Journal: Scientific Reports

Article Title: 5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells

doi: 10.1038/s41598-017-04756-9

Figure Lengend Snippet: 5T4-28Z CAR gene modification promotes the anti-NPC activity of CIK cells in a 5T4-dependent manner. Target cells consisted of 5T4 low-expressing S18 and S26 cells and their spheroid cells with an intermediate or high level of 5T4 expression. Effector cells included 5T4-28Z-CIK, 5T4-28Z-CIK (NKG2D-), C-28Z-CIK and NT-CIK cells. ( a–d ) Briefly, 1 × 10 4 target cells per well were co-cultured with different effector cells at E/T ratios of 10/1, 5/1 and 2.5/1 for 4 h. The cytotoxicity of effector cells was examined with LDH release assays. Values in the line graphs represent the mean ± SD of three parallel wells. ( e ) Then, 2 × 10 4 target cells per well were co-incubated with 1 × 10 5 effector cells per well for 24 h. Effector cells cultured alone in medium served as the negative control. IFN-γ production of the effector cells was assessed using ELISAs. Values in the histogram represent the mean ± SD of three parallel wells. ( f ) In addition, 2 × 10 5 target cells per well were co-cultured with 1 × 10 6 effector cells per well for 5 h in the presence of 1 × Protein Transport Inhibitor Cocktail and anti-human CD107α-APC antibody or IgG isotype control. Effector cells cultured alone served as the negative control. Degranulation of effector cells was evaluated using FACS. Values presented in the histogram represent the mean ± SD of triplicate samples. ( g ) S26 cells or S26 spheroids labelled with CM-Dil were co-cultured with 5T4-28Z-CIK cells and monitored using an inverted fluorescence microscope with climate control. Images captured intermittently are displayed. The results are representative of at least three independent experiments. * indicates p < 0.05 (one-way ANOVA).

Article Snippet: Next, 50 μl of supernatant per well was collected to measure LDH release using a cytotoxicity LDH Assay Kit-WST ® (Dojindo) according to the manufacturer’s specifications.

Techniques: Modification, Activity Assay, Expressing, Cell Culture, Incubation, Negative Control, Control, Fluorescence, Microscopy

Journal: BMC oral health

Article Title: Keratinocyte necroptosis promotes the progression of radiation-induced oral mucositis.

doi: 10.1186/s12903-025-06278-7

Figure Lengend Snippet: Fig. 4 Pretreatment with RIPK3 inhibitor or MLKL inhibitor reduces radiation-induced cell death and inflammation. (A) Lactate dehydrogenase (LDH) release was measured in HaCaT cells cultured for 2.5 days post-irradiation with a dose of 12 Gy, in the presence or absence of the RIPK3 inhibitor GSK’872 or the MLKL inhibitor GW806742X. (B) Flow cytometry analysis of HaCaT cells stained with propidium iodide (PI) and annexin V, and (C) quantification of double-positive cells (PI and annexin V staining). (D) Representative fluorescent images of Hoechst 33342 (blue) and PI (red) double staining. Scale bar: 100 μm. (E, F) The mRNA expression levels of inflammatory cytokines by qRT-PCR, including IL-1β, IL-6, and TNF-α. Results are shown as ± SD means of three replicates from three independent experiments. Statistical significance was determined using one-way ANOVA and LSD post hoc tests. *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Cytotoxicity was evaluated using the Lactate Dehydrogenase (LDH) Assay Kit (Beyotime).

Techniques: Cell Culture, Irradiation, Flow Cytometry, Staining, Double Staining, Expressing, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism

doi: 10.3390/biomedicines9091192

Figure Lengend Snippet: Cells from condensed areas of the cell sheet (CS) are characterized by an increased content of the cytochrome-C oxidase IV component (COX-IV) and higher lactate dehydrogenase (LDH) activity. ( A ) Western blotting of the mitochondrial ETC COX-IV subunit in lysates of ML, total CS and microdissected condensed or scattered areas. Graphs present a densitometry analysis of the COX-IV content normalized to β-actin (densitometry analysis). ( B ) LDH activity (LDHact) in the lysates of microdissected condensed areas and scattered areas. DNA amounts in the respective cell lysates were used for the normalization of LDH activity to the cell number. Control data was obtained from lysates of the A431 cell line that increased the proportion of anaerobic glycolysis and the THP-1 cell line with the major input of oxidative phosphorylation but not glycolysis. The band numbers indicate individual biological replicate samples. In the figure graphs and histograms, significant differences were marked by * ( p < 0.05) and ** ( p < 0.005).

