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: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: Rapamycin suppresses the lung metastasis of circulating B16 melanoma cells. (a) Influence of rapamycin on B16 cell apoptosis. B16 cells were treated with 0.5 or 5 μg/mL rapamycin for different time periods. The cells were harvested and stained with phycoerythrin (PE)‐conjugated Annexin V for apoptosis detection by flow cytometry. (b,c) Rapamycin suppresses lung metastasis of B16 cells. B16 cells were treated with rapamycin (0.5 μg/mL) in vitro for 24 h, and then i.v. injected into mice. Twenty‐one days later, the mice were sacrificed. The data shown are the representative photograph of lungs from three independent experiments (b); and the number of lung tumor nodules (c). (d) Long‐term survival of mice after injection of rapamycin‐treated B16 cells. The survival period of tumor‐bearing mice in the rapamycin group was significantly prolonged, compared with that in the control group (n = 16; P < 0.001, Kaplan–Meier analysis). (e) Rapamycin inhibited the migration of B16 cells. The migration of rapamycin‐treated B16 cells by Transwell assay was determined as described in the Materials and Methods. The data shown are the representative from three reproducible experiments. (f) Rapamycin administration inhibited the lung metastasis of circulating B16 cells. Mice were administrated with 0, 1.25, 2.5, and 5 mg/kg rapamycin by i.p. injection once daily for 7 days. Four hours after the first injection of rapamycin, the mice were injected with 2 × 105 B16 tumor cells. Twenty‐one days later, the mice were sacrificed and the number of lung tumor nodules was counted.

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: Staining, Flow Cytometry, In Vitro, Injection, Control, Migration, Transwell Assay

Journal: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: Autophagy is not involved in the suppression of B16 metastasis by rapamycin. (a) Effect of rapamycin on autophagy in B16 cells. B16 cells were treated with rapamycin (0.5 μg/mL) in vitro. Twenty‐four hours later, the cells were harvested to determine LC3 by Western blotting. (b) Inhibition of autophagy by beclin 1 knockdown did not alter the effect of rapamycin on B16 lung metastasis. Beclin 1 siRNA‐ or control siRNA‐expressing B16 cells, treated with rapamycin, were injected into mice via the tail vein. Twenty‐one days later, mice were sacrificed and the lung tumor nodules were counted. PBS‐treated B16 cells were used as positive control. (c) Knockdown of Beclin 1 did not inhibit the suppression of rapamycin on B16 cell migration in vitro. Beclin 1 siRNA‐ or control siRNA‐expressing B16 cells were treated with rapamycin. The Transwell assay was determined as described in the Materials and Methods. (d) Autophagy inhibitors did not inhibit rapamycin‐mediated suppression of B16 cell lung metastasis. B16 cells were treated with rapamycin plus autophagy inhibitor wortmannin (WM, 100 nm) or 3‐methyladenine (3‐MA, 3 mm) for 24 h. The treated cells were injected into mice. Twenty‐one days later, the mice were sacrificed and the number of lung tumor nodules was enumerated. The data shown are the representative from two independent experiments.

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: In Vitro, Western Blot, Inhibition, Knockdown, Control, Expressing, Injection, Positive Control, Migration, Transwell Assay

Journal: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: Rapamycin inhibits αv integrin signaling of B16 cells. (a,b) Rapamycin down‐regulated the expression of αv integrin in B16 cells. B16 cells were treated with rapamycin in vitro. Twenty‐four hours later, the cells were harvested for the detection of αv and β3 expression by real‐time RT‐PCR (a); or 24 h later, the cells were used for Western blot analysis (b). (c) Rapamycin inhibited the activation of focal adhesion kinase (FAK). B16 cells were treated with rapamycin for 24 h. The phosphorylated FAK and total FAK were analyzed by Western blotting. (d) Assay of cdc2 expression. The rapamycin‐treated cells were used for total RNA isolation and cell lysate preparation. The expression of cdc2 was analyzed by real‐time RT‐PCR (bottom) and Western blotting (up). (e,f) Assay of matrix metalloproteinase (MMP)‐2 and MMP‐9 production. B16 cells were cultured in the presence or absence of rapamycin for 24 h. MMP‐2 and MMP‐9 in supernatants were detected by zymography assay (e). The relative mRNA levels of MMP‐2 and MMP‐9 were detected by real‐time RT‐PCR (f).

