and Fer-1 ( N =3, n =9) on cell death (G361: at 48 h; SK-MEL-28: at 72 h). ( b ) Effect of BAY on viability (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) in the absence and presence of the ferroptosis inhibitor Fer-1 and the RIPK1 inhibitors Nec-1 and Nec-1s. ( c ) Effect of BAY on cell viability (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) in cells transfected with siCTRL, siRIPK1-no. 1 and siRIPK1-no. 2. ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N=3, n =6) transfected with siCTRL, siMLKl-no. 1 and siMLKL-no. 2. Statistics: Significant differences relative to the marked conditions are indicated by * P <0.05 and *** P <0.001. In panels b–d, significant differences ( P <0.05) between conditions are marked by symbols (a, b, &, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of GPX4 knockdown/overexpression on the BAY-induced reduction in cell viability. ( a ) Effect of BAY on the number of MBB-negative (GSH-depleted) cells in the vehicle- and BAY-treated condition (G361: at 12 h; SK-MEL-28: at 24 h; N =3, n =9). Higher bars reflect a reduction in the number of MBB-positive cells. ( b ) Effect of BAY on reactive oxygen species (ROS) levels (at 24 h; N =3, n =3) in cells transfected with the empty or GPX4-OE vector. ( c ) Cellular lipid peroxidation (at 24 h) in vehicle-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), TOC-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), BAY-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), BAY+TOC-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), empty vector-transfected cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8), GPX4-OE-transfected cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8), empty vector-transfected-+BAY-treated cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8) and GPX-OE transfected-+BAY-treated cells (G361: N =3, n =8; SK-MEL-28: N =3, n =8). ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siGPX4, empty vector and GPX4-OE. Statistics: Significant differences relative to vehicle (panel a), empty vector (panel b) and the marked conditions (panel c) are indicated by * P <0.05, ** P <0.01, * P <0.01 and *** P <0.001. In panel d, significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Over Expression, Transfection, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) Schematic of the HT-SRFA with Renilla luciferase dual split protein (DSP). Fusion of effector cells, CHO-DSP1, transiently expressing VZV glycoproteins, gB and gH[TL]-gL, with target cells, MeWo-DSP2, reconstitutes the Renilla luciferase DSP. Effector and target cells were seeded into 384-well plates, and treated with compound libraries for 48 hrs before measuring fusion efficiency; cell viability was assessed by CellTiter-Glo. Positive controls were wells without drug; negative controls had effector cells transfected with empty vectors; medium only controls were included for cell viability. (B) Scatter plot of cell fusion and cell viability derived from the HT-SRFA. The Y-axis and X-axis indicate fusion efficiency and cell viability values normalized to the mean of positive controls. The mean of the percentage (% positive ctrl) from two biological replicates are shown: blue circles are all 4,846 compounds screened, red and black circles are positive and negative controls and the black crosses are medium only. Grey and orange boxes show ±3 CV % of positive controls for fusion efficiency and cell viability respectively. (C) Frequency (%) of compounds that affect cell fusion identified by the HT-SRFA (left), compound classes of fusion enhancers (center) and inhibitors (right), and number of compounds in each class.

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Luciferase, Expressing, Transfection, Derivative Assay

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p < 0.05; ****, p < 0.0001).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Expressing, Cell Culture, Fluorescence, Microscopy, Translocation Assay, Activity Assay, Activation Assay, Staining, Binding Assay, Western Blot, Transfection, Control, Whisker Assay