Article Snippet: The enzymatic activity of lactate dehydrogenase (LDH) in condensed and scattered areas of CS was assayed in the samples obtained immediately after laser microdissection using a Lactate Dehydrogenase (LDH) Assay Kit (Fluorometric) (ab197000, Abcam, Cambridge, UK) according to the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot

Journal: PloS one

Article Title: Low levels of GSTA1 expression are required for Caco-2 cell proliferation.

doi: 10.1371/journal.pone.0051739

Figure Lengend Snippet: Figure 5. Distinct doses of NaB differently affect cell proliferation and AlkP and GSTA1 enzyme activities. Preconfluent Caco-2 cells were treated with NaB (1 mM and 10 mM) in serum-free media. (A) Cellular proliferation was assessed from 24–96 h. Asterisks depict significant differences between control and NaB treatments (*, p#0.05; **, p#0.01 and ***, p#0.001). (B) Cytotoxicity was determined in preconfluent and postconfluent Caco-2 cells treated with 1 mM and 10 mM NaB at 48 h. Cytotoxicity measured LDH release and presented as % cytotoxicity. (C) AlkP activity (mmol/mg/min) and (D) GSTA1 activity (nmol/mg/min) was determined. Values represent the mean 6 S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p#0.001). doi:10.1371/journal.pone.0051739.g005

Article Snippet: Cells were treated with NaB (1 mM and 10 mM) for 24 h to 72 h. LDH release was measured at 544/590 nm using a FluoStar OPTIMA fluorimeter (BMG Labtech).

Techniques: Control, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage *

doi: 10.1074/jbc.M110.197483

Figure Lengend Snippet: TDP-43 induces death of motor neuronal NSC34 cells and primary cortical neurons. A–C, NSC34 cells, seeded on 6-well plates at 1 × 105/well, were co-infected with LacZ or TDP-43 virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. Cell mortality was assessed by microscopic views of cells attached to the dishes (B) and FACS analysis (C) at 48 h after infection. TDP-43 was detected with immunoblot (IB) analysis with TDP43-C antibody (A). TDP43-C antibody was raised against the C-terminal fragment of “human” TDP-43. D and E, primary cortical neurons, seeded on 96-well plates at 5 × 104 cells/well, were infected with LacZ or TDP-43 virus at a m.o.i. of 100 (WST-8 assay) or 400 (LDH assay and Calcein assay). These viruses are able to express proteins without co-expression of cre-recombinase. Cell mortality (LDH assay) and cell viability (WST-8 assay and Calcein assay) were assessed at 5 and 7 days after infection, respectively (D). Data were analyzed with Student's t test. Immunoblot (IB) analyses were performed with antibody to TDP43-C or cleaved caspase-3 (E).

Article Snippet: 24 h after the medium replacement, LDH release from cells was measured with an LDH assay kit (Wako).

Techniques: Infection, Western Blot, Lactate Dehydrogenase Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage *

doi: 10.1074/jbc.M110.197483

Figure Lengend Snippet: TDP-43-induced death is associated with the increase of Bim expression and the decrease of Bcl-xL expression. A and B, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with LacZ or TDP-43 virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. After infection, some cells were co-incubated with 100 μm Z-Asp-CH2-DCB. At 24 h, medium was replaced with DMEM/N2 supplement with or without 100 μm Z-Asp-CH2-DCB. 24 h after medium replacement, LDH release was measured (A). Immunoblot (IB) analyses were performed with antibodies to cleaved-poly(ADP-ribose) polymerase (PARP), Bim, Bcl-xL, and TDP43-C (B). The ratio of immunoreactive density of Bim and Bcl-xL to that of GAPDH was measured using ImageJ software. C and D, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were infected with LacZ or TDP-43 virus at a m.o.i. of 400. These viruses are able to express proteins without co-expression of cre-recombinase. 24 h after infection, medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, immunoblot (IB) analyses were performed with antibodies to TDP43-C, cleaved caspase-3, Bim, Bcl-xL, Bcl-2, and Mcl-1 (C) and quantitative real-time PCR analysis for Bim, Bcl-xL, Bcl-2, and Mcl-1 were performed (D). Data were analyzed with Student's t test. E, NSC34 cells, seeded on 48-well plates at 2.5 × 104 cells/well, were transfected with 0.1 μg/well of the Bim-luc vector in association with 0.1 μg/well of pEF1-Myc/His-vec or pEF1-TDP-43. At 48 h after transfection, luciferase activity was measured by dual-luciferase assays. Data were analyzed with Student's t test. Immunoblot analysis was performed with TDP43-C antibody.