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Activation Assay, Isolation, Cell Culture, Zymography

Journal: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: Down‐regulation of αv integrin is required for rapamycin‐mediated suppression of B16 metastasis. (a) Construction of αv integrin‐overexpressing B16 cell line. αv Integrin‐expressing plasmid was transfected to B16 cells. The stably transfected cells were used to analyze αv integrin expression by Western blotting. (b) Rapamycin did not inhibit the exogenous expression of αv integrin. (c) Overexpression of αv integrin partly reversed the suppression of rapamycin in lung metastasis. αv‐Overexpressing or mock B16 cells, treated with rapamycin, were injected into mice via the tail vein. Twenty‐one days later, the mice were sacrificed and the lung tumor nodules were counted. (d) Comparison of the effect on B16 cell metastasis among rapamycin, CH50, and αv integrin antagonist. B16 cells were treated with rapamycin (0.5 μg/mL), CH50 (10 μg/mL), or high‐dose CH50 (25 μg/mL); or αv integrin antagonist (10 μg/mL) or high dose (25 μg/mL) for 24 h. Then the cells were injected to the mice. Twenty‐one days later, the mice were sacrificed and the lung tumor nodules were counted.

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: Expressing, Plasmid Preparation, Transfection, Stable Transfection, Western Blot, Over Expression, Injection, Comparison

Journal: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: Rapamycin suppresses tumor metastasis by up‐regulating apoptosis signaling. (a) Inhibition of apoptosis declined the effect of rapamycin on B16 cell metastasis. B16 cells were treated with rapamycin in the presence or absence of zVAD (10 μm), and then injected to mice via the tail vein. Twenty‐one days later, the mice were sacrificed and the lung tumor nodules were counted. (b,c) Rapamycin increased the apoptosis potential of B16 cells. B16 cells were treated with rapamycin for 24 h. The cells were harvested for RNA isolation. The expressions of Bcl‐2, Bcl‐xL, Bax, and Bid were determined by real‐time RT‐PCR (b); or the cells were used for protein analysis by Western blotting (c).

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: Inhibition, Injection, Isolation, Quantitative RT-PCR, Western Blot

Journal: Cancer Science

Article Title: Rapamycin inhibits lung metastasis of B16 melanoma cells through down‐regulating alphav integrin expression and up‐regulating apoptosis signaling

doi: 10.1111/j.1349-7006.2009.01412.x

Figure Lengend Snippet: S6K1 is involved in the up‐regulation of Bax and Bid, but not in the down‐regulation of αv integrin in B16 cells. (a) siRNA silenced S6K1 expression. After stable transfection of S6K1 siRNA vector into the B16 cell line, S6K1 mRNA and protein expression was examined by RT‐PCR and Western blotting. (b–d) The influence of S6K1 knockdown on the expressions of Bax, Bid, and αv integrin in B16 cells. S6K1 or control siRNA‐expressing B16 cells were cultured in the presence or absence of rapamycin. Twenty‐four hours later, cells were harvested for RNA isolation. The real‐time RT‐PCR was performed to analyze the expression of Bax (b), Bid (c), and αv integrin (d). (e) The inhibition of mTOR and S6K1 by rapamycin. B16 cells were cultured in the presence or absence of rapamycin. Twenty‐four hours later, cells were harvested to determine mTOR and S6K1 by Western blotting.

Article Snippet: Mouse melanoma tumor cell line B16 F1 was purchased from the China Center for Type Culture Collection (CCTCC; Wuhan, China), and cultured according to the guidelines.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Control, Cell Culture, Isolation, Quantitative RT-PCR, Inhibition

Journal: Saudi Journal of Biological Sciences

Article Title: Quercetin-3-O-β- d -glucopyranosyl-(1 → 6)-β- d -glucopyranoside suppresses melanin synthesis by augmenting p38 MAPK and CREB signaling pathways and subsequent cAMP down-regulation in murine melanoma cells

doi: 10.1016/j.sjbs.2015.03.009

Figure Lengend Snippet: Effect of QCGG on (A) intracellular cAMP accumulation in B16F10 cells. Cells were exposed to α-MSH (100 nM) alone or with QCGG (0–100 μg/ml) for 48 h. Intracellular cAMP levels were measured using an enzyme immunoassay protocol; (B) expression levels of MAPKs, phosphorylated-MAPKs, CREB, and phosphorylated-CREB in B16 cells. Cells were exposed to α-MSH (100 nM) alone or with QCGG (0–100 μg/ml) for 48 h. Protein expression was determined by Western blotting. Equal protein loading was checked by GAPDH. Representatives of three independent experiments are shown. Results shown are means ± SEM and are representative of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 compared with the control.

Article Snippet: B16F10 mouse melanoma cells (CRL 6323) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control