Journal: Molecular Autism

Article Title: TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling

doi: 10.1186/s13229-019-0311-3

Figure Lengend Snippet: Characterization of TSC1 iPSC-derived NPCs. a All TSC1 NPC lines (Het, Null, Corr-WT) express expected neural progenitor markers SOX2 (upper panel) and NESTIN (panel below). DAPI in blue, SOX2 in green, and NESTIN in green. Scale bar = 100 μm. Immunostaining was performed at least 3 times. b TSC1-Het and Null NPCs display increased cell size compared to TSC1-Corr-WT as shown in bright field images ( a ) and by forward scatter FACS analysis; n = 3. c As expected, TSC1-Null and -Het NPCs show dose-dependent increased mTORC1 signaling (pS6 readout) compared to Corr-WT. Protein expression was quantified and normalized to the Corr-WT NPCs, n = 6, mean values ± s.e.m. are shown, * p < 0.01, ** p < 0.001 calculated with Student’s t test. d Proliferation rate of NPC lines was quantified at day 0 (D0, equal cell seeding), and live cell numbers were assessed at D2, D3, and D5. Mutant TSC1 NPCs (Het and Null) revealed genotype-dependent increased proliferation compared to Corr-WT. Data was normalized to Corr-WT at D0, mean values ± S.D. of three separate experiments are shown,* p < 0.01, ** p < 0.001 calculated with Student’s t test. e , f MAP2 immunostaining showed genotype-dependent increased neurite outgrowth (number and length) in TSC1 mutant NPCs, which were quantified using a custom image analysis pipeline and HCA Vision imaging software creating neurite segmentation (representative panel shown for the DMSO-treated NPCs). Analysis on n = 6 field images per treatment with approximately 50 cells per field. Data normalized to DMSO treated Corr-WT NPCs. Mean values + s.e.m. are shown. * p < 0.05, ** p < 0.001, **** p < 0.0001, n.s. = not significant, calculated with Welch’s t test (GraphPad Prism 7.05). g TSC1 NPCs (Corr-WT, Het, and Null-clone B) were treated with 100 nM of rapamycin for 24 h or with DMSO and analyzed by flow cytometry using the forward scatter height (FSC-H) gating. For each cell line the blue curve represents the rapamycin-treated cells and the red curve represents the DMSO control cells. A shift in the curves shows a cell size difference. N = 3. h Proliferation rate of NPC lines after treatment with a vehicle control (DMSO) or rapamycin (100nM) was quantified at day 0 (D0, equal cell seeding), and live cell numbers were assessed at D2, D3, D4, and D5. No significant differences were observed between DMSO or rapamycin-treated NPCs in all cell types at all time point; n = 3. Mean values ± S.D. of three separate experiments are shown, data was normalized to the Corr-WT treated with DMSO at D0

Article Snippet: = not significant, calculated with Welch’s t test (GraphPad Prism 7.05). g TSC1 NPCs (Corr-WT, Het, and Null-clone B) were treated with 100 nM of rapamycin for 24 h or with DMSO and analyzed by flow cytometry using the forward scatter height (FSC-H) gating.

Techniques: Derivative Assay, Immunostaining, Expressing, Mutagenesis, Imaging, Software, Flow Cytometry, Control

Journal: Vaccines

Article Title: Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

doi: 10.3390/vaccines8020318

Figure Lengend Snippet: Immunization of BALB/c mice with TERT DNA, TERT variant with a C-terminal hemagglutinin tag (TERT-HA DNA), and empty vector followed by booster immunization with given plasmids mixed with DNA encoding firefly luciferase (Luc DNA), with follow up of luciferase expression by in vivo imaging. Scheme of the immunization ( A ); results of in vivo bioluminescence imaging of booster sites at days 1–12 post injection, example of 3 mice—one from TERT, one from TERT-HA, and one from empty vector group ( B ); dynamics of bioluminescence signal change in TERT, TERT-HA DNA, and empty vector-immunized mice on days 1–12 post booster injection; each line of different colors corresponds to one site of injection (two per mouse) ( C ); relative average level of bioluminescence signal for each group on days 1 to 12 post booster injection ( D ). Bioluminescence signal is represented by the total flux from site of immunization, mean ± SD. Analyzed by ordinary two-way ANOVA with Dunnett’s multiple comparison test, ** − p < 0.01; **** − p < 0.0001; ns—not significant.

Article Snippet: Total photon flux from the site of coinjection of TERT DNA or TERT-HA DNA and Luc DNA was compared using ordinary two-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism 6, San Diego, CA, USA).

Techniques: Variant Assay, Plasmid Preparation, Luciferase, Expressing, In Vivo Imaging, In Vivo, Imaging, Injection, Comparison

Journal: Vaccines

Article Title: Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

doi: 10.3390/vaccines8020318

Figure Lengend Snippet: Generation of tumors by 4T1luc2 clones expressing rtTERT. Tumor growth rate was assessed using total fluorescence signal from the site of injection of 2500 ( A ), 5000 ( B ), and 10,000 ( C ) cells. Tumor volume was evaluated by total fluorescence signal from the site of cell injection by day 16 ( D ) or by calipering at day 21 ( E ). Histochemical characterization of the solid tumors formed by the parental 4T1luc2 cells ( F ) and their derivatives expressing rtTERT 4T1luc2_rtTERT_C6 ( G ); 4T1luc2_rtTERT_H9 ( H ) after ectopic implantation into BALB/c mice (H&E staining, magnification 200×). Results of tumor growth ( A – C ) were analyzed using RM two-way ANOVA with Dunnett’s multiple comparison test. Data on tumor volumes ( D , E ) were analyzed using Kruskal–Wallis with Dunn’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant.

Article Snippet: Total photon flux from the site of coinjection of TERT DNA or TERT-HA DNA and Luc DNA was compared using ordinary two-way ANOVA with Dunnett’s multiple comparison test (GraphPad Prism 6, San Diego, CA, USA).

Techniques: Clone Assay, Expressing, Fluorescence, Injection, Staining, Comparison