These effects were sustained for 48 h subsequent N-Shh stimulation. of proliferation after treatment with different focus of Gefitinib one agent, SANT-1 one agent or the mix of SANT-1 and Gefitinib in A549 cells. (DOCX) pone.0149370.s004.docx (13K) GUID:?28BBDBBC-7249-4AE7-B0C6-DEFFEFDBE4DE S5 Desk: The organic date from the proliferation results following treatment with different focus of Gefitinib one agent, SANT-1 one agent or the mix of Gefitinib and SANT-1 in H1975 cells analyzed by factorial analysis. (DOCX) pone.0149370.s005.docx (14K) GUID:?1AF0B07E-16E5-4225-B3FF-9D6F7375ED52 S6 Desk: The consequences of proliferation after treatment with different focus of Gefitinib one agent, SANT-1 one agent or the mix of SANT-1 and Gefitinib in H1975 cells. (DOCX) pone.0149370.s006.docx (13K) GUID:?822EFE2C-6C95-404F-A7A3-EF8C131A89CB Data Availability StatementAll relevant data are inside the paper and its own Supporting Information data files. Abstract Aberrant activation from the hedgehog (Hh) signaling pathway continues to be implicated in the epithelial-to-mesenchymal changeover (EMT) and tumor stem-like cell (CSC) maintenance; both processes can lead to tumor treatment and progression resistance in a number of types of individual cancer. Hh cooperates using the epidermal development aspect receptor (EGFR) signaling pathway in embryogenesis. We discovered that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung tumor (NSCLC) cells, although it was turned on in EGFR-TKI-resistant NSCLC cells inappropriately, followed by EMT ABCG2 and induction overexpression. Upregulation of Hh signaling through extrinsic SHH publicity downregulated E-cadherin appearance and raised ABCG2 and Snail appearance, leading to gefitinib tolerance (and beliefs < 0.05 were considered to indicate statistical significance in all full cases. Results Distinctions in Hh signaling pathway activity between EGFR-TKI-sensitive and -resistant NSCLC cells To assess Hh signaling pathway distinctions between EGFR-TKI-sensitive and -resistant NSCLC cells, three NSCLC cell lines, Computer9, H1975, and A549, harboring different mutations and various in awareness to TKIs, had been used. First, appearance of GLI1, a marker of activation from the Hh signaling pathway, was dependant on immunocytochemistry. As proven in Fig 1A, GLI1 was portrayed in the nuclei of EGFR-TKI-resistant cell lines A549 and H1975, but GLI1 appearance was harmful in the EGFR-TKI-sensitive cell range PC9. We verified this total result by Q-PCR and American blot evaluation. As proven in Fig 1C and 1B, GLI1 was portrayed at an extremely low level in Computer9 weighed against H1975 and A549 cells and respectively). Prior research indicated that Hh signaling regulates EMT via upregulation from the transcription aspect downregulation and Snail of E-cadherin[27, 28]. The stem cell marker ABCG2 is a primary target from the Hh signaling pathway[29] also. To help expand clarify the Hh pathway distinctions between -resistant and EGFR-TKI-sensitive cells, these three essential downstream focus on genes were analyzed by American blotting. We discovered that Snail appearance was significantly weaker in the EGFR-TKI-sensitive Computer9 cell range weighed against the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.001). E-cadherin appearance in Computer9 cells was quite high, while its appearance was very weakened in the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.008 and = 0.001; Fig 2D and S1 and S2 Dining tables). These results present that aberrant activation from the Shh signaling pathway qualified prospects to EGFR-TKI level of resistance in NSCLC cells. To examine the molecular systems root the contribution of Shh signaling to EGFR-TKI level of resistance in NSCLC cells, we analyzed Snail, E-cadherin, and ABCG2 appearance at 0, 24, and 48 h after treatment of Computer9 cells with