Journal: International Journal of Molecular Sciences

Article Title: The Expression Profile of mRNA and tRNA Genes in Splenocytes and Neutrophils after In Vivo Delivery of Antitumor Short Hairpin RNA of Indoleamine 2,3- Dioxygenase

doi: 10.3390/ijms21186703

Figure Lengend Snippet: The effect of indoleamine 2,3-dioxygenase (IDO) short-hairpin RNA (shRNA) treatment on LLC1 tumor-bearing mice. ( a ) 2 × 10 5 LLC1 cells were subcutaneously injected into female inbred C57BL/6JNarl mice. Then, 50 μg of Scramble IDO shRNA (Scr IDOsh) or IDO shRNA (IDO sh) was treated by intramuscular injection at 7 and 14 days after LLC1 injection. The total splenocytes at day 16 and magnetic bead-enriched neutrophils at day 18 were collected, and subsequently subjected to RNA sequencing. ( b ) Mean tumor volume at day 16 ( n = 7). ( c ) The tumor-infiltrating Ly6g + cells. The labels sample 1 and sample 2 indicate that the images were taken from two independent murine tumors of each group. ( d ) Quantification of Ly6g + cells for immunohistochemistry analysis ( n = 4).

Article Snippet: LLC1 (LL/2) was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: shRNA, Injection, RNA Sequencing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: The Expression Profile of mRNA and tRNA Genes in Splenocytes and Neutrophils after In Vivo Delivery of Antitumor Short Hairpin RNA of Indoleamine 2,3- Dioxygenase

doi: 10.3390/ijms21186703

Figure Lengend Snippet: The expression of inflammatory molecules. ( a ) The expression of inflammatory molecules of total splenocytes, determined by RNA sequencing and ( b ) by QPCR. To detect the secreted protein levels, splenocytes were collected at 16 days after LLC1 injection. After 24-h culture, these molecules were detected by magnetic fluorescence microsphere immunoassay (MAGPIX) analysis. The expression of ( c ) IFN-γ, ( d ) IL-10, ( e ) TNF-α, ( f ) IL-1α, ( g ) CCL5, and ( h ) S100A9 was shown. ( i ) The expression of inflammatory molecules of splenic neutrophils determined by RNA sequencing and ( j ) by QPCR. *, **, and *** A statistically significant difference when compared with scramble IDO shRNA group (* p < 0.05; ** p < 0.01; *** p < 0.001). n = 3–4 in each experiment, except to RNA sequencing data. IFN-γ, interferon-γ; IL-10, interlukein-10; TNF-α, tumor necrosis factor- α; TGF-β, tumor transforming growth factor-β; CCL5, CC chemokine ligand 5; S100A9, S100 calcium-binding protein A9; Arg1, arginase 1.

Article Snippet: LLC1 (LL/2) was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Expressing, RNA Sequencing, Injection, Fluorescence, shRNA, Binding Assay

Journal: Polymers

Article Title: WSG, a Glucose-Rich Polysaccharide from Ganoderma lucidum , Combined with Cisplatin Potentiates Inhibition of Lung Cancer In Vitro and In Vivo

doi: 10.3390/polym13244353

Figure Lengend Snippet: WSG enhances the cytotoxic effects of cisplatin on A549 and LLC1 cells. ( A ) A549 and LLC1 cells were subjected to 24 h combination treatment using different concentrations of WSG and cisplatin. A crystal violet staining assay was conducted for cell viability examination. Each sample in the combination treatment group was normalized against each untreated control. ( B , C ) CI and DRI of the combination treatment in the A549 and LLC1 cells, determined using CompuSyn software. ( D ) WSG reduced the IC 50 of cisplatin in A549 and LLC1 cells. The IC 50 values of cisplatin were measured using CompuSyn Software. Significant differences between the treatment and control groups are presented (*** p < 0.001). ( E ) Left: the schematic design for the in vitro cell viability assay of WSG (0–360 μg/mL)→cisplatin (5 μM) sequential treatment. Right: The viability of the cells was determined by using the crystal violet assay. ( F ) Left: the schematic design for the in vitro cell viability assay of cisplatin (5 μM)→WSG (0–360 μg/mL) sequential treatment. Right: The viability of the cells was determined by using the crystal violet assay. Each sample in the combination treatment group was normalized against each untreated control. Error bars indicated SD.

Article Snippet: Human lung adenocarcinoma A549 cells, murine LLC1 cells, human lung fibroblast MRC-5, and murine macrophages Raw264.7 were acquired from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan).