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) FKBP1A shRNA knockdown (KD) in MeWo cells. FKBP1A Transcript levels detected by RT-qPCR in MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells were normalized to housekeeping gene PGM1 and presented as a percentage of control cells, with mean ± SEM from three independent experiment (two-tailed, unpaired t-test, ****, p < 0.0001). Western blot of FKBP1A protein (anti-FKBP1A). FKBP1A band density was normalized to α-tubulin in control and FKBP1A KD cells respectively. The ratio of the two values was presented as a percentage of control cells. A representative picture, and mean ± SEM of the ratio from three independent experiments are shown. (B) Quantification of calcineurin phosphatase activity in FKBP1A KD cells using the NFATC1 nuclear translocation assay. MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells transiently expressing GFP-NFATC1 were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min before ionomycin (Iono, 1 μM; 30 min). Fluorescence microscopy images of GFP-NFATC1 (green), nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. Red arrowheads indicate nucleus localization ( N ), white arrowhead indicates diffused localization in both nucleus and cytoplasm ( N/C ), and white arrows indicate cytoplasm localization ( C ). Box and whisker plots of GFP-NFATC1 positive cells. The percentage of GFP positive cells in control and FKBP1A KD cells with GFP-NAFTC1 localization to N, N/C, and C. Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 15 fields of view, 13–22 cell per field of view) from two independent experiments. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each treatment condition (Untreated, DMSO+Iono, and Pim+Iono) compared to that in the control cells, and significantly different pairs are shown (****, p < 0.0001). (C and D) Effect of FKBP1A KD on VZV gB/gH-gL-mediated fusion and response to pimecrolimus. Box and whisker plots for cell fusion quantified by the SRFA in MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells transfected with plasmids to express VZV gB/gH[TL]-gL and co-cultured with MeWo-DSP2 cells, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM) for 48 hrs. Cell fusion efficiency when untreated was compared between FKBP1A KD cells and control cells, shown as a percentage of control cells (C), and the response to pimecrolimus was evaluated by normalization to the untreated (% medium) per control and FKBP1A KD cell line respectively (D). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) from three independent experiments. Dots are outliers. Statistical differences were evaluated by unpaired, nonparametric Mann-Whitney test (C) (ns, not significant) or two-way ANOVA (D) (ns, not significant; ****, p < 0.0001).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: shRNA, Knockdown, Quantitative RT-PCR, Control, Two Tailed Test, Western Blot, Activity Assay, Nuclear Translocation Assay, Expressing, Fluorescence, Microscopy, Staining, Whisker Assay, Transfection, Cell Culture, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) CNB1 shRNA knockdown (KD) in MeWo cells. CNB1 transcript levels in MeWo-DSP1 control cells or CNB1 KD MeWo-DSP1 cells detected by RT-qPCR were normalized to housekeeping gene PGM1 and presented as a percentage of control cells, with mean ± SEM from two independent experiment (two-tailed, unpaired t-test, ****, P < 0.0001). Western blot of CNB1 protein (anti-CNB1). CNB1 band density was normalized to α-tubulin in control and CNB1 KD cells respectively. The ratio of the two values was presented as a percentage of control cells. A representative picture, and mean ± SEM of the ratio from two independent experiments are shown. (B and C) Effect of CNB1 KD on VZV gB/gH-gL-mediated fusion and response to pimecrolimus. Box and whisker plots for cell fusion quantified by the SRFA in MeWo-DSP1 control cells or CNB1 KD MeWo-DSP1 cells transfected with plasmids to express VZV gB/gH[TL]-gL and co-cultured with MeWo-DSP2 cells, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM) for 48 hrs. Cell fusion efficiency when untreated was compared between CNB1 KD cells and control cells, shown as a percentage of control cells (B), and the response to pimecrolimus was evaluated by normalization to the untreated (% medium) per control and CNB1 KD cell line respectively (C). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) from two independent experiments. Statistical differences were evaluated by unpaired, nonparametric Mann-Whitney test (B) (**, p < 0.01) or two-way ANOVA (C) (ns, not significant; *, p < 0.05; ****, p < 0.0001).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: shRNA, Knockdown, Control, Quantitative RT-PCR, Two Tailed Test, Western Blot, Whisker Assay, Transfection, Cell Culture, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus ( N ), diffused in nucleus and cytosol ( N/C ), or localized in cytosol ( C ). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p < 0.0001).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Fluorescence, Microscopy, Expressing, Staining, Whisker Assay, Infection

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) Confocal microscopy of LifeAct-tGFP MeWo cells infected with pOka-TK-RFP, treated with DMSO or pimecrolimus (Pim; 10 μM). Representative plaques captured from live-cell confocal microscopy at 24 and 30 hpi from and Movies , that show pOka-TK-RFP infected cells (red), LifeAct-tGFP labeled actin filaments (green), and nuclei stained with Hoechst 33342 (blue). Scale bars = 100 μm. (B) Frequency of nuclei in syncytia at 40 min intervals from 24 to 30 hpi for plaques in (A). Coefficient of determination (R 2 ) was calculated to determine the linear relationship of increased nuclei frequency in syncytia during the 6 hour window of infection. (C) Scatter dot plots of increased nuclei frequency in syncytia from 24 to 30 hpi. Mean ± SEM for plaques (n = 8) from two independent live-cell confocal microscopy experiment. Statistical significance was evaluated by an unpaired, nonparametric Mann-Whitney test (*, p < 0.05).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Confocal Microscopy, Infection, Labeling, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: Plaque sizes of VZV pOka in MeWo cells untreated (medium; Med), treated with DMSO or pimecrolimus (Pim; 10 μM) at 4 dpi. (A) Immunohistochemistry staining of VZV plaques with the mean plaque size per condition. Scale bars = 0.3 mm. (B) Box and whisker plots of plaque sizes (mm 2 ). Boxes show the 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) from three independent experiments (n = 60). Dots are outliers. Statistical differences were evaluated by one-way ANOVA (ns, not significant; ****, p < 0.0001). (C) Box and whisker plots of MeWo cells, MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells at 4 dpi with VZV pOka, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM). Percentage of plaque size normalized to the untreated (% medium). Boxes show the 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean ( + ) from two independent experiments (n = 60). Dots are outliers. Statistical differences were evaluated by two-way ANOVA (ns, not significant; ****, p < 0.0001). (D) Frequency and total area of plaques in the presence of pimecrolimus. MeWo cells infected with pOka were untreated (medium), treated with DMSO or pimecrolimus (10 μM) for 4 days, plaque frequency and total plaque area were analyzed by ImageJ automatic particle counting. Representative result from ≥3 independent experiments is shown, with input Immunohistochemistry images (scale bars = 2 mm), processed images and measurements listed.