N-Shh (0.5 g/mL) by Western blotting. As proven in Fig 2E, after contact with N-Shh for 24 h, the manifestation of Snail was raised (= 0.003), and ABCG2 manifestation was markedly upregulated in Personal computer9 cells (= 0.008). These results were suffered for 48 h pursuing N-Shh excitement. These results verified that hyperactivation of Hh signaling added to EGFR-TKI level of resistance in NSCLC cells through activation from the EMT changeover as well as the ABCG2 upregulation. Hh inhibition reversed EMT induction and reduced ABCG2 manifestation in EGFR-TKI-resistant NSCLC cells Following, to further measure the molecular systems of Hh signaling in EGFR-TKI-resistant NSCLC cells, we analyzed GLI1, Snail, E-cadherin, and ABCG2 manifestation at 0, 24, and 48 h after treatment of the EGFR-TKI-resistant cell lines H1975 and A549 with SANT-1 (40 M). The full total outcomes indicated that after treatment with SANT-1 for 24 h, GLI1 manifestation was downregulated (= 0.003 respectively); after treatment with SANT-1 for 48 h, Snail manifestation was nearly absent in H1975 cells (Fig 3A and 3B). Conversely, E-cadherin manifestation was elevated considerably pursuing treatment of EGFR-TKI-resistant cell lines with SANT-1 for 48 h (= 0.252 and = 0.187.However, the mean rank of EGFR-TKI-sensitive cells was 5.25, that of secondary resistant mutation cells was 4.88, as well as the mean rank of KRAS mutation cells was 9.38. factorial evaluation. (DOCX) pone.0149370.s005.docx (14K) GUID:?1AF0B07E-16E5-4225-B3FF-9D6F7375ED52 S6 Desk: The consequences of proliferation after treatment with different focus of Gefitinib solitary agent, SANT-1 solitary agent or the mix of Gefitinib and SANT-1 on H1975 cells. (DOCX) pone.0149370.s006.docx (13K) GUID:?822EFE2C-6C95-404F-A7A3-EF8C131A89CB Data Availability StatementAll relevant data are inside the paper and its own Supporting Information documents. Abstract Aberrant activation from the hedgehog (Hh) signaling pathway continues to be implicated in the epithelial-to-mesenchymal changeover (EMT) and tumor stem-like cell (CSC) maintenance; both procedures can lead to tumor development and treatment level of resistance in a number of types of human being tumor. Hh cooperates using the epidermal development element receptor (EGFR) signaling pathway in embryogenesis. We discovered that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung tumor (NSCLC) cells, although it was inappropriately turned on in EGFR-TKI-resistant NSCLC cells, followed by EMT Ubenimex induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH publicity downregulated E-cadherin manifestation and raised Snail and ABCG2 manifestation, leading to gefitinib tolerance (and ideals < 0.05 were thought to indicate statistical significance in every cases. Results Variations in Hh signaling pathway activity between EGFR-TKI-sensitive and -resistant NSCLC cells To assess Hh signaling pathway variations between EGFR-TKI-sensitive and -resistant NSCLC cells, three NSCLC cell lines, Personal computer9, H1975, and A549, harboring different mutations and various in level of sensitivity to TKIs, had been used. First, manifestation of GLI1, a marker of activation from the Hh signaling pathway, was dependant on immunocytochemistry. As demonstrated in Fig 1A, GLI1 was indicated in the nuclei of EGFR-TKI-resistant cell lines A549 and H1975, but GLI1 manifestation was adverse in the EGFR-TKI-sensitive cell range Personal computer9. We verified this result by Q-PCR and Traditional western blot evaluation. As demonstrated in Fig 1B and 1C, GLI1 was indicated at an extremely low level in Personal computer9 Ubenimex weighed against H1975 and A549 cells and respectively). Earlier research indicated that Hh signaling regulates EMT via upregulation from the transcription element Snail and downregulation of E-cadherin[27, 28]. The stem cell marker