Journal: Current biology : CB

Article Title: Stable transfection in protist Corallochytrium limacisporum identifies novel cellular features among unicellular animals relatives.

doi: 10.1016/j.cub.2021.06.061

Figure Lengend Snippet: Figure 2. Stable transfection of Corallochy- trium limacisporum by nucleofection (A) Transfection constructs: mCherry fused to pu- romycin resistance gene (pac) under the control of either promoter variant: C. limacisporum actin (Pact, CAMP) or a-tubulin (Patub, CTMP) pro- moters and in both cases with the terminator from the actin gene of Abeoforma whisleri (Awhi_actT). (B) Steps of the protocol. (a) Cells at the expo- nential phase of growth (1.5 3 106 cells) are washed with 13 PBS from culture medium. (b) Cells are mixed with transfection construct (10 mg) and carrier DNA (40 mg) and 20 mL of Lonza P3 commercial buffer and subjected to transfection program in Lonza 4D-Nucleofector System (code EN-138). (c) Cells are then transferred to 1 mL of culture medium (marine broth) for recovery period of 48 h. (d) Puromycin at the concentration of 300 mg/mL is added for selection and enrichment of positive cells. (e) After 48 h, cells are transferred to marine agar plates supplemented with puro- mycin and grown for 5–7 days. (f) Pink-rounded clonal colonies can be isolated for further manip- ulations and preserved at 80C. (C and D) Examples of established clonal lines of C. limacisporum India transfected with CAMP plasmid and grown in the presence of 300 mg/mL puromycin either on solid medium, scale bar 5 mm (C) or in liquid medium, scale bar is 10 mm (D). Wild- type (not-transfected) lines do not grow in the se- lective medium and they don’t show mCherry fluorescence in the non-selective medium. See also Figures S1A and S1B for information on plasmid integration, Figure S1C for transfection efficiency, Figures S1D and S1E for evaluation of promoter strength, and Figures S1F and S1G for stability.

Article Snippet: The final constructs were named CAMP (Addgene #104446) and CTMP (Addgene #129560), respectively (Figure 2A).

Techniques: Stable Transfection, Transfection, Construct, Control, Variant Assay, Concentration Assay, Selection, Isolation, Plasmid Preparation

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 1. Localization of CTMP in mitochondria. (A) Cytoplasm and mitochondria fractions were isolated from A549 cells. Total lystates, the cytosolic fraction, and mitochondria were analyzed by Western blotting (COXIV-mitochondria marker and α-tubulin-cytosolic marker). (B) A549 cells were transiently co-transfected with pEGFP-N1-CTMP and pDsRed-Mito for 24 h, and cells were observed using confocal microscopy. (C) A549 cells stably expressing pDsRed-Mito were infected with lentivirus-CTMP for 24 h and immunostained with anti-CTMP antibody. (D) The mitochondria fraction from A549 cells was isolated and treated with the indicated reagents. Samples were separated into supernatant (S) and precipitate (P) fractions, and then analyzed by immunoblotting with the indicated antibodies (cytochrome c, mitochondria membrane space protein; and COXIV, mitochondria membrane protein). (E) Immunofluorescence assay of apoptosis-inducing factor (AIF). A549 cells were grown on glass coverslips and transiently transfected with pEGFP-N1-CTMP. The cells were then stained with anti-AIF antibody (scale bar, 10 µm).

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Isolation, Western Blot, Marker, Transfection, Confocal Microscopy, Stable Transfection, Expressing, Infection, Membrane, Immunofluorescence, Staining

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 2. CTMP induces apoptosis in A549 cells. (A) A549 cells were infected with lentivirus-CTMP for 24 h and treated with 1 µM staurosporine for the indi cated times (6 and 8 h). (B) A549 cells were infected with lentivirus-CTMP for 24 h and treated with 0.2 µM actinomycin D for the indicated times (12 and 24 h). (C) Western blot analysis was performed to measure CTMP expression in A549 cells after siRNA transfection for the indicated times. Right panel, densitometric analysis of CTMP expression in A549 cells after siRNA transfection for 48 h. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values and Western blot analysis measuring the levels of cleaved PARP and active caspase-3 was performed. (D) ELISA assay of active caspase-3. (E) ELISA assay of cleaved PARP. CON, untreated A549 cells; VEC, A549 cells infected with the lentivirus expression vector (VEC); CTMP, A549 cells infected with lentivirus-CTMP (CTMP). (F) Quantitative analysis of TUNEL-positive cells. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Infection, Western Blot, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, TUNEL Assay

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 3. CTMP inhibits OPA1 expression and changes mitochondria morphology. (A) Western blot analysis of MFN1, MFN2, and DRP-1. A549 cells were infected with lentivirus-VEC/CTMP and cultured for 48 h before analysis. (B) A549 cells were infected with lentivirus-CTMP, treated with 0.2 µM CCCP (carbonyl cyanide 3-chlorophenylhydrazone), and transfected with siRNA specific for CTMP. (C) The mitochondrial ultrastructure was analyzed by TEM. (D) A549 cells stably expressing mitochondrion-targeted pDsRed-Mito were infected with lentivirus expression vector and lentivirus-CTMP for 48 h and observed with confocal microscopy (scale bar, 10 nm).

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Infection, Cell Culture, Transfection, Stable Transfection, Plasmid Preparation, Confocal Microscopy

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 4. CTMP reduces mitochondria membrane potential that leads to cytochrome c release. (A) CTMP-infected cells were stained using 5 µg/ml JC-1 and analyzed by flow cytometry. (B) Western blot analysis of CTMP and cytochrome c in mitochondria. (C and D) The bands of interest were further analyzed by densitometry. Each bar represents the mean ± SE (n=3), *p<0.05 and **p<0.01 compared to the corresponding control values.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Membrane, Infection, Staining, Flow Cytometry, Western Blot, Control

Journal: International journal of oncology

Article Title: Carboxyl-terminal modulator protein induces apoptosis by regulating mitochondrial function in lung cancer cells.

doi: 10.3892/ijo.2011.1319

Figure Lengend Snippet: Figure 5. CTMP inhibits heat-shock proteins. A549 cells were infected with lentivirus-VEC/CTMP and cultured for 48 h. (A) Western blot analysis of Hsp27 and Apaf-1. (B) Immunofluorescence assay of Hsp27. (C) FACS analysis of Apaf-1 in CTMP-infected cells.

Article Snippet: A549 cells were grown to 70% confluence, 10 nM of chemically synthesized siRNA targeting CTMP or non-targeting 20-25 nt siRNA (control siRNA) were transfected using Santa Cruz transfection reagent according to the manufacturer's instructions (Santa Cruz Biotechnology).

Techniques: Infection, Cell Culture, Western Blot, Immunofluorescence