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Immunohistochemistry, Staining, Whisker Assay, Control, Infection

Journal: PLoS Pathogens

Article Title: Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

doi: 10.1371/journal.ppat.1009022

Figure Lengend Snippet: (A) Consensus docking motifs on calcineurin substrates for calcineurin binding. PxIxIT and LxVP motifs from 20 previously verified substrates of calcineurin were used to generate the consensus motif by WebLogo 3. Frequency of amino acids at each position is represented by the height of the letter. Chemical properties of amino acids are represented by the color of the letter: green, polar; purple, neutral; blue, basic; red, acidic; black, hydrophobic. (B) Heat map of uniquely phosphorylated proteins detected in MeWo cells treated with pimecrolimus (10 μM) for 2 hrs. Total cellular proteins were extracted in the presence of phosphatase inhibitor and subject to Zr-IMAC phosphopeptide enrichment, followed by orbitrap mass spectrometry. Three biological samples were analyzed for each condition and the relative spectra of uniquely phosphorylated proteins detected in all three pimecrolimus treated samples but not in DMSO samples are shown in the heat map. Nuclear factor of activated T cells cytoplasmic 1 (NFATC1); ETS transcription factor (ELK1); phosphatase and actin regulator 2 (PHACTR2); interleukin enhancer binding factor 3 (ILF3); nuclear receptor coactivator 1 (NCOA1); transmembrane channel-like protein 8 (TMC8); methyl-CpG binding domain protein 1 (MBD1). Previously known calcineurin substrates are highlighted with asterisks (*). (C) Potential docking motif for calcineurin binding on newly identified phosphoproteins located by Clustal alignment using the consensus motif. Conserved amino acids on PxIxIT and LxVP motif (red), conservative substitutions (blue), docking motif on previously known substrates of calcineurin (boxed).

Article Snippet: Human melanoma MeWo cells (HTB-65, ATCC), MeWo-DSP1 cells derived from MeWo cells that express DSP 1-7, MeWo-DSP2 expressing the DSP 8-11 [ ], MeWo-DSP1 cells expressing shRNA against FKBP1A, MeWo-DSP1 cells expressing shRNA against CNB1, and MeWo LifeAct-tGFP cells expressing a short peptide fused with tGFP that binds to F-actin [ ] were propagated in minimal essential medium (MEM, Corning) supplemented with 10% FBS, nonessential amino acids (1X, Corning), penicillin (100 U/ml, Gibco), streptomycin (100 μg/ml, Gibco), and amphotericin B (0.5 μg/ml, Corning).

Techniques: Binding Assay, Phospho-proteomics, Mass Spectrometry

Journal: The journal of gene medicine

Article Title: Improved in vivo gene transfer into tumor tissue by stabilization of pseudodendritic oligoethylenimine-based polyplexes.

doi: 10.1002/jgm.1430

Figure Lengend Snippet: Figure 4. Reporter gene expression of stabilized BPEI (c/p ratio 0.8) and HD O (c/p ratio 1/1) polyplexes. (a) K562 cells; (b) Neuro2a cells; (c) B16F10 cells and (d) PC3 cells. LPEI was applied as positive control at the optimized N/P ratio of 6, which represents a c/p ratio of 0.8. Crosslinking degrees are indicated in the x-axis. For crosslinked polyplexes, solid bars indicate DSS-linked particles at the indicated crosslinking degrees; crossbred bars indicate DSP-linked particles at indicated crosslinking degrees. Left: transfection efficiency is expressed as RLU/well and shown as the mean ± SD of three independent measurements, each performed in triplicate. Right: metabolic activity of cells 24 h after transfection is demonstrated; experiments were performed in parallel

Article Snippet: B16F10 murine melanoma cells (kindly provided by I. J. Fidler, Texas Medical Center, Houston, TX, USA) and murine neuroblastoma Neuro2a cells (ATCC CCI-131) were cultured in Dulbecco’s modified Eagle’s medium (1 g/l glucose) supplemented with 10% FCS.

