Journal: bioRxiv

Article Title: Membrane-associated polymerases deliver most of the actin subunits to a lamellipodial network

doi: 10.1101/2025.03.24.645090

Figure Lengend Snippet: A. Schematic representation of the route actin takes into filaments through the actin-binding protein profilin. The covalently linked actin-profilin probe can enter filaments directly from the cytosol and by first binding to the proline-rich region of NPFs like WAVE1. A similar probe that contains a modified profilin that can still incorporate into filaments but cannot bind proline-rich domains (actin-profilin MUT ) only enters the filament directly from the cytosol. B. Illustration of actin-profilin orientations for linked constructs using previously published structures (PDB:2BTF) with either 10, 16 or 30 alternating glycine-serine residues between the N terminus of β-actin and the C-terminus of profilin. EGFP is linked to the N terminus of β-actin for probe visualization. C. Fluorescence of B16F1 cells transiently expressing indicated constructs. Scale bar represents 5µm. D. Averaged linescans at the leading edge of cells expressing actin-profilin probes with different linker lengths, where the cell edge = 0. The bands depict 95% confidence intervals. For all conditions, n = 100 lines drawn from 10 cells. Lines are normalized to the cell mean intensity. Only the probe with the longest linker shows a similar increase at the leading edge to eGFP-actin. E. Quantification of mobile fraction of recovery curve after photobleaching per cell indicating that both actin and actinGS 30 profilin readily encorporate into filaments, as they both exhibit substantial nonmobile fractions. Number of cells per experiment per round for both A and B are as follows with cell number indicated in parenthesis: eGFP (37,23,22) eGFP-actin WT (22,27), eGFP-profilin1 (11,9,26), β-actinGS 10 profilin (23,18), β-actinGS 16 profilin (21,12,23), β-actinGS 30 profilin (16,35,14). One-way ANOVA and Tukey’s multiple comparison tests are comparisons with eGFP.

Article Snippet: B16F1 (ATCC-CRL-6323) mouse melanoma cells were cultured in DMEM/F12 with 4.5 g/mL glucose and supplemented with 10% fetal bovine serum (FBS, Life Technologies Certified, US) and penicillin/streptomycin.

Techniques: Binding Assay, Modification, Construct, Fluorescence, Expressing, Comparison

Journal: bioRxiv

Article Title: Membrane-associated polymerases deliver most of the actin subunits to a lamellipodial network

doi: 10.1101/2025.03.24.645090

Figure Lengend Snippet: A. Cartoon illustrating actin polymerization with the WAVE1 regulatory complex with the proline-rich region intact (left) and a WAVE1 with its proline-rich region mutated so that profilin cannot bind (right). B. Diagram of the PVCA domain of WAVE1 depicting modifications made to the proline-rich domain to alter binding of profilin-actin (profilin-binding deficient). C. Fluorescence of B16F1 depleted of both WAVE1 and 2 isoforms (WAVE1/2 double knockout) and rescues transiently expressing indicated constructs. B16F1 wild-type cells are shown for comparison. EGFP-WAVE1 with a modified proline-rich domain (WAVE1 MUT ) is shown in comparison to the intact domain (WAVE1 WT ), with magnified insets of the leading edge. The wildtype WAVE1 rescues the typical lamellipodia phenotype, while cells with the WAVE1 MUT that cannot bind profilin exhibit many filopodia, more similar to the double knockout cells. Scale bar represents 5 µm for inset, 10 µm for whole image. D. Principal component analysis of cell shape for various cell lines. The cells rescued with wild type WAVE1 appear more similar to wild type B16F1 cells, while cells transiently expressing only the WAVE1 MUT construct are intermediate between the wild type and double-knockout cells. The first principal component (PC1, x-axis) explains 84.1% of the variation in the data, while the second principal component (PC2, y-axis) represents 15.9% of the variation. Confidence ellipses are drawn at 95% for each group. Means for each group are displayed by larger sizes and representative outlines of cell shape for each mean are displayed in the corners. E. Filopodia number per cell. B16F1 wildtype cells (16, 14, 15), double-knockout cells (16, 37), and double-knockout cells transiently expressing either WAVE1 MUT (10,10,12,23) or WAVE1 WT (8,15,20). F. Filopodia length in microns, n= filopodia number per round and are as follows: (58,88,69), (290,536), (79,99,11), and (52,20,60), respectively. Replicate means are represented by large dots superimposed on cell-level data. One-way ANOVA and Tukey’s multiple comparison tests were performed on replicate means.

Article Snippet: B16F1 (ATCC-CRL-6323) mouse melanoma cells were cultured in DMEM/F12 with 4.5 g/mL glucose and supplemented with 10% fetal bovine serum (FBS, Life Technologies Certified, US) and penicillin/streptomycin.