ABCG2 can be a direct focus on from the Hh signaling pathway[29]. To help expand clarify the Hh pathway variations between EGFR-TKI-sensitive and -resistant cells, these three essential downstream focus on genes were analyzed by European blotting. We discovered that Snail manifestation was substantially weaker in the EGFR-TKI-sensitive Personal computer9 cell range weighed against the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.001). E-cadherin manifestation in Personal computer9 cells was quite high, while its manifestation was very fragile in the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.008 and = 0.001; Fig 2D and S1 and S2 Dining tables). These results display that aberrant activation from the Shh signaling pathway qualified prospects to EGFR-TKI level of resistance in NSCLC cells. To examine the molecular systems root the contribution of Shh signaling to EGFR-TKI level of resistance in NSCLC cells, we analyzed Snail, Ubenimex E-cadherin, and ABCG2 manifestation at 0, 24, and 48 h after treatment of Personal computer9 cells with N-Shh (0.5 g/mL) by Western blotting. As demonstrated in Fig 2E, after contact with N-Shh for 24 h, the manifestation of Snail was raised (= 0.003), and ABCG2 manifestation was markedly upregulated in Personal computer9 cells (= 0.008). These results were suffered for 48 h pursuing N-Shh excitement. These results verified that hyperactivation of Hh signaling added to EGFR-TKI level of resistance in NSCLC cells through activation from the EMT changeover as well as the ABCG2 upregulation. Hh inhibition reversed EMT induction and reduced ABCG2 manifestation in EGFR-TKI-resistant NSCLC cells Following, to further measure the molecular systems of Hh signaling in EGFR-TKI-resistant NSCLC cells, we analyzed GLI1, Snail, E-cadherin, and ABCG2 manifestation at 0, 24, and 48 h after treatment of the EGFR-TKI-resistant cell lines H1975 and A549 with SANT-1 (40 M). The outcomes indicated that after treatment with SANT-1 for 24 h, GLI1 manifestation was downregulated (= 0.003 respectively); after treatment with SANT-1 for 48 h, Snail manifestation was nearly absent in H1975 cells (Fig 3A and 3B). Conversely, E-cadherin manifestation was elevated considerably pursuing treatment of EGFR-TKI-resistant cell lines with SANT-1 for 48 h (= 0.252 and = 0.187 respectively). Nevertheless, colonies very hardly shaped in the group put through mixed SANT-1 and gefitinib treatment (< 0.001). These total results indicate that SANT-1 and gefitinib may have a synergistic effect in EGFR-TKI-resistant.E-cadherin was moderately or strongly expressed generally in most NSCLC cells (9/12, = 0.555). (14K) GUID:?BDCDB362-0B21-4244-Abdominal61-2077C2E8C992 S4 Desk: The consequences of proliferation after treatment with different focus of Gefitinib solitary agent, SANT-1 solitary agent or the mix of Gefitinib and SANT-1 on A549 cells. (DOCX) pone.0149370.s004.docx (13K) GUID:?28BBDBBC-7249-4AE7-B0C6-DEFFEFDBE4DE S5 Desk: The uncooked date from the proliferation results following treatment with different focus of Gefitinib one Rabbit Polyclonal to PDRG1 agent, SANT-1 one agent or the mix of Gefitinib and SANT-1 in H1975 cells analyzed by factorial analysis. (DOCX) pone.0149370.s005.docx (14K) GUID:?1AF0B07E-16E5-4225-B3FF-9D6F7375ED52 S6 Desk: The consequences of proliferation after treatment with different focus of Gefitinib one agent, SANT-1 one agent or the mix of Gefitinib and SANT-1 on H1975 cells. (DOCX) pone.0149370.s006.docx (13K) GUID:?822EFE2C-6C95-404F-A7A3-EF8C131A89CB Data Availability StatementAll relevant data are inside the paper and its own Supporting Information data files. Abstract Aberrant activation from the hedgehog (Hh) signaling pathway continues to be implicated in the epithelial-to-mesenchymal changeover (EMT) and cancers stem-like cell (CSC) maintenance; both procedures can lead to