Techniques: Gene Expression, Positive Control, Transfection, Activity Assay

Journal: Oncotarget

Article Title: Multi-modal effects of 1B3, a novel synthetic miR-193a-3p mimic, support strong potential for therapeutic intervention in oncology

doi: 10.18632/oncotarget.27894

Figure Lengend Snippet: Cells were transfected with either 1B3 or ‘negative’ miRNA control (3A1) in the presence of RNAiMAX transfection reagent. Non-transfected cells (mock) were also included to define baseline readout. ( A ) Nuclei count was determined 96 h after transfection with 10-nM of 1B3 by staining with Hoeschst-33342 and imaging using the Thermo CellInsite Automated Imager. Values were normalized to mock transfected cells. The dashed line represents the mock value of 1. ( B ) Caspase activation was measured at 48 or 72 h after transfection with 10-nM of 1B3 using Caspase-Glo 3/7 assay (Promega). Values were normalized to mock transfected cells. The time point with the highest caspase activation is shown. The dashed line represents the mock value of 1. ( C ) Cell cycle was assayed at 48, 72 or 96 h after transfection with 10-nM of either 3A1 or 1B3, and image-based DNA content analysis used to determine the proportion in each phase of the cell cycle. Values were normalized to mock transfected cells and the optimal time point shown. Proportion in each cell cycle phase for A2058 (at 72 h; D ), Hep3B (at 48 h; E ), H1299 (at 48 h; F ), and HUH-7 (at 72 h; G ) after transfection with 10-nM of either mock, 3A1 or 1B3. All values are the mean of at least three independent replicates. Error bars show standard deviation.

Article Snippet: The human cell lines Hep3b (HCC), SNU-449 (HCC), H1299 (NSCLC), H1975 (NSCLC), H460 (NSCLC), A2058 (melanoma), BT-549 (TNBC), HCT116 (colon), PANC-1 (pancreatic), and A549 (NSCLC) cells were obtained from ATCC.

Techniques: Transfection, Control, Staining, Imaging, Activation Assay, Caspase-Glo Assay, Standard Deviation

Journal: Oncotarget

Article Title: Multi-modal effects of 1B3, a novel synthetic miR-193a-3p mimic, support strong potential for therapeutic intervention in oncology

doi: 10.18632/oncotarget.27894

Figure Lengend Snippet: Effect of 1B3 on cell proliferation, apoptosis, cell cycle, senescence, and migration in a panel of human cancer cell lines

Article Snippet: The human cell lines Hep3b (HCC), SNU-449 (HCC), H1299 (NSCLC), H1975 (NSCLC), H460 (NSCLC), A2058 (melanoma), BT-549 (TNBC), HCT116 (colon), PANC-1 (pancreatic), and A549 (NSCLC) cells were obtained from ATCC.

Techniques: Migration, Cell Counting

Journal: Oncotarget

Article Title: Multi-modal effects of 1B3, a novel synthetic miR-193a-3p mimic, support strong potential for therapeutic intervention in oncology

doi: 10.18632/oncotarget.27894

Figure Lengend Snippet: Human melanoma A2058 tumor cells (5 × 10 6 ) in 0.1-mL PBS were injected subcutaneously in the flank BALBc/nude mice. After eight days, animals were randomized to experimental groups based on subcutaneous tumor size (median ~150 mm 3 ; established subcutaneous tumor model). Animals were then treated with either vehicle (PBS, i.p.; black) or INT-1B3 (3 mg/kg/administration, i.v.; green) daily for five days the first week, then twice weekly for the next two weeks. Subcutaneous tumor volumes (expressed as mm 3 ) were calculated throughout the study based on caliper-aided tumor measurements and applying the following formula: (length × width 2 )/2. ( A ) Time-dependent evolution of tumor volumes (median values expressed as mm 3 ). ( B ) on Day 22, individual tumor volumes, median (horizontal bar) and interquartile range (vertical error bar), are presented for the indicated experimental groups. A 2-tailed non-parametric Mann–Whitney statistical analysis was performed for INT-1B3 ( p = 0.0062) as compared to PBS-treated group, with n = 7 and 5 for PBS and INT-1B3 groups, respectively.

Article Snippet: The human cell lines Hep3b (HCC), SNU-449 (HCC), H1299 (NSCLC), H1975 (NSCLC), H460 (NSCLC), A2058 (melanoma), BT-549 (TNBC), HCT116 (colon), PANC-1 (pancreatic), and A549 (NSCLC) cells were obtained from ATCC.