Techniques: Staining, Control, Software, In Vitro, Viability Assay, Crystal Violet Assay

Journal: Polymers

Article Title: WSG, a Glucose-Rich Polysaccharide from Ganoderma lucidum , Combined with Cisplatin Potentiates Inhibition of Lung Cancer In Vitro and In Vivo

doi: 10.3390/polym13244353

Figure Lengend Snippet: Co-treatment with WSG and cisplatin suppresses lung tumor growth in mice bearing LLC1 cells. ( A ) Experimental schematic. LLC1-bearing mice ( n = 5 in each group) were intraperitoneally injected with cisplatin and WSG. ( B ) Tumor volume was monitored for the indicated times. ( C ) Tumor weights were determined after each intervention. The bars represent means ± standard deviations. Significant differences between the treatment and control groups are presented (* p < 0.05, *** p < 0.001).

Article Snippet: Human lung adenocarcinoma A549 cells, murine LLC1 cells, human lung fibroblast MRC-5, and murine macrophages Raw264.7 were acquired from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan).

Techniques: Injection, Control

Journal: Polymers

Article Title: WSG, a Glucose-Rich Polysaccharide from Ganoderma lucidum , Combined with Cisplatin Potentiates Inhibition of Lung Cancer In Vitro and In Vivo

doi: 10.3390/polym13244353

Figure Lengend Snippet: Co-treatment with WSG and cisplatin suppresses tumorigenesis in the lung tissues of mice injected with LLC1 cells. ( A ) Experimental schematic. LLC1 cells were injected through the lateral tail veins of the mice ( n = 5). The LLC1-bearing mice were intraperitoneally injected with cisplatin and WSG. ( B ) At the end of the treatment, lung tissue was collected. One of five tissue samples is presented. As indicated by the blue arrows, tumor lesions were observed on the surface of the lung tissues. Lower panels of the lung sections under H&E staining (20× magnification). Bar scale: 2 mm. ( C ) Tumors (nodules) in the lung lesions. ( D ) Tumor volume (mm 3 ) of the lung lesions. Each bar represents the mean ± standard deviation. Significant differences between the treatment and control groups are presented (* p < 0.05, ** p < 0.01, *** p < 0.001). ( E ) Combination treatment increased the survival rate of LLC1-bearing mice ( n = 8).

Article Snippet: Human lung adenocarcinoma A549 cells, murine LLC1 cells, human lung fibroblast MRC-5, and murine macrophages Raw264.7 were acquired from the Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan).

Techniques: Injection, Staining, Standard Deviation, Control

Journal: Redox Biology

Article Title: Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth

doi: 10.1016/j.redox.2019.101405

Figure Lengend Snippet: Nitro-oleate increased proliferation and migration of LLc1 cells through inhibition of sEH and EET signalling (A) 5 μM or 10 μM nitro-oleate reduced sEH activity. In contrast, sEH activity was not reduced by the non-electrophile oleate (N=12 per group from 3 independent experiments). The time graph shows that sEH-dependent hydrolysis of PHOME to 6-methoxy-2-naphtaldehyde (6M2N), which is fluorescent, is linear over 60 min. (B-D) Real-time monitoring of LLc1 cell proliferation (cell index) and proliferation rates calculated from these measurements. (B) 5 μM or 10 μM nitro-oleate stimulated proliferation compared to untreated or DMSO-treated control cells (N=4 independent experiments). (C) In addition, oleate did not stimulate cell proliferation (N=4 independent experiments). (D) The increased proliferation induced by 5 μM nitro-oleate was normalized by the EET antagonist 14,15-EE-5(Z)-E (N=4 independent experiments). (E) Real-time monitoring of LLc1 cells trans-well migration (cell index) and migration rates calculated from these measurements. 5 μM nitro-oleate stimulated transmigration compared to untreated or vehicle (DMSO)-treated control cells (N=4 independent experiments). *P < 0.05 significantly different compared to untreated control. #P < 0.05 significantly different compared to the nitro-oleate treated group.

Article Snippet: The LLc1 cell line (ECACC 90020104) was purchased from the European collection of authenticated cell cultures, and cultured in DMEM containing 2 mM l -glutamine, 10% fetal bovine serum and antibiotics (Invitrogen).