tumor development and treatment level of resistance in a number of types of individual cancer tumor. Hh cooperates using the epidermal development aspect receptor (EGFR) signaling pathway in embryogenesis. We discovered that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancers (NSCLC) cells, although it was inappropriately turned on in EGFR-TKI-resistant NSCLC cells, followed by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH publicity downregulated E-cadherin appearance and raised Snail and ABCG2 appearance, leading to gefitinib tolerance (and beliefs < 0.05 were thought to indicate statistical significance in every cases. Results Distinctions in Hh signaling pathway activity between EGFR-TKI-sensitive and -resistant NSCLC cells To assess Hh signaling pathway distinctions between EGFR-TKI-sensitive and -resistant NSCLC cells, three NSCLC cell lines, Computer9, H1975, and A549, harboring different mutations and various in awareness to TKIs, had been used. First, appearance of GLI1, a marker of activation from the Hh signaling pathway, was dependant on immunocytochemistry. As proven in Fig 1A, GLI1 was portrayed in the nuclei of EGFR-TKI-resistant cell lines A549 and H1975, but GLI1 appearance was detrimental in the EGFR-TKI-sensitive cell series Computer9. We verified this result by Q-PCR and Traditional western blot evaluation. As proven in Fig 1B and 1C, GLI1 was portrayed at an extremely low level in Computer9 weighed against H1975 and A549 cells and respectively). Prior research indicated that Hh signaling regulates EMT via upregulation from the transcription aspect Snail and downregulation of E-cadherin[27, 28]. The stem cell marker ABCG2 can be a direct focus on from the Hh signaling pathway[29]. To help expand clarify the Hh pathway distinctions between EGFR-TKI-sensitive and -resistant cells, these three essential downstream focus on genes were analyzed by American blotting. We discovered that Snail appearance was significantly weaker in the EGFR-TKI-sensitive Computer9 cell series weighed against the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.001). E-cadherin appearance in Computer9 cells was quite high, while its appearance was very vulnerable in the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.008 and = 0.001; Fig 2D and S1 and S2 Desks). These results present that aberrant activation from the Shh signaling pathway network marketing leads to EGFR-TKI level of resistance in NSCLC cells. To examine the molecular systems root the contribution of Shh signaling to EGFR-TKI level of resistance in NSCLC cells, we analyzed Snail, E-cadherin, and ABCG2 appearance at 0, 24, and 48 h after treatment of Computer9 cells with N-Shh (0.5 g/mL) by Western blotting. As proven in Fig 2E, after contact with N-Shh for 24 h, the appearance of Snail was raised (= 0.003), and ABCG2 appearance was markedly upregulated in Computer9 cells (= 0.008). These results were suffered for 48 h pursuing N-Shh arousal. These results verified that hyperactivation of Hh signaling added to EGFR-TKI level of resistance in NSCLC cells through activation from the EMT changeover as well as the ABCG2 upregulation. Hh inhibition reversed EMT induction and reduced ABCG2 appearance in EGFR-TKI-resistant NSCLC cells Following, to further measure the molecular systems of Hh signaling in EGFR-TKI-resistant NSCLC cells, we analyzed GLI1, Snail, E-cadherin, and ABCG2 appearance at 0, 24, and 48 h after treatment of the EGFR-TKI-resistant cell lines H1975 and A549 with SANT-1 (40 M). The outcomes indicated that after treatment with SANT-1 for 24 h, GLI1 appearance was downregulated (= 0.003 respectively); after treatment with SANT-1 for 48 h, Snail appearance was nearly absent in H1975 cells (Fig 3A and 3B). Conversely, E-cadherin appearance was elevated considerably pursuing treatment of EGFR-TKI-resistant cell lines with SANT-1 for 48 h (= 0.252 and = 0.187 respectively). Nevertheless, colonies very hardly produced in the group put through mixed SANT-1 and gefitinib treatment (< 0.001). These total results indicate that SANT-1 and gefitinib may have a synergistic effect in EGFR-TKI-resistant NSCLC cells. To verify this, we treated the EGFR-TKI-resistant NSCLC cell lines A549 and.Conversely, Snail was negative or weakly expressed generally in most tissues (11/12, = 0.658) (Desks ?