Journal: ACS Nano

Article Title: A Thermogenetic Tool Employing Elastin-like Polypeptides for Controlling Programmed Cell Death

doi: 10.1021/acsnano.5c07332

Figure Lengend Snippet: Heat-activated human CASP8 in HEK293T cells. (A) Gene design. A fluorescent marker, mTagBFP2, along with a self-cleavage peptide, P2A, is fused upstream the ELP­[V60]-CASP8 (left) or ELP­[V60]-CASP8_C360A mutant (right). (B) Fluorescence images of HEK293T cells expressing mTagBFP2-ELP­[V60]-CASP8 or mTagBFP2-ELP­[V60]-CASP8_C360A mutant with Annexin XII staining at different temperatures for 24 h. The blue color represents mTagBFP2 and the green color represents Annexin XII signals. (C) A graph of the fluorescence ratio of Annexin XII/mTagBFP2 against temperature after 24 h of incubation. (D) Cytotoxicity assay of HEK293T cells expressing mTagBFP2-ELP­[V60]-CASP8 or mTagBFP2-ELP­[V60]-CASP8_C360A mutant at different temperatures for 24 h. Cell cytotoxicity at different times of HEK293T cells expressing (E) mTagBFP2-ELP­[V60]-CASP8 or (F) mTagBFP2-ELP­[V60]-CASP8_C360A mutant. ns indicates no significant difference, while * ( p < 0.05), ** ( p < 0.01), and *** ( p < 0.001) denote statistically significant differences, as determined using a two-tailed Student’s t -test. Data show mean ± SD ( n = 5). Bright field (BF) and fluorescence images of HEK293T cells coexpressing the CASP8 indicator and (G) mTagBFP2-ELP­[V60]-CASP8 or (H) mTagBFP2-ELP­[V60]-CASP8_C360A mutant. Time-lapse imaging of HEK293T cells coexpressing the CASP8 indicator and (I) mTagBFP2-ELP­[V60]-CASP8 or (K) mTagBFP2-ELP­[V60]-CASP8_C360A mutant at 30 and 39 °C. Arrows indicate CASP8-activating cells. Scale bar, 200 μm for panel (B) and 50 μm for panels (G,H,I,K).

Article Snippet: We purchased plasmids encoding ELP [V60] containing the sequence (VPGVG) 60 (plasmid #67013), human caspase 8 (plasmid #11817), mCherry (plasmid #124429), and mTagBFP2 (plasmid #55327) from Addgene.

Techniques: Marker, Mutagenesis, Fluorescence, Expressing, Staining, Incubation, Cytotoxicity Assay, Two Tailed Test, Imaging

Journal: ACS Nano

Article Title: A Thermogenetic Tool Employing Elastin-like Polypeptides for Controlling Programmed Cell Death

doi: 10.1021/acsnano.5c07332

Figure Lengend Snippet: Local heat activation of human CASP8 at single cells. (A) Schematic illustration of the optical heating using 1470 nm laser combined with FLIM-based thermometry for precise temperature control and measurement in single cells. (B) Calibration of fluorescence lifetime of mCherry in the CASP8 indicator as a function of medium temperature. The temperature sensitivity of mCherry was determined to be 0.018 ns/°C, which is based on the slope of the linear fitting curve. (C) Optical heating of single HEK293T cells expressing the CASP8 indicator. The cell temperature was calculated using the calibration curve shown in panel (B) and plotted as a function of the 1470 nm laser power. (D) Cytotoxicity of HEK293T cells expressing mTagBFP2-ELP­[V60]-CASP8 after incubation at 39 °C for various durations. The inset shows an enlarged view from 0 to 120 min. The * denotes statistically significant differences ( p < 0.05), as determined using two-tailed Student’s t -test. Data show mean ± SD ( n = 5). (E) Optical heating of HEK293T cells coexpressing mTagBFP2-ELP­[V60]-CASP8 and the CASP8 indicator, with corresponding FLIM images shown in (F). Blue arrows indicate CASP8-activating cells. (G) Optical heating of HEK293T cells coexpressing mTagBFP2-ELP­[V60]-CASP8_C360A mutant and the CASP8 indicator, with its FLIM images shown in (H). Cells were maintained at 30 °C under 5% CO 2 . Color bars indicate fluorescence lifetime (τ). Scale bars, 20 μm. Illustrations were created with BioRender.com.

Article Snippet: We purchased plasmids encoding ELP [V60] containing the sequence (VPGVG) 60 (plasmid #67013), human caspase 8 (plasmid #11817), mCherry (plasmid #124429), and mTagBFP2 (plasmid #55327) from Addgene.

Techniques: Activation Assay, Control, Fluorescence, Expressing, Incubation, Two Tailed Test, Mutagenesis

Journal: ACS Nano

Article Title: A Thermogenetic Tool Employing Elastin-like Polypeptides for Controlling Programmed Cell Death

doi: 10.1021/acsnano.5c07332

Figure Lengend Snippet: Local heat activation of human CASP8 at single cells. (A) Schematic illustration of the optical heating using 1470 nm laser combined with FLIM-based thermometry for precise temperature control and measurement in single cells. (B) Calibration of fluorescence lifetime of mCherry in the CASP8 indicator as a function of medium temperature. The temperature sensitivity of mCherry was determined to be 0.018 ns/°C, which is based on the slope of the linear fitting curve. (C) Optical heating of single HEK293T cells expressing the CASP8 indicator. The cell temperature was calculated using the calibration curve shown in panel (B) and plotted as a function of the 1470 nm laser power. (D) Cytotoxicity of HEK293T cells expressing mTagBFP2-ELP­[V60]-CASP8 after incubation at 39 °C for various durations. The inset shows an enlarged view from 0 to 120 min. The * denotes statistically significant differences ( p < 0.05), as determined using two-tailed Student’s t -test. Data show mean ± SD ( n = 5). (E) Optical heating of HEK293T cells coexpressing mTagBFP2-ELP­[V60]-CASP8 and the CASP8 indicator, with corresponding FLIM images shown in (F). Blue arrows indicate CASP8-activating cells. (G) Optical heating of HEK293T cells coexpressing mTagBFP2-ELP­[V60]-CASP8_C360A mutant and the CASP8 indicator, with its FLIM images shown in (H). Cells were maintained at 30 °C under 5% CO 2 . Color bars indicate fluorescence lifetime (τ). Scale bars, 20 μm. Illustrations were created with BioRender.com.

Article Snippet: We purchased plasmids encoding ELP [V60] containing the sequence (VPGVG) 60 (plasmid #67013), human caspase 8 (plasmid #11817), mCherry (plasmid #124429), and mTagBFP2 (plasmid #55327) from Addgene.

Techniques: Activation Assay, Control, Fluorescence, Expressing, Incubation, Two Tailed Test, Mutagenesis