Techniques: Migration, Inhibition, Activity Assay, Control, Transmigration Assay

Journal: Redox Biology

Article Title: Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth

doi: 10.1016/j.redox.2019.101405

Figure Lengend Snippet: Nitro-oleate-induced tumor growth is dependent on C521 in vitro and in vivo (A) LLc1 cells engineered to stably overexpress WT or C521S sEH do so, as detected by immunoblotting with an antibody to the hydrolase or the V5 tag that the recombinant proteins also carry. The immunoblots are representative of 3 independent experiments. (B-C) 5 μM nitro-oleate decreased sEH activity and stimulated proliferation compared to untreated or oleate-treated LLc1 cells overexpressing WT, but not C521S, sEH (N=4 independent experiments). *P < 0.05 significantly different compared to untreated WT sEH LLc1 cells. (D-E) 5 mg/kg/day nitro-oleate, oleate or vehicle was administered to mice after their implantation with LLc1 cells overexpressing WT or C521S sEH. Nitro-oleate, but not oleate nor vehicle, stimulated growth in mice with LLc1 cells overexpressing WT, but not C521S, sEH. Nitro-oleate, but not oleate, selectively reduced activity in cells overexpressing the WT hydrolase (N=6–8 per experimental group from 3 independent experiments). (F) IL-1β, IL-6, IL-10, IL-12p35 and IL-12p40 abundance in tumor tissue was decreased in LLc1 cells overexpressing WT sEH upon exposure to nitro-oleate. These cytokines were lower in abundance basally in tumor tissue derived from cells expressing C521S sEH compared to those overexpressing WT hydrolase. In contrast to tumors originating from cells expressing WT hydrolase, those derived from cells expressing C521S sEH did not change their cytokine abundance when exposed to nitro-oleate (N=6–8 per experimental group from 3 independent experiments). *P < 0.05 significantly different compared to LLc1 cells expressing WT sEH exposed to vehicle.

Article Snippet: The LLc1 cell line (ECACC 90020104) was purchased from the European collection of authenticated cell cultures, and cultured in DMEM containing 2 mM l -glutamine, 10% fetal bovine serum and antibiotics (Invitrogen).

Techniques: In Vitro, In Vivo, Stable Transfection, Western Blot, Recombinant, Activity Assay, Derivative Assay, Expressing

Journal: Redox Biology

Article Title: Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth

doi: 10.1016/j.redox.2019.101405

Figure Lengend Snippet: Impact of nitro-oleate on LLc1 tumor growth (A-C) Temporal measurement of tumor volume in WT or C521S sEH KI mice during treatment with vehicle or 5 mg/kg/day nitro-oleate after implantation with LLc1 cells. The area under the curve for tumor volume was calculated from these data. Nitro-oleate stimulated tumor growth compared to vehicle in WT mice, although this failed to reach statistical significance at the 5% level (P=0.07). In contrast, there was no difference in tumor volume with or without nitro-oleate treatment in KI mice (N=12–15 mice were studied per experimental group). (D) Tumor sEH expression was not different between genotypes or altered by nitro-oleate treatment. (E) sEH activity in tumor tissue harvested from WT mice was lower in those from mice exposed to nitro-oleate compared to those only administered vehicle. In contrast, the nitro-lipid did not modulate hydrolase activity present in tumors isolated from KI mice (N=12–15 mice were studied per experimental group). (F) sEH activity and tumor volume showed a negative correlation in WT mice exposed to nitro-oleate (P < 0.03). This correlative association was absent in KI treated with the nitro-lipid, as well as either genotype administered vehicle alone. (G) EET/DHET ratios in plasma of WT or KI mice treated with or without nitro-oleate. The concentration of plasma the EET/DHET for the 11 (12) or 8 (9) regioisomers showed a trend towards increasing in WT, but not KI, mice harbouring tumors exposed to nitro-oleate (N=3 mice per experimental group) (H) More isolectin B4 and CD31 positive cells were detected in tumor sections isolated from WT mice exposed to nitro-oleate compared to vehicle. In contrast, nitro-lipid treatment did not increase expression in tumors implanted in C521S sEH KI mice (N=4 mice per experimental group). (I) Plasma IL-1β and IL-12p70 was significantly decreased in WT mice exposed to nitro-oleate, with a similar downward trend observed for IL-6 and IL-10. This plasma cytokine-lowering by nitro-oleate was not observed in the KI mice, but it was notable they had basally low levels, comparable to those in the vehicle control group (N=4 mice per experimental group). Cytokine expression in tumor tissue showed a similar trend to their expression in plasma. *P < 0.05 significantly different compared to WT or vehicle control group. #P < 0.05 significantly different compared to vehicle treated KI group. The biochemical and histological analyses reported in panels D-I were performed on samples collected on day 16 of the protocol.

Article Snippet: The LLc1 cell line (ECACC 90020104) was purchased from the European collection of authenticated cell cultures, and cultured in DMEM containing 2 mM l -glutamine, 10% fetal bovine serum and antibiotics (Invitrogen).

Techniques: Expressing, Activity Assay, Isolation, Clinical Proteomics, Concentration Assay, Control