Techniques: Injection, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical model for evaluating human TCRs against chimeric syngeneic tumors

doi: 10.1136/jitc-2024-009504

Figure Lengend Snippet: Hybrid DMβ-TCR redirects HLA-A2 transgenic mouse T cells against NY-peptide pulsed B16-A2Kb:NYprot melanoma target cells. ( A ) Schematic of a hybrid A2/NY TCR expressed by an HLA-A2 transgenic murine T cell engaging NY-peptide pulsed B16 tumor cells gene-modified to express a chimeric HLA-A2 molecule comprising α3 of H-2Kb to facilitate mouse CD8-coreceptor engagement. These cells also express the full NY protein but it is not processed and presented. ( B ) Schematic of primary mouse CD8 + T-cell activation, transduction, and expansion. ( C ) Transduction efficiency of mouse CD8 + T cells with hybrid TCRs evaluated by an anti-Vβ13.1 antibody staining (top) and specific tetramer (bottom), (representative data of n>5 donors). ( D ) IncuCyte live-cell imaging to evaluate killing by hybrid TCR-T cells at an E:T ratio of 2:1 of the B16-A2Kb:NYprot cell line peptide-loaded with 10 µM of either NY-peptide or MelanA-peptide. Cytotox red is added in the co-culture to visualize and quantify target cell death (n=3 donors). ( E ) Flow cytometric analysis of B16 and B16-A2Kb:NYprot cells co-cultured or not in the presence of IFN-γ overnight and cell-surface stained for HLA-A2. Statistical analysis by two-way ANOVA ( D ), with correction for multiple comparisons by post hoc Tukey’s test. ****p<0.0001. ns, not significant; ANOVA, analysis of variance; E:T, Effector:Target; MHC, major histocompatibility complex; UTD, untransduced.

Article Snippet: Human embryonic kidney (HEK-293T), Phoenix-Ecotropic (ECO), human melanoma A375 (HLA-A*0201 + NY-ESO-1 + ), mouse melanoma B16 (H-2Kb + ) and mouse colon carcinoma MC38 (H-2Kb + ) cell lines were purchased from the ATCC.

Techniques: Transgenic Assay, Modification, Activation Assay, Transduction, Staining, Live Cell Imaging, Co-Culture Assay, Cell Culture, Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical model for evaluating human TCRs against chimeric syngeneic tumors

doi: 10.1136/jitc-2024-009504

Figure Lengend Snippet: Hybrid A2/NY TCR-T cells are reactive against B16 tumor cells gene-modified to express a single chain trimer of A2Kb and the NY peptide. ( A ) Schematic of the functional single chain trimer (SCT) in which human β2m is connected by GS linkers to both human α1 and the NY-peptide. The H74L amino acid substitution is depicted in pink on the α1-chain. ( B ) Schematic of the retroviral vector encoding the SCT. ( C ) Anti-human β2m antibody staining of STC-engineered B16 tumor cells. ( D ) Cytotoxicity of B16-A2Kb:NY cells (left) by TCR-T cells at an Effector to Target ( E:T ) ratio of 2:1, followed by IncuCyte live cell imaging using Cytotox red dye (n=3 donors). B16-A2Kb:MelanA cells were used as a control (right). ( E ) Cytokine and chemokine secretion by TCR T cells post 24 hours co-culture with B16-A2Kb:NY (left) and B16-A2Kb:MelanA (right) tumor cells at E:T ratio=1:1 (n=3 donors) measured with CBA. Tumor cells alone were used as a control. Statistical analysis by two-way ANOVA ( D ), with correction for multiple comparisons by post hoc Tukey’s test. ****p<0.0001. ANOVA, analysis of variance; E:T, Effector:Target; UTD, untransduced.

Article Snippet: Human embryonic kidney (HEK-293T), Phoenix-Ecotropic (ECO), human melanoma A375 (HLA-A*0201 + NY-ESO-1 + ), mouse melanoma B16 (H-2Kb + ) and mouse colon carcinoma MC38 (H-2Kb + ) cell lines were purchased from the ATCC.

Techniques: Modification, Functional Assay, Retroviral, Plasmid Preparation, Staining, Live Cell Imaging, Control, Co-Culture Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical model for evaluating human TCRs against chimeric syngeneic tumors