(Desks11 and ?and22). Correlation evaluation showed that E-cadherin appearance was significantly negatively correlated with Snail appearance (= 0.582, = 0.047). treatment with different focus of Gefitinib one agent, SANT-1 one agent or the mix of Gefitinib and SANT-1 on H1975 cells. (DOCX) pone.0149370.s006.docx (13K) GUID:?822EFE2C-6C95-404F-A7A3-EF8C131A89CB Data Availability StatementAll relevant data are inside the paper and its own Supporting Information data files. Abstract Aberrant activation from the hedgehog (Hh) signaling pathway continues to be implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human malignancy. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (and values < 0.05 were considered to indicate statistical significance in all cases. Results Differences in Hh signaling pathway activity between EGFR-TKI-sensitive and -resistant NSCLC cells To assess Hh signaling pathway differences between EGFR-TKI-sensitive and -resistant NSCLC cells, three NSCLC cell lines, PC9, H1975, and A549, harboring different mutations and differing in sensitivity to TKIs, were used. First, expression of GLI1, a marker of activation of the Hh signaling pathway, was determined by immunocytochemistry. As shown in Fig 1A, GLI1 was expressed in the nuclei of EGFR-TKI-resistant cell lines A549 and H1975, but GLI1 expression was unfavorable in the EGFR-TKI-sensitive cell line PC9. We confirmed this result by Q-PCR and Western blot analysis. As shown in Fig 1B and 1C, GLI1 was expressed at a very low level in PC9 compared with H1975 and A549 cells and respectively). Previous studies indicated that Hh signaling regulates EMT via upregulation of the transcription factor Snail and downregulation of E-cadherin[27, 28]. The stem cell marker ABCG2 is also a direct target of the Hh signaling pathway[29]. To further clarify the Hh pathway differences between EGFR-TKI-sensitive and -resistant cells, these three important downstream target genes were examined by Western blotting. We found that Snail expression was considerably weaker in the EGFR-TKI-sensitive PC9 cell line compared with the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.001). E-cadherin expression in PC9 cells was quite high, while its expression was very poor in the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.008 and = 0.001; Fig 2D and S1 and S2 Tables). These findings show that aberrant activation of the Shh signaling pathway leads to EGFR-TKI resistance in NSCLC cells. To examine the molecular mechanisms underlying the contribution of Shh signaling to EGFR-TKI resistance in NSCLC cells, we examined Snail, E-cadherin, and ABCG2 expression at 0, 24, and 48 h after treatment of PC9 cells with N-Shh (0.5 g/mL) by Western blotting. As shown in Fig 2E, after exposure to N-Shh for 24 h, the expression of Snail was elevated (= 0.003), and ABCG2 expression was markedly upregulated in PC9 cells (= 0.008). These effects were sustained for 48 h following N-Shh stimulation. These results confirmed that hyperactivation of Hh signaling contributed to EGFR-TKI resistance in NSCLC cells through activation of the EMT transition and the ABCG2 upregulation. Hh inhibition reversed EMT induction and decreased ABCG2 expression in EGFR-TKI-resistant NSCLC cells Next, to further assess the molecular mechanisms of Hh signaling in EGFR-TKI-resistant NSCLC cells, we examined GLI1, Snail, E-cadherin, and ABCG2 expression at 