Article Snippet: 24 h after the medium replacement, LDH release from cells was measured with an LDH assay kit (Wako).

Techniques: Expressing, Infection, Incubation, Western Blot, Software, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage *

doi: 10.1074/jbc.M110.197483

Figure Lengend Snippet: TDP-43 induces cell death by up-regulating Bim. A and B, NSC34 cells, seeded on 6-well plates at 7 × 104 cells/well, were transfected with 5 nm si-control (Cont.), siBim#1, or siBim#2 using Lipofectamine 2000 reagent. 24 h after transfection, cells were infected with LacZ or TDP-43 virus at a m.o.i. of 800. All samples were co-infected with cre-recombinase virus at a m.o.i. of 40. 24 h after infection, medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, LDH release was measured (A). Immunoblot (IB) analyses were performed with antibodies to TDP43-C, Bim, and cleaved caspase-3 (B).

Article Snippet: 24 h after the medium replacement, LDH release from cells was measured with an LDH assay kit (Wako).

Techniques: Transfection, Infection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage *

doi: 10.1074/jbc.M110.197483

Figure Lengend Snippet: TDP-43-induced death is associated with the down-regulation of Bcl-xL. A and B, treatment with MG132 inhibits TDP-43-induced cell death. NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with LacZ or TDP-43 virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced by DMEM/N2 supplement with or without 2 μm MG132. 24 h after medium replacement, LDH release was measured (A). Immunoblot (IB) analyses were performed with antibodies to Bcl-xL, cleaved caspase-3, Bim, and TDP43-C. The ratio of immunoreactive density of Bcl-xL to that of GAPDH was measured for each lane using ImageJ software (B). C and D, Bcl-xL overexpression inhibits TDP-43-induced cell death. NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with LacZ or TDP-43 virus at a m.o.i. of 400 in association with LacZ or Bcl-xL virus at a m.o.i. of 400. All samples were co-infected with cre-recombinase virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced by DMEM/N2 supplement in the presence or absence of 0.02 μm staurosporine (STS). 24 h after medium replacement, LDH release was measured (C). Immunoblot analyses were performed with antibodies to cleaved caspase-3, Bim, Bcl-xL, and TDP43-C (D).

Article Snippet: 24 h after the medium replacement, LDH release from cells was measured with an LDH assay kit (Wako).

Techniques: Infection, Western Blot, Software, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage *

doi: 10.1074/jbc.M110.197483

Figure Lengend Snippet: Cleavage of TDP-43 leads to attenuation of TDP-43-induced death. A, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with the indicated virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced with DMEM/N2 supplement with or without 2 μm MG132. 24 h after medium replacement, LDH release from cells was measured. B, immunoblot (IB) analyses were performed with antibodies to cleaved caspase-3, Bim, Bcl-xL, and TDP43-C. #, GAPDH band. C and D, U2OS cells, seeded on 6-well plates at 5 × 104/well, were co-infected with TDP-43-(1–414) or TDP-43-(170–414) virus at a m.o.i. of 100 for TDP-43-(1–414) or 800 for TDP-43-(170–414) in association with cre-recombinase or LacZ virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, LDH release was measured (D). Immunoblot analysis was performed with antibodies to TDP43-C (C). <, TDP-43-(1–414); ≪, TDP-43-(170–414). E and F, NSC34 cells, seeded on 6-well plates at 1 × 105/well, were co-infected with TDP-43-wt or -D89E/D169E virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, LDH release was measured (F). Immunoblot analysis was performed with antibody to TDP43-C (E). <, CTF35; #, an uncharacterized protein, migrating slightly slower than CTF35. exp, exposure. G and H, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with an indicated adenovirus at a m.o.i. of 400. All samples were co-infected with cre-recombinase virus at a m.o.i. of 40. 24 h after infection, cell medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, LDH release from cells was measured (G). Immunoblot analysis was performed with antibodies to cleaved caspase-3, Bim, Bcl-xL, and TDP43-C (H).

Article Snippet: 24 h after the medium replacement, LDH release from cells was measured with an LDH assay kit (Wako).

Techniques: Infection, Western Blot