doi: 10.1136/jitc-2024-009504

Figure Lengend Snippet: Hybrid DMβ-T cells exert control of B16-A2Kb:NY tumors and exhibit a superior fitness profile to V49I- and wtc51m-T cells post-transfer. ( A ) Schematic of ACT studies to evaluate tumor control (top) and immune cell infiltration over time (days; D ) (bottom). ( B ) Control of subcutaneous B16-A2Kb:NY tumors in HLA-A2/H-2Kb C57BL/6 transgenic mice, following ACT of mouse CD45.2 + CD8 + TCR-T cells (n≥7 mice/group). ( C ) Mouse survival post-ACT (n≥7 mice/group). ( D ) Number of intratumoral adoptively transferred CD45.2 + CD8 + TCR T cells 3 days post-ACT (timepoint 1) (n≥5 mice/ group). ( E ) Phenotypic analysis of markers for function, inhibition, and activation of adoptively transferred intratumoral CD45.2 + CD8 + TCR-T cells (n≥5 mice/ group), shown as percentage of the T cells. ( F ) Number of intratumoral adoptively transferred CD45.2 + CD8 + TCR-T cells 6 days post-ACT (time point 2) (n=6 mice/ group). ( G ) Percentage expression of inhibitory cell-surface receptors by intratumoral CD45.2 + CD8 + TCR-T cells 6 days post-ACT (n=6 mice/ group). ( B–G ) Representative data of two independent experiments. Statistical analysis by two-way ANOVA ( B ), Mantel-Cox ( C ) or one-way ANOVA ( D–G ), with correction for multiple comparisons by post hoc Tukey’s test ( B–G ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant, ACT, adoptive cell transfer; ANOVA, analysis of variance.

Article Snippet: Human embryonic kidney (HEK-293T), Phoenix-Ecotropic (ECO), human melanoma A375 (HLA-A*0201 + NY-ESO-1 + ), mouse melanoma B16 (H-2Kb + ) and mouse colon carcinoma MC38 (H-2Kb + ) cell lines were purchased from the ATCC.

Techniques: Control, Transgenic Assay, Inhibition, Activation Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO inhibits ASC speck formation during AIM2 inflammasome activation. ( A – D ) LPS (100 ng/mL)-primed BMDMs were pre-treated with CO for 30 min, then transfected with poly(dA:dT) (1 μg/mL) for 2 h. Western blots of ( A ) ASC translocation from Triton X-100 soluble fraction to insoluble fraction, ( B ) ASC oligomerization in the insoluble fraction (pellet), and ( C ) ASC speck images at 400× magnification upon stimulation with poly(dA:dT). ASC and nuclei are depicted in red and blue (DAPI), respectively. White arrowheads point to ASC speck. ( D ) Quantification of cells with ASC specks showed the percentage of positive cells. At least three fields and more than 100 cells were counted for each condition. Data represent the means ± SDs of three independent experiments; ** p < 0.01.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Activation Assay, Transfection, Western Blot, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO inhibits AIM2 speck formation resulting from poly(dA:dT) stimulation. ( A , B ) HEK293T cells were transfected with a FLAG-tagged AIM2 expression vector 24 h prior to the experiment. Cells were pre-treated with CO (10 μg/mL) for 30 min, then stimulated with poly(dA:dT) (1 μg/mL) for 4 h. ( A ) AIM2 speck images at 400× magnification upon stimulation with poly(dA:dT) in cells expressing FLAG-tagged AIM2. AIM2 and nuclei are depicted in red and blue (DAPI), respectively. White arrowheads point to AIM2 speck. ( B ) Statistical analysis of the AIM2 specks showing the percentage of positive cells. At least three fields and more than 100 cells were counted for each condition. Data represent the means ± SDs of three independent experiments; *** p < 0.001.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO reduces IMQ-induced serum IL-17A elevation and the expression of AIM2 inflammasome components in skin tissue. CO and Aldara cream were applied as described in C to obtain serum and prepare skin tissue extracts. ( A ) Serum IL-17A levels measured by an ELISA assay. Data represent the means ± SDs; *** p < 0.001. ( B ) Western blot analysis for the levels of AIM2 inflammasome components in skin extracts.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Expressing, Cream, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: MAGEC2 interacts with TRIM28 in melanoma cell lines. a Endogenous MAGEC2 binds with TRIM28 in A375 cells. A375 cells (1 × 10 8 ) were lysed and immunoprecipitated with mIgG, anti-MAGEC2 or anti-TRIM28 antibodies and immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Mouse IgG (mIgG) was used as a negative control for the immunoprecipitation. b Endogenous MAGEC2 binds with TRIM28 in Hs 695 T cells. Hs 695 T cells (1 × 10 8 ) were lysed and immunoprecipitated with anti-mIgG or anti-TRIM28 antibodies and immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Mouse IgG (mIgG) was used as a negative control for the immunoprecipitation. The experiments were repeated at least three times

Article Snippet: Human melanoma cell line A375 was purchased from ATCC (USA; ATCC® CRL-1619TM), human pancreatic cancer cell line AsPC1 and lung cancer cell line A549 were from National Infrastructure of Cell Line Resource (China; Catalog #: 3111C0001CCC000214 and 3111C0001CCC000002, respectively).