0, 24, and 48 h after treatment of the EGFR-TKI-resistant cell lines H1975 and A549 with SANT-1 (40 M). The results indicated that after treatment with SANT-1 for 24 h, GLI1 expression was downregulated (= 0.003 respectively); after treatment with SANT-1 for 48 h, Snail expression was almost absent in H1975 cells (Fig 3A and 3B). Conversely, E-cadherin expression was elevated significantly following treatment of EGFR-TKI-resistant cell lines with SANT-1 for.E-cadherin was moderately or strongly expressed in most NSCLC tissues (9/12, = 0.555). analysis. (DOCX) pone.0149370.s005.docx (14K) GUID:?1AF0B07E-16E5-4225-B3FF-9D6F7375ED52 S6 Table: Ubenimex The effects of proliferation after treatment with different concentration of Gefitinib single agent, SANT-1 single agent or the combination of Gefitinib and SANT-1 on H1975 cells. (DOCX) pone.0149370.s006.docx (13K) GUID:?822EFE2C-6C95-404F-A7A3-EF8C131A89CB Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human malignancy. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (and values < 0.05 were considered to indicate statistical significance in all cases. Results Differences in Hh signaling pathway activity between EGFR-TKI-sensitive and -resistant NSCLC cells To assess Hh signaling pathway differences between EGFR-TKI-sensitive and -resistant NSCLC cells, three NSCLC cell lines, PC9, H1975, and A549, harboring different mutations and differing in sensitivity to TKIs, were used. First, expression of GLI1, a marker of activation of the Hh signaling pathway, was determined by immunocytochemistry. As shown in Fig 1A, GLI1 was expressed in the nuclei of EGFR-TKI-resistant cell lines A549 and H1975, but GLI1 expression was negative in the EGFR-TKI-sensitive cell line PC9. We confirmed this result by Q-PCR and Western blot analysis. As shown in Fig 1B and 1C, GLI1 was expressed at a very low level in PC9 compared with H1975 and A549 cells and respectively). Previous studies indicated that Hh signaling regulates EMT via upregulation of the transcription factor Snail and downregulation of E-cadherin[27, 28]. The stem cell marker ABCG2 is also a direct target of the Hh signaling pathway[29]. To further clarify the Hh pathway differences between EGFR-TKI-sensitive and -resistant cells, these three important downstream target genes were examined by Western blotting. We found that Snail expression was considerably weaker in the EGFR-TKI-sensitive PC9 cell line compared with the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.001). E-cadherin expression in PC9 cells was quite high, while its expression was very weak in the EGFR-TKI-resistant cell lines H1975 and A549 (= 0.008 and = 0.001; Fig 2D and S1 and S2 Tables). These findings show that aberrant activation of the Shh signaling pathway leads to EGFR-TKI resistance in NSCLC cells. To examine the molecular mechanisms underlying the contribution of Shh signaling to EGFR-TKI resistance in NSCLC cells, we examined Snail, E-cadherin, and ABCG2 expression at 0, 24, and 48 h after treatment of PC9 cells with N-Shh (0.5 g/mL) by Western blotting. As shown in Fig 2E, after exposure to N-Shh for 24 h, the expression of Snail was elevated (= 0.003), and ABCG2 expression was markedly upregulated in PC9 cells (= 0.008). These effects were sustained for 48 h following N-Shh stimulation. These results confirmed that hyperactivation of Hh signaling contributed to EGFR-TKI resistance in NSCLC cells through activation of the EMT transition and the ABCG2 upregulation. Hh inhibition reversed EMT induction and decreased ABCG2 expression in EGFR-TKI-resistant NSCLC cells Next, to further assess the molecular mechanisms of Hh signaling in EGFR-TKI-resistant NSCLC cells, we examined GLI1, Snail, E-cadherin, and ABCG2 expression at 0, 24, and 48 h after treatment of the EGFR-TKI-resistant cell lines H1975 and A549 with SANT-1 (40 M). The results.