Fig 1: Compound 6d induces mitochondrial-mediated intrinsic apoptosis by targeting Bcl-2. (A) CRC cells were treated with indicated concentrations of compound 6d for 8 h and then stained with the Annexin V-FITC/PI kit. The stained cells were analyzed with flow cytometry analysis. (B) Compound 6d regulates the expression of apoptosis-related proteins. CRC cells were treated with different concentrations of compound 6d (0, 5, 10, 15 μmol/L) for 8 h. The release of cytosol cytochrome c, expression of the BCL2 family proteins Bcl-2 and Bax, the cleavage levels of activated caspase 3 and poly (ADP-ribose) polymerase (PARP), were estimated to determine whether or not the intrinsic apoptotic pathway is involved in the anti-tumor effect. β-actin level was used as internal control for equal protein loading. (C) Compound 6d treatment increased cytosolic cytochrome c levels. Control and compound 6d (15 μM)-treated cells were subjected to cell fractionation using kit to separate cytoplasmic and mitochondrial fraction, and then cytosolic and mitochondrial cytochrome c levels were detected through western blotting, respectively. The translocase of outer mitochondrial membrane 20 homolog (Tomm 20) was used as a mitochondrial marker. (D) Compound 6d inhibits the interaction between exogenous Bcl-2 and endogenous Bax, but not the interaction of Bcl-xL and Bax. After treatment, lysates from HCT116 cells transfected with HA-Bcl-2 or HA-Bcl-xL were pulled down with anti-HA, followed by WB with corresponding primary antibodies. **** p < 0.0001 versus vehicle. (E) In vitro binding assay was performed using recombinant His-Bcl-2 and His-Bax proteins. Anti-Bcl-2 antibody was used for pull-down after incubating recombinant proteins with compound 6d, AT101 and GX15-070, and then anti-His antibody was employed for WB. Both AT101 and GX15-070 were used as positive control.
Fig 2: HDAC6 is a direct substrate of caspase 3 and is cleaved by caspase 3 in response to compound 6d. (A) HDAC6 is cleaved by caspases in the presence of compound 6d. A cleaved caspase substrate motif (DE (T/S/A)D) antibody was used to identify endogenous levels of caspase-cleaved proteins with a C-terminal aspartic acid residue. IP was performed using an anti-HDAC6 antibody, and was then subjected to WB with the cleaved caspase substrate motif (DE (T/S/A)D) antibody. (B) Compound 6d promotes the punctuated accumulation of caspase 3 and co-localization between caspase 3 and HDAC6. After 12 h of incubation with compound 6d, HCT116 cells were fixed, permeabilized, and stained with anti-HDAC6 antibody (green), anti-caspase 3 antibody (red), and DAPI (blue). Scale bar, 10 μm. (C) HDAC6 is a direct substrate of caspase 6. In vitro cleavage experiment was employed using exogenous expressed HDAC6 and cleaved caspase 3, the active form of caspase 3. The samples were analyzed using WB with corresponding antibodies after 2 h of incubation. FL: full-length of HDAC6; P140: a ~140 kDa long-cleaved band of HDAC6; P17: a ~17 kDa short-cleaved band of HDAC6. (D) Full-length of HDAC6 was recovered in the presence of caspase 3 inhibitor. HCT116 cells were treated with compound 6d for 4 h, and these cells were subsequently exposed in the absence and presence of the caspase 3 inhibitor Z-DEVD-FMK (20 μM). Following that, the lysates were collected and subjected to WB with indicated antibodies. β-tubulin was loaded as a control. (E) Caspase 3 depletion eliminates the effect of compound 6d on HDAC6 cleavage. HCT116 cells were transfected with caspase 3 shRNA and scramble shRNA, and were then subjected to WB after treatment with or without compound 6d for 4 h. (F) HDAC6 cleavage induced by compound 6d depends on the presence and activation of caspase 3. Different cancer cells, such as MCF7 (absence of caspase 3), Hep3B, A549, U87, and HCT116, were treated with indicated concentrations of compound 6d, and then subjected to WB using the corresponding antibodies. “*” represented non-specific band.
Fig 3: Multicolor MIP imaging of nitrile chameleons provides evidence of caspase-phosphatase cooperation in apoptosis.(a) Simultaneous visualization of phosphatase and caspase-3/7 activity profile in Dox-pretreated SJSA-1 cells.(b) Colocalization analysis of the mapping in (a).(c) Study of spatial interaction between phosphatase and caspase-3/7 in Dox-pretreated SJSA-1 cells.(d) Intensity plot of Phos-CN(P) along the arrows in (c).(e) Correlation scatterplot of caspase-3/7 and phosphatase activity in Dox-pretreated SJSA-1 cells
Fig 4: Real-time MIP imaging of nitrile chameleons generates the activity maps of caspase 3/7 and phosphatase in living cells.(a) Schematic illustration of the principle of enzyme activity mapping by real-time MIP imaging of nitrile chameleons(b) MIP images of phosphatase activity profile in living SJSA-1 cells(c) Pinpointed MIP spectrum (indicated by arrow)(d) Quantification of MIP signal intensity of Phos-CN(S) and Phos-CN(P) in cell(e) MIP images of phosphatase activity profile in phosphatase inhibitor-pretreated SJSA-1 cells(f) Quantification of MIP signal intensity of Phos-CN(S) and Phos-CN(P) in phosphatase inhibitor-pretreated SJSA-1 cells(g) Statistic of product-to-substrate ratio ([P]/[S]) in the cells from PIC-pretreated and PIC-free groups.(h) MIP images of caspase 3/7 activity profile in Doxorubicin-pretreated SJSA-1 cells(i) Pinpoint MIP spectrum (indicated by arrow)(j) Quantification of MIP intensity of Casp-CN(S) and Casp-CN(P) in Dox-pretreated cells and the cells from Dox-free control group
Fig 5: Development of nitrile chameleons for mapping specific enzyme activity.(a) Molecular structures of Casp-CN(S) and the enzymatic product Casp-CN(P)(b) Molecular structures of Phos-CN(S) and the enzymatic product Phos-CN(P)(c) MIP spectra of Casp-CN(S) and the enzymatic product Casp-CN(P), 50 mM in DMSO(d) MIP spectra of Phos-CN(S) and the enzymatic product Phos-CN(P), 50 mM in DMSO(e) and (f) MIP signal intensity of (e) Casp-CN(P) and (f) Phos-CN(P) at different concentration concentrations in DMSO.(g) Time-dependent formation of Casp-CN(P) catalyzed by active caspase 3 (25 U/mL)(h) Time-dependent formation of Phos-CN(P) catalyzed by alkaline phosphatase (ALP, 0.5 U/mL)(i) TEM images of the nano-assemblies formed by Casp-CN(S) before and after the addition of active caspase 3 (25 U/mL, 6 h)(j) TEM images of the nano-assemblies formed by Phos-CN(S) before and after the addition of alkaline phosphatase (ALP, 0.5 U/mL 6 h)
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