Techniques: Immunoprecipitation, Negative Control

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: Knockdown of TRIM28 reduces MAGEC2 protein level and does not affect MAGEC2 mRNA level in melanoma cells. a , b Knockdown of TRIM28 decreases MAGEC2 protein level in A375 and Hs 695 T cells. TRIM28-specific siRNAs or control siRNA (si-NC) were transfected into A375 ( a ) or Hs 695 T cells ( b ) for 48 h, and cell lysates were immunoblotted with anti-MAGEC2 or anti-TRIM28 antibodies. Expression levels of GAPDH are indicated as an internal control. c , d Knockdown of TRIM28 does not affect the expression levels of other members of MAGE family in A375 cells ( c ) or Hs 695 T cells ( d ). A375 or Hs 695 T cells were transfected with TRIM28-specific siRNA for 48 h, and cell lysates were immunoblotted with indicated antibodies. e , f , g , h Knockdown of TRIM28 does not affect MAGEC2 mRNA level in A375 ( e , f ) or Hs 695 T cells ( g , h ). e , f Expression of MAGEC2 mRNA was examined in TRIM28-specific siRNA transfected-A375 cells by conventional PCR (E) or quantitative real-time PCR ( f ). g , h Knockdown of TRIM28 does not affect MAGEC2 mRNA level in Hs 695 T cells. Expression of MAGEC2 mRNA was examined in TRIM28-specific siRNA transfected-Hs 695 T cells by conventional PCR ( g ) or quantitative real-time PCR ( h ). Data are represented as mean ± SD. Each experiment was repeated three times

Article Snippet: Human melanoma cell line A375 was purchased from ATCC (USA; ATCC® CRL-1619TM), human pancreatic cancer cell line AsPC1 and lung cancer cell line A549 were from National Infrastructure of Cell Line Resource (China; Catalog #: 3111C0001CCC000214 and 3111C0001CCC000002, respectively).

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

Journal: BMC Cancer

Article Title: Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

doi: 10.1186/s12885-018-4844-1

Figure Lengend Snippet: TRIM28 protects MAGEC2 from proteasomal degradation. a , b Downregulation of TRIM28 promotes the proteasome-mediated degradation of MAGEC2 in A375 cells ( a ) or Hs 695 T cells ( b ). TRIM28 specific siRNAs or si-NC were transfected into Hs695 T or A375 cells. Twenty-four hours after transfection, cells were treated with PS-341 (10 μM), MG132 (20 μM) or vehicle (DMSO) for 8 h before immunoblotting with indicated antibodies. c Lysosome inhibitor does not affect the level of MAGEC2 protein in TRIM28 knockdown Hs 695 T cells or A375 cells. TRIM28 specific siRNAs or si-NC was transfected into A375 (left panel) or Hs695 (right panel) T cells. Twenty-four hours after transfection, cells were treated with CQ (20 μM) or vehicle (DMSO) for 24 h before immunoblotting with indicated antibodies. Detection of p62 is served as a positive control for treatment with CQ. Each experiment was repeated at least three times

Article Snippet: Human melanoma cell line A375 was purchased from ATCC (USA; ATCC® CRL-1619TM), human pancreatic cancer cell line AsPC1 and lung cancer cell line A549 were from National Infrastructure of Cell Line Resource (China; Catalog #: 3111C0001CCC000214 and 3111C0001CCC000002, respectively).

Techniques: Transfection, Western Blot, Knockdown, Positive Control

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

Article Title: Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

doi: 10.4049/jimmunol.167.1.98

Figure Lengend Snippet: FIGURE 2. RT-PCR analysis of HLA-D gene transcription in the four melanoma cell lines and in the BUA cell line. The number of amplification cycles is given for each primer set in Table I. cDNAs either from cells treated for 48 h with 250 U/ml IFN-g (1) or from untreated cells (2) and the PCR products were analyzed on ethidium bromide-stained agarose gels. To assess that equal amounts of cDNA were used, a 16-cycle amplification was performed on the cDNAs with GAPDH primers, and the correspond- ing Dig-labeled PCR products were stained with CSPD.

Article Snippet: The HT144 (ATCC: HTB-63), 42/95, M74, and A375 (ATCC: CRL1619) human melanoma cell lines were kindly provided by E. Tartour (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 255, Curie Institute, Paris, France).

Techniques: Reverse Transcription Polymerase Chain Reaction, Staining, Labeling

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

Article Title: Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

doi: 10.4049/jimmunol.167.1.98

Figure Lengend Snippet: FIGURE 1. Cell surface expression of the HLA-DR molecules in the 42/95 and HT144 melanoma cell lines examined by indirect immunoflu- orescence. The cells were either treated with 250 U/ml IFN-g for 48 h or untreated as indicated on the figure (light histograms). The dark histogram corresponds to the isotypic control. The L243 mAb was used to detect the expression of HLA-DR molecules by cytofluorometry.

