Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. Cells were seeded in equal proportion (200,000 each, ~ 30% confluence) in a 25 cm 2 culture flask and allowed to evolve for ~30 days. Self-organization of endothelial cells in 3D aggregates and accumulation of cancer cells around these structures was observable after ~7 days (80-90% confluence). After ~21 days, clearly delineated 3D networked constructs incorporating the vast majority of cancer cells were observable.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. Cells were seeded in a proportion of HBEC-5i (180,000 cells)/cancer cells (~20,000) in a 25 cm 2 culture flask and allowed to evolve for ~30 days. Self-organization of endothelial cells in 3D aggregates and accumulation of cancer cells in these structures was observable after ~7 days. After ~21 days, clearly delineated 3D networked constructs were observable.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : HBEC-5i/SK-OV-3 (turbo GFP/green) model; Bottom panel : HBEC-5i/MDA-MB-231 (turbo FP602/red) model. HBEC-5i cells (~200,000) were seeded in a 25 cm 2 culture flask and allowed to evolve until they formed 3D networked structures (~10 days), time when ~200,000 cancer cells were added to the flask. The migration of SK-OV-3 and MDA-MB-231 cells toward HBEC-5i structures was observable already after 2 days of co-culture. The evolution of endothelial/cancer constructs was monitored for an additional 3 weeks.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Migration, Co-Culture Assay, Construct

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: (A,D) Control wells with red fluorescent SK-BR-3 cells seeded in the upper inserts; ( B,C,E,F ) Experimental wells with red fluorescent SK-BR-3 cells seeded in the upper inserts and HBEC-5i in the bottom wells; (G,J) Control wells green fluorescent SK-OV-3 cells seeded in the upper inserts; ( H,I,K,L ) Experimental wells with green fluorescent SK-OV-3 cells seeded in the upper inserts and HBEC-5i in the bottom wells. The images were taken from the bottom wells after 5 days incubation.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Control, Incubation

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel: HBEC-5i/SK-OV-3 (turbo GFP/green) model; (A) Green fluorescent SK-OV-3 cells (FITC filter); (B) Dead cells (red) visualized by PI staining (TRITC filter); (C) Merged image of transmitted, FITC- and TRITC-filtered images of co-cultured cells. Bottom panel: HBEC-5i/MDA-MB-231 (turbo FP602/red) model; (D) Red fluorescent MDA-MB-231 cells (TRITC filter); (E) Dead cells (green) visualized by staining with EasyProbe dye (FITC filter); (F) Merged image of transmitted, TRITC- and FITC-filtered images of co-cultured cells. Images were acquired after 28 days of co-culture.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Staining, Cell Culture, Co-Culture Assay

Journal: PLOS One

Article Title: Scaffold-free 3D-cell co-culture model system for the study of metastatic cancer in the brain TME

doi: 10.1371/journal.pone.0349061

Figure Lengend Snippet: Top panel : monoculture controls of HBEC-5i and SK-BR-3 cells treated with Lapatinib (10 µM); (A,B) Dead cells (green) in HBEC-5i monocultures; (C,D) Dead cells (yellow) in red fluorescent SK-BR-3 monocultures. Bottom panel : Co-culture HBEC-5i/SK-BR-3 (turbo FP602/red) cell model treated with Lapatinib (1 and 10 µM). (E-H) Dead cells (yellow) in the endothelial/cancer co-culture system. EasyProbe green dye (FITC filter) was used to visualize the dead cells in all cultures.

Article Snippet: HBEC-5i human brain endothelial cells were obtained from ATCC (Manassas, VA).

Techniques: Co-Culture Assay

Journal: bioRxiv

Article Title: Molecular cloning of a novel, nervous system-specific RGS6 isoform lacking canonical G protein regulatory effects and with dominant negative actions

doi: 10.64898/2026.05.08.723811

Figure Lengend Snippet: Molecular cloning of novel RGS6 splice forms from human brain. ( A ) Splicing diagram of known RGS6 splice forms. Location of primers used for PCR-based amplification of RGS6 transcripts from a human whole brain cDNA library are shown in red. ( B ) Representative plasmid restriction digest (right) or nested PCR (down) indicating positive hits whose size is larger than known RGS6 splice forms. Each gel has a confirmed RGS6Lα1(+GGL) transcript (largest known splice form) indicated as well as yellow asterisks denoting transcripts encoding RGS6LA3α1(+GGL) which encodes RGS6B.

Article Snippet: Full-length cDNAs encoding novel RGS6 splice forms were amplified from a Marathon ready human brain cDNA library (Clontech; Mountain View, CA, USA) using a PCR-based strategy.

Techniques: Molecular Cloning, Amplification, cDNA Library Assay, Plasmid Preparation, Nested PCR

Journal: bioRxiv

Article Title: Molecular cloning of a novel, nervous system-specific RGS6 isoform lacking canonical G protein regulatory effects and with dominant negative actions

doi: 10.64898/2026.05.08.723811

Figure Lengend Snippet: RGS6B is an RGS6L(+GGL) isoform containing A3 and the α terminal exon (RGS6LA3α1(+GGL)). ( A ) Schematic diagram of the exon splicing scheme comparing RGS6Lα1(+GGL) (RGS6L) and RGS6LA3α1(+GGL) (RGS6B). mRNA sequence conservation of exon A3 between mouse and human is depicted below with the consensus sequence from 100 vertebrate species extracted by PhyloP. PCR amplification of exon A3 containing mRNA transcripts from select mouse tissues ( B ) or human cDNA libraries ( C ). Plasmids encoding RGS6Lα1, RGS6Lα2 and RGS6LA3α1 (all +GGL) are used as negative and positive controls, respectively. LNG, lung; KDN, kidney; LVR, liver; SPL, spleen; CTX, cortex; MB, midbrain; CRB, cerebellum; HIP, hippocampus; MSC, muscle; HRT, heart; PNC, peripheral nervous system; BR, whole brain. ( D ) HEK293T cells were transfected with a plasmid encoding RGS6LA3α1(+GGL) to confirm co-migration with mouse RGS6B via immunoblotting. ( E ) shRNA or miRNA constructs targeting the A3 exon were introduced into mouse primary cortical astrocytes. The ratio of RGS6B: RGS6L was determined via immunoblot and quantified from 4 independent experiments. α Tubulin serves as a loading control for immunoblots. Data were analyzed by one sample t-test to detect deviation of each group from 100% (scramble RNAi control). *P<0.05, ***P<0.001. Data are expressed as mean ± S.E.M.

Article Snippet: Full-length cDNAs encoding novel RGS6 splice forms were amplified from a Marathon ready human brain cDNA library (Clontech; Mountain View, CA, USA) using a PCR-based strategy.

Techniques: Sequencing, Amplification, Transfection, Plasmid Preparation, Migration, Western Blot, shRNA, Construct, Control