Fig 1: dAdo generated by tNT triggers caspase-3-dependent death of mouse macrophage RAW 264.7 cells. (a), In all the groups in the presence of 100 µM dCF (orange columns), the viability of RAW 264.7 cells in the tNT+ dAMP group (65.66%, 63 mM dAMP pretreated with 6 µM tNT at 37°C for 2 h) was significantly lower than that of cells in the H2O (95.33%), 6 µM tNT (86.00%), 63 mM dAMP (76.33%), or working buffer (81.33%) groups. There was no significant difference of cell viability between the tNT+ dAMP group and the 55 µM dAdo group (66.66%, as a positive control) in the presence of dCF. A significant difference of cell viability was observed in tNT+ dAMP groups (with or without dCF) and dAdo groups (with or without dCF). The viability of RAW 264.7 cells was measured by the trypan blue staining. Statistical significance was examined by one-way ANOVA and Dunnett’s multiple comparisons test. ns, not significant; *** P < 0.001; n = 3. (b), Western blot analysis showed that the band of active caspase-3 was detected in RAW 264.7 cells treated with 55 µM dAdo and 100 µM dCF for 12 h, 16 h, or 24 h. Jurkat cell extract treated with cytochrome c in vitro, obtained from Cell Signaling Technology (#9663), was used as a positive control. (c), The band of active caspase-3 was also detected in RAW 264.7 cells treated with dAMP that had been incubated with tNT in the presence of dCF for 12 h. ß-actin was used as a loading control. Experiments were done in triplicate.
Fig 2: Effects of CPT on the invasiveness of human bladder cancer cells in vitro. (A) Western blot detection of PDH and PDK4 in T24 and J82 cells treated with CPT for 48 h. α-tubulin was used as the internal control. (B) Effects of CPT on the viability of spheroids formed from T24 and J82 cells. Bars indicate the mean ± SD. Adenosine triphosphate content was assessed using CellTiter-Glo® 3D assays. **P<0.001 compared with untreated cells using Tukey's test. Effects of CPT on the invasiveness of (C) T24 and (D) J82 cells. Each sample was assayed in triplicate, and the bars represent the mean ± SD. Images of T24 cells invading the Matrigel membrane. Images were captured at ×100 magnification. **P<0.001 compared with untreated cells using Tukey's test. (E) Western blot analysis of caspase-3 cleavage in T24 and J82 cells treated with CPT. Untreated (−) or cytC-treated Jurkat cell extracts (caspase-3 control cell extracts, Cell Signaling Technology, Inc.) were used as negative or positive controls, respectively. α-tubulin was used as the internal control. (F) Apoptotic cytotoxicity of CPT in T24 and J82 cells. Stacked bars indicate the cell cycle profiles after treatment with CPT for 24 h; the sub-G1 fraction, presented as the black area on the top of each bar, indicates the proportion of the apoptotic cells. CPT, cryptotanshinone; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4; p-, phosphorylated; cytC, cytochrome C.
Fig 3: FAK controls necroptotic cell death in macrophages during Mtb infection. (A) THP-1, THP-FAKi, and THP-FAK+ macrophages were infected with Mtb at an MOI of 10, and cell lysates were prepared at the indicated days post-infection (d.p.i.). Total and cleaved caspase 3, and total FAK protein levels were analyzed by western blotting. Non-infected THP-1 cell lysates were used as a negative control for caspase 3 activation, while lysates containing cleaved caspase-3 (#9663, Cell Signaling Technologies) were used as a positive control. ß-tubulin was used as loading control. (B) THP-1, THP-FAKi, and THP-FAK+ macrophages were infected as in (A) and at the indicated days post-infection, cells were stained with annexin V and FVS780. Stained cells were analyzed by flow cytometry to quantify the percentage of healthy, apoptotic, and necrotic cells as described in the Methods. (C) THP-1, THP-FAKi, and THP-FAK+ macrophages were infected with Mtb at an MOI of 10, and lactate dehydrogenase (LDH) released in culture supernatants were assessed using the CYQUANT LDH kit at indicated days post-infection. The amount of LDH in the supernatant is proportional to the measured absorbance at 490 nm. (D) THP-1, THP-FAKi and THP-FAK+ macrophages were mock treated or pre-treated with 1 µg/ml LPS for 4 h, followed by treatment with 5 µM nigericin for 24 h Macrophage viability was assessed using the Cell Titre-Glo assay. (E–G) THP-1, THP-FAKi, and/or THP-FAK+ macrophages were mock treated or pre-treated with (E) necrostatin-1s (Nec-1s, 10 µM), (F) GSK-872 (5 µM), or (G) necrosulfonamide (NSA, 10 µM) for 24 h Cells were then infected with Mtb at an MOI of 10 for 6 days, and cell viability was assessed using the Cell Titer-Glo assay. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, ***p < 0.001, ns, non-significant.
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