Techniques: Binding Assay, Fluorescence, Double Knockout, Expressing, Construct, Comparison, Modification

Journal: Gene therapy

Article Title: IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response.

doi: 10.1038/sj.gt.3301012

Figure Lengend Snippet: Figure 1 In vitro invasion and migration capacity of tumor cells. Vector- transduced (CT26-B and B16-B) and IL-10 gene-transduced (CT26-IL10 and B16-IL10) cells and clones (CT26-2F, CT26-3E, B16-1A and B16- 3B) were evaluated for their capacity to invade Matrigel (a) or to migrate through filters of 8 mm pore size (b) as described in Materials and methods. Columns (bars) correspond to means (± s.e.) of migrated cells in 10–12 high-power fields. The asterisks indicate that the differences with the controls are statistically significant (P , 0.01–0.05).

Article Snippet: The CT26 mouse colon carcinoma cell line34 obtained from the MD Anderson Cancer Research Center (Houston, TX, USA) and the B16 (F1) mouse melanoma cell line purchased from the American Type Culture Collection (ATCC, CRL 6323; Rockville, MD, USA) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum, 2 mm glutamine, 2.5 units/ml penicillin and 2.5 mg/ml streptomycin.

Techniques: In Vitro, Migration, Plasmid Preparation, Clone Assay, Pore Size

Journal: Gene therapy

Article Title: IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response.

doi: 10.1038/sj.gt.3301012

Figure Lengend Snippet: Figure 2 Kinetics of tumor growth in athymic nude mice. Mice were injected s.c. with 5 × 105 vector-transduced cells or IL-10 gene-transduced CT26 cells. (a) Corresponds to mice injected with CT26-B and CT26-IL10 cells; (b) corresponds to mice injected with B16-B and B16-IL10 cells. One representative experiment out of two with similar results is shown. Each group contained eight to 10 animals.

Article Snippet: The CT26 mouse colon carcinoma cell line34 obtained from the MD Anderson Cancer Research Center (Houston, TX, USA) and the B16 (F1) mouse melanoma cell line purchased from the American Type Culture Collection (ATCC, CRL 6323; Rockville, MD, USA) were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum, 2 mm glutamine, 2.5 units/ml penicillin and 2.5 mg/ml streptomycin.

Techniques: Injection, Plasmid Preparation

Journal: Pharmacognosy Magazine

Article Title: Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

doi: 10.4103/pm.pm_121_17

Figure Lengend Snippet: Treatment with different concentrations of Viwithan showed cytotoxic effect on B16F1 cell line at different time points of incubation. Cells were treated with different concentrations of Viwithan for 24, 48, and 72 h. All data are expressed as mean of three independent experimental observations

Article Snippet: The cancer cell lines used in the study such as HepG2, HeLa, HCT-116, Ehrlich-Lettre ascites carcinoma (EAC), and B16F1 cells mouse melanoma cell lines were procured from National Centre for Cell Science, Pune.

Techniques: Incubation

Journal: Pharmacognosy Magazine

Article Title: Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

doi: 10.4103/pm.pm_121_17

Figure Lengend Snippet: Treatment with different concentrations of Viwithan showed cytotoxic effect on B16F1 cell line after 48 h incubation. Cells were treated with different concentrations of Viwithan for 48 h. All data are expressed as mean of three independent experimental observations

Article Snippet: The cancer cell lines used in the study such as HepG2, HeLa, HCT-116, Ehrlich-Lettre ascites carcinoma (EAC), and B16F1 cells mouse melanoma cell lines were procured from National Centre for Cell Science, Pune.

Techniques: Incubation

Journal: Pharmacognosy Magazine

Article Title: Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

doi: 10.4103/pm.pm_121_17

Figure Lengend Snippet: Morphological changes observed in Viwithan treated B16F1 cells. Cells were treated with 220, 180, and 100 μg/ml of Viwithan for (A) 24, (B) 48, and (C) 72 h, respectively

Article Snippet: The cancer cell lines used in the study such as HepG2, HeLa, HCT-116, Ehrlich-Lettre ascites carcinoma (EAC), and B16F1 cells mouse melanoma cell lines were procured from National Centre for Cell Science, Pune.

Techniques:

Journal: Pharmacognosy Magazine

Article Title: Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

doi: 10.4103/pm.pm_121_17

Figure Lengend Snippet: Apoptotic changes observed in Viwithan-treated B16F1 cells stained with acridine orange and ethidium bromide. Cells were treated with 220 and 180 μg/ml of Viwithan for (A) 24 and (B) 48 h, respectively

Article Snippet: The cancer cell lines used in the study such as HepG2, HeLa, HCT-116, Ehrlich-Lettre ascites carcinoma (EAC), and B16F1 cells mouse melanoma cell lines were procured from National Centre for Cell Science, Pune.

Techniques: Staining