Article Snippet: The HT144 (ATCC: HTB-63), 42/95, M74, and A375 (ATCC: CRL1619) human melanoma cell lines were kindly provided by E. Tartour (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 255, Curie Institute, Paris, France).

Techniques: Expressing, Control

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

Article Title: Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

doi: 10.4049/jimmunol.167.1.98

Figure Lengend Snippet: FIGURE 4. RT-PCR analysis of CIITA transcript expression in the dif- ferent melanoma cell lines and in the BUA cell line through a 33-cycle amplification procedure. cDNAs were prepared from cells either untreated (2) or treated (1) with 250 U/ml IFN-g for 6 h. The amplification products were detected on ethidium bromide-stained gels. To assess that equal amounts of cDNAs were used, a 16-cycle amplification was performed on the cDNAs with GAPDH-specific primers, and the Dig-labeled amplifica- tion products were stained with CSPD.

Article Snippet: The HT144 (ATCC: HTB-63), 42/95, M74, and A375 (ATCC: CRL1619) human melanoma cell lines were kindly provided by E. Tartour (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 255, Curie Institute, Paris, France).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Labeling

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

Article Title: Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter.

doi: 10.4049/jimmunol.167.1.98

Figure Lengend Snippet: FIGURE 5. RT-PCR analysis of IRF-1 transcript expression in the dif- ferent melanoma cell lines and in the BUA cell line. cDNA quantification with GAPDH-specific primers was performed on Dig-labeled amplified products.

Article Snippet: The HT144 (ATCC: HTB-63), 42/95, M74, and A375 (ATCC: CRL1619) human melanoma cell lines were kindly provided by E. Tartour (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 255, Curie Institute, Paris, France).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Labeling

Journal: PeerJ

Article Title: lncRNA H19 is involved in TGF- β 1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT Signaling Pathway

doi: 10.7717/peerj.3950

Figure Lengend Snippet: (A) Expression of TGF-β1 gene in inflammatory MAC-T cells was analyzed by RT-qPCR. (B) Expression of EMT markers (E-cadherin, N-cadherin, pan-cytokeratins, vimentin, β-catenin and α-SMA), ECM protein (albumin, MMP9, collagen type I and fibronectin ) in MAC-T cells were analyzed by western blotting. (C) Expression of H19 in MAC-T cells upon TGF-β1 treatments was analyzed by RT-qPCR. Untreated MAC-T cells were used as a control. GAPDH was used as an internal control. ∗ p < 0.01 vs control.

Article Snippet: The membranes were blocked with 10% non-fat milk and incubated with anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-N-cadherin (Bioss, Beijing, China), albumin (Bioss), matrix metalloproteinase 9 (MMP9) (Bioss), vimentin (Bioss), pan-cytokeratin (a wide spectrum cytokeratin antibody, Bioss), fibronectin (Bioss), β-catenin (Sigma) and anti-GAPDH (TransGene) antibodies at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: PeerJ

Article Title: lncRNA H19 is involved in TGF- β 1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT Signaling Pathway

doi: 10.7717/peerj.3950

Figure Lengend Snippet: (A) Information for the lentviral vector CD513B-1. BamHI and NotI sites in CD513B-1 were used to construct the expression plasmid. (B) Fluorescence microscopy examination of MAC-T cells transfected with lentviral vectors. GFP expression was observed by fluorescence microscopy. Up, MAC-T cells infected with CD513B-1 or CD513B-1-H19 were detected in bright field; down, MAC-T cells infected with CD513B-1 or CD513B-1-H19 were detected in dark field. (C) Analysis of H19 expression in MAC-T cells with empty vector or CD513B-1-H19 by RT-qPCR. (D) The expression of EMT marker proteins (E-cadherin, N-cadherin, pan-cytokeratin, β-catenin, vimentin and α-SMA) and ECM proteins (albumin, collagen 1, fibronectin and MMP9) were evaluated in H19-overexpressing MAC-T cells by western blotting. Control vector-transduced cells were used as a control. GAPDH was used as an internal control. ∗ p < 0.05 vs control.

Article Snippet: The membranes were blocked with 10% non-fat milk and incubated with anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-N-cadherin (Bioss, Beijing, China), albumin (Bioss), matrix metalloproteinase 9 (MMP9) (Bioss), vimentin (Bioss), pan-cytokeratin (a wide spectrum cytokeratin antibody, Bioss), fibronectin (Bioss), β-catenin (Sigma) and anti-GAPDH (TransGene) antibodies at 4 °C overnight.

Techniques: Plasmid Preparation, Construct, Expressing, Fluorescence, Microscopy, Transfection, Infection, Quantitative RT-PCR, Marker, Western Blot