Journal: Molecules

Article Title: Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

doi: 10.3390/molecules21050570

Figure Lengend Snippet: Antiproliferative activity of tested compounds on VEGFR-2 dependent PC-3 cells. ( a ) Dose-response curves of active inhibitors ( 1 and 3 ) and their corresponding caged compounds ( 4 and 5 ) were determined without UV irradiation. Cell growth was measured 48 h after incubation with the compounds. The caged derivatives do not exhibit cytotoxic activity: the TGI mark is not reached even at high concentrations; ( b ) Cells were incubated for 1 h with the compounds and then irradiated at 365 nm (1.1 kW/m²) for 5 min. Cell growth was determined 48 h after incubation with the compounds. After irradiation, the caged derivatives show similar ( 5 ) or even left shifted ( 4 ) dose-response curves as that of active compounds. GI50 = 50% growth inhibition; TGI = total growth inhibition; LC 50 = 50% lethal concentration; n = 4. Error bars represent standard deviation. The effect of UV irradiation on the PC-3 cells is shown in .

Article Snippet: PC-3 cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Activity Assay, Irradiation, Incubation, Inhibition, Concentration Assay, Standard Deviation

Journal: Molecules

Article Title: Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

doi: 10.3390/molecules21050570

Figure Lengend Snippet: Antiproliferative activity of tested compounds in cellular growth assays with PC-3 cells, without and after irradiation at 365 nm (1.1 kW/m 2 ). The GI 50 values (50% cellular growth inhibition) are presented. “Not reached”: The tested compound did not inhibit the cell growth to 50%.

Article Snippet: PC-3 cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Activity Assay, Irradiation, Inhibition

Journal: Molecules

Article Title: Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

doi: 10.3390/molecules21050570

Figure Lengend Snippet: Microscopic images of stained PC-3 cells. The investigated compounds 3 and 5 are shown in green. Both compounds were applied at 10 µM concentrations. The cell nuclei were counterstained with 1 µg/mL DAPI and are marked blue. The dashed line represents 20.3 µm. ( a ) Staining with carbazole 3 ; ( b ) Staining with caged compound 5 .

Article Snippet: PC-3 cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: Staining

