Innovative subsidy schemes under a limited budget: how and when do they matter

Inspired by the global budget concept in health care, we propose an innovative subsidy scheme in which governments do not commit the subsidy amount in advance but determine it by the end of the subsidy period. The performance of the traditional and proposed subsidy schemes is evaluated in an oligopoly without competition, where each manufacturer decides its product quality and production quantity under the diseconomies of scale in production. Both types of schemes can take into account product quality or not. Our results show that compared to the traditional scheme, the proposed scheme drives manufacturers to reduce production quantity but improves product quality and brings them higher profits. Furthermore, the scheme results in greater social welfare when the subsidy budget is large. Finally, we conduct robustness checking by considering the market competition and absence of the diseconomies of scale, respectively. The result shows that the main findings in the basic model continue to hold, while there are additional findings. In the first scenario, as the market competition intensity increases, our proposed scheme becomes more preferable to the manufacturers and the government. In the second scenario, subsidy schemes that take product quality into account are never preferable.</p

The secretion of CXCL16 by lung cancer cell lines <i>in vitro</i>.

<p>Digested A549, H292 and 95D cells were seeded in 24-well plates (500 µl/well) at a density of 5×10⁵/ml, respectively. Supernatants of the cell cultures were collected at 24, 36, 48, 60, 72, 96 and 100 h of culture. An ELISA assay was performed to examine the release of the soluble CXCL16 in cultured A549, H292 and 95D cells <i>in vitro</i>. As shown in Fig. 4, three kinds of lung cancer cell lines all secreted CXCL16 spontaneously in a time-dependent manner, despites of a difference in the concentration of CXCL16 in the culture medium. The experiments were repeated three times. Error bars depict the standard error of the mean.</p

The membrane expression of CXCR6 and CXCR4 in lung cancer cell lines <i>in vitro</i>.

<p>Flow cytometry (FCM) was used to detect the membrane expression of CXCR6 in lung cancer cell lines. The cells of 1×10⁵ were counted and the dilution ratio of (phycoerythrin)-CXCR6 monoclonal antibody and PE-CY5-CXCR4 monoclonal antibody were 1∶10 and 1∶5, respectively. The histogram demonstrated the average expression proportion of CXCR6 and CXCR4 in A549, H292 and 95D cells, respectively. The FCM pictures were representative of the experiments. The percentage of membrane CXCR6-positive cells in A549, H292 and 95D was 52.4±5.80, 56.03±11.42 and 34.8±6.17, respectively. Furthermore, the membrane expression of CXCR4 was also observed in A549, H292 and 95D cells at the same time. The experiments were repeated three times and the images were representative of the experiments. Error bars depict the standard error of the mean.</p

CXCL16-CXCR6 and CXCL12-CXCR4 were co-expressed in human lung cancer.

<p>Immunohistochemistry was performed to determine the protein expression of CXCL16-CXCR6 and CXCL12-CXCR4 in human primary lung cancer tissues. The antibodies used in this experiments were mouse anti-human CXCR4 (25 µg/ml), mouse anti-human CXCR6 (25 µg/ml), mouse anti-human CXCL12 (20 µg/ml) and goat anti-human CXCL16 (20 µg/ml) antibodies. It was demonstrated in Fig. 1 that a specific brown-coloured staining for CXCL16-CXCR6 and CXCL12-CXCR4 in the cytoplasm and membrane of human different pathological types of lung cancer cells. Moderate-to-strong, brown-coloured staining for CXCL16 and CXCR6 was also observed in normal lung tissues, but the positive expression was mainly restricted to the alveolar epithelial cells and inflammatory cells. There was no evidence for nonspecific staining with the control antibody. The pictures were the representative of the experiments. AC: adenocarcinomas; SC: squamous carcinomas; BC: bronchoalveolar carcinoma; Con: normal lung tissues; Villi: human first-trimester villous tissues, as a positive control. Magnification, ×200.</p

The expression pattern of CXCL16-CXCR6 and CXCL12-CXCR4 in different pathological types of lung cancer.

<p>According to the immunostaining intensity score and the percentage of positive cells, the results of immunochemistry analysis were classified as follows: <2, negative expression; 2–3, weak expression; 4–5, moderate expression, and 6–7 as strong expression. AC: adenocarcinomas; SC: squamous carcinomas; ASC: adenosquamous carcinomas; BC: bronchoalveolar carcinoma.</p

Effects of CXCL16-CXCR6 on A549 cell viability <i>in vitro</i>.

<p>The MTT assay was applied to evaluate the effects of CXCL16-CXCR6 on cell viability <i>in vitro</i>. A: A549 cells from blank-control, shRNA-control and CXCR6-shRNA (2819-1) groups were seeded in 96-well flat-bottom microplates with a density of 2×10³ cells/well. The <i>in vitro</i> viability was observed at 24, 48, 72, 96 and 120 h respectively. B: After starved with 1640 without FCS for 12 h, the cells from blank-control, shRNA-control or CXCR6-shRNA (2819-1) groups were treated with control medium (1640) or CXCL16 at a concentration of 100 ng/mL for 48 h. Then the <i>in vitro</i> viability was recorded by MTT assay. The experiments were repeated three times. Error bars depict the standard error of the mean. ^aP<0.01, compared with the corresponding control.</p

Effects of CXCL16 on PCNA expression of lung cancer cell lines <i>in vitro</i>.

<p>After starved for 12(100 ng/mL) or a combination of CXCL16 with CXCL16 neutralizing antibody (100 ng/mL) for anther 48 h. Then, the effects of CXCL16 stimulation on PCNA (proliferating cell nuclear antigen) expression was detected by flow cytometry. As shown in Fig. 6, there were no significant changes in PCNA level in various experimental groups (P>0.05, compared with the control). The images are representative of the experiments and the results were reproducible and consistent in three lung cancer cell lines. Error bars depict the standard error of the mean.</p

CXCL16-CXCR6 and CXCL12-CXCR4 proteins were co-expressed in human lung cancer cell lines <i>in vitro</i>.

<p>Immunocytochemistry was conducted to detect the protein expression of CXCL16-CXCR6 and CXCL12-CXCR4 in human lung cancer cell lines. The antibodies used in this experiments were mouse anti-human CXCR4 (25 µg/ml), mouse anti-human CXCR6 (25 µg/ml), mouse anti-human CXCL12 (20 µg/ml) and goat anti-human CXCL16 (20 µg/ml) antibodies. Positive brown-coloured staining for both CXCL16 and CXCR6 was clearly observed in the cytoplasm and cytomembrane of A549, H292 and 95D cells, respectively. Furthermore, CXCL12 and CXCR4 were also co-expressed in the three lung cancer cell lines. No background staining was observed in the isotype control. A: The images were representative of the experiments; B: The relative expression intensity of CXCL16-CXCR6 and CXCL12-CXCR4 in A549, H292 and 95D cells. Tro: human primary cultured trophoblast cells as a positive control. Magnification, ×200.</p

CXCR6 was effectively down-regulated by siRNA techonology.

<p>To silence the CXCR6 gene expression, we transfected A549 cells with phU6/GFP/Neo plasmid containing short hairpin RNA (shRNA) molecules targeted against CXCR6, with the usage of Lipofectamine 2000 (Invitrogen). The sequences for three shRNA oligonucleotides were: (CXCR6-2819-1): 5′-ctGAG GAC AAT TCC AAG ACT T-3′ (sense) and 5′-AAG TCT TGG AAT TGT CCT CAG-3′ (Anti-sense); (CXCR6-2820-2) 5′-ctCAC CAT GAT TGT CTG CTA T-3′ (sense) and 5′-ATA GCA GAC AAT CAT GGT GAG-3′ (Anti-sense); (CXCR6-2821-1) 5′-gcTTG CTC ATC TGG GTG ATA T-3′ (sense) and 5′-ATA TCA CCC AGA TGA GCA AGC-3′ (Anti-sense). Efficiency of RNA interference against CXCR6 was validated by western blot. It was shown in Fig. 7 that CXCR6 protein was substantially expressed in A549 cells (bank-control, without any treatment) and the RNA interference technology effectively inhibited CXCR6 expression in A549 cells. Lane 1: Blank-control; Lane 2: RNA-control; Lane 3: CXCR6 shRNA-(2821-1); Lane 4: CXCR6 shRNA-(2820-2); Lane 5: CXCR6 shRNA-(2819-1).</p

CXCL16 induces migration and invasion of lung cancer cell lines <i>in vitro</i>.

<p>Invasion assay was employed to investigate the effects of CXCL16-CXCR6 axis on the invasive ability of A549, 95D and H292 cells <i>in vitro</i>. Firstly, the isolated A549, H292 or 95D cells (1×10⁵/200 µl serum-free 1640) were plated in the upper chamber, and treated with CM, CXCL16 (100 ng/ml) or a combination of CM or CXCL16 with CXCL16 neutralization antibody(100 ng/mL). Secondly, the A549 cells, from the blank-control, phU6/GFP/Neo-CXCR6 and phU6/GFP/Neo group, were seeded on the upper chamber at a density of (1×10⁵/200 µl serum-free 1640), then treated with CXCL16 (100 ng/ml) or CM. The cells migrated to the lower surface were counted and the invasive index was calculated as the proportion of the migrated cells of the experiment group to that of its own control. Error bars depict the standard error of the mean. Con: the control; CXCL16: treated with 100 ng/ml CXCL16; antiCXCL16: treated with 100 ng/mL CXCL16 neutralizing antibody; CM: conditioned medium for A549, 95D or H292; Blank-control: without any treatment, shRNA-control: phU6/GFP/Neo; CXCR6-shRNA: phU6/GFP/Neo-CXCR6 (2819-1). ^aP<0.01 compared to the vehicle control; ^bP<0.05, ^cP<0.01, compared to the CXCL6 alone; ^dP<0.05, ^eP<0.01compared to the CM treatment group.</p