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A Cell viability assay of PC9 cells treated for 5 d with pemetrexed (Pem) and gefitinib (1 µM; Gef). The mean ± SEM of n = 6 is shown (representative of three independent experiments). B PC9 cells containing a pool of EGFR-T790M barcoded cells were treated with gefitinib (1 µM) or pemetrexed (50 nM) for 6 d. The EGFR-T790M mean ± SEM is shown ( n = 4; representative of three independent experiments). C PC9, HCC4006 or HCC827 NSCLC cells containing EGFR-T790M barcoded subpopulations were treated with gefitinib (1 µM) or sorafenib (5 µM; Soraf) for 5 d. The EGFR-T790M mean ± SEM is shown ( n = 4; representative of three independent experiments). D PC9, H1975, and YU-1150 cells containing EGFR-C797S-barcoded subpopulations were treated with osimertinib (0.1 µM; Osim) alone or with sorafenib (5 µM) for 10 d. The EGFR-C797S mean ± SEM is shown ( n = 4; representative of three independent experiments). E PC9 and H1975 cells described in ( E ) were treated with osimertinib (1 µM or 0.1 µM, respectively) alone or with sorafenib (5 µM) for 20 d or 15 d. The cells were then fixed and stained ( n = 3; representative of two and three independent experiments). EGFR-scores of responders ( R ) and non-responders (NR) from a cohort of renal-cell carcinoma ( F ) and thyroid cancer patients ( G ) treated with sorafenib. H H358 cells containing a KRAS-G12D-barcoded subpopulation were treated with sotorasib (10 nM; Sotor) alone or with sorafenib (5 μM) for 12 d. The KRAS-G12D mean ± SEM is shown ( n = 4, representative of three independent experiments). p = 0.4857. I Ceritinib-resistant H3122 cells labeled with VIRHD lentivirus were mixed with parental cells (1:50) and treated with ceritinib (50 nM; Cerit) alone or with sorafenib (5 µM) for 7 d. VIRHD-cells mean ± SEM is shown ( n = 5, representative of three independent experiments). J EGFR-C797S CRISPR-barcoded PC9 cells were treated with osimertinib (0.1 µM) alone or with sorafenib (5 µM), sunitinib (1 µM; Sun), regorafenib (2 µM; Reg), lenvatinib (1 µM; Lenv), or cabozantinib (5 µM; Cab) for 7 d (left) or 10 d (right). EGFR-C797S mean ± SEM is shown ( n = 4–5; representative of three independent experiments). K PC9 cells were treated with sorafenib (5 μM) for 72 h, and cell cycle was analyzed by FACS. The mean ± SEM of four independent experiments is shown. L PC9 cells were treated with sorafenib (5 μM) for 72 h, and percentage of apoptotic cells was measured by annexin V staining. The mean ± SEM of three independent experiments is shown. ns not significant. Statistics are: B , C , D , H , I , J matched Mann-Whitney, two-tailed; F , G matched Student t -test, two-tailed; K , L matched two-way ANOVA with the Tukey correction. N refers to biological replicates. Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Viability Assay, Staining, Labeling, CRISPR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A PC9 cells containing subpopulations of EGFR-G724S, ERBB2-ex20ins, KRAS-G12D, BRAF-V600E or PIK3CA-E545K cells were treated as indicated with osimertinib (0,1 μM; Osim), sorafenib (5 μM; Soraf) or trametinib (10 nM; Tram) for 7 d to 20 d. The mean ± SEM of the mutant barcodes is shown ( n = 4; representative of three independent experiments). B BRAF-V600E osimertinib-resistant YUX-1024 and YU-1150 cells were mixed with parental cells (1:100) and treated for 7 d with osimertinib (0.1 μM) alone or with sorafenib (5 μM). The BRAF-V600E mean ± SEM is shown ( n = 4; representative of three independent experiments). C PC9 cells containing a EML4-ALK-barcoded subpopulation were treated for 10 d with osimertinib (0.1 µM), sorafenib (5 µM), or crizotinib (500 nM; Criz). The EML4-ALK mean ± SEM is shown ( n = 5; representative of three independent experiments). D PC9 cells overexpressing ERBB2-ex20ins or MET were mixed with parental cells (1:100) and treated with osimertinib (0.1 μM) alone or with sorafenib (5 μM) for 10 d or 15 d. The mean fraction ± SEM of ERBB2-ex20ins- or MET-overexpressing cells is shown ( n = 4; representative of three independent experiments). E Lentiviral-labeled, osimertinib-resistant HCC827-GR6 cells were mixed with parental HCC827 (1:100) and treated for 6 d with osimertinib (0.1 µM) alone or with sorafenib (5 µM). HCC827-GR6 mean fraction ± SEM is shown ( n = 4; representative of three independent experiments). F PC9 cells transduced with empty or inducible-SNAI2 vectors were pre-treated with or without doxycycline (1 µg/ml; Dox) for 7 d, mixed with parental PC9 (1:100) and treated for 7 d with osimertinib (0.1 µM) alone or with sorafenib (5 µM). The mean fraction ± SEM of vector-labeled cells is shown ( n = 4; representative of three independent experiments). LIM1215 cells containing EGFR-G465R ( G ) or KRAS-G12D ( H ) CRISPR-barcodes were treated for 6 d with cetuximab (20 µg; Cetux) or osimertinib (1 µM), alone or with sorafenib (5 μM). The barcode mean fraction ± SEM is shown ( n = 3 for EGFR-G465R; n = 4 for KRAS-G12D; representative of three independent experiments). Statistics calculated by Mann–Whitney test, two-tailed. “ N ” refers to biological replicates. Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Mutagenesis, Labeling, Transduction, Plasmid Preparation, CRISPR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A PC9 cells were treated with sorafenib (5 µM; Soraf) or trametinib (50 nM; Tram), followed by immunoblot (representative of three independent experiments). B YUX-1024 and YU-1150 PDCs were treated for 2 h with sorafenib (5 µM), osimertinib (0.1 µM; Osim), eFT-508 (1 µM; eFT), or trametinib (50 nM), followed by immunoblot (representative of three independent experiments). C PC9 cells containing inducible constitutively active MKNK2 (CA-MKNK2; Flag-tagged) were pre-treated with or without doxycycline (0.1 μg/ml; Dox) for 12 h, treated for 1 h with sorafenib, eFT-508 (20 μM) or trametinib (1 μM), followed by immunoblot (representative of four independent experiments). D Flag-eIF4E was incubated with recombinant active MKNK2 (rMKNK2) and the indicated inhibitors, followed by immunoblot (representative of three independent experiments). E PC9 cells were treated with sorafenib (5 µM) or trametinib (50 nM) for 6 h, followed by immunoblot (representative of three independent experiments). F PC9 cells were treated with or without sorafenib (5 µM), followed by immunoblot (representative of four independent experiments). G YU-1150 and YUX-1024 PDCs were treated with sorafenib (5 µM) for 3 d, followed by immunoblot (representative of three independent experiments). H PC9 cells were treated with sorafenib (5 µM) in the presence or the absence of cycloheximide (20 µg/mL; CHX) for 1 d, followed by immunoblot (representative of three independent experiments). I PC9 cells were treated with sorafenib (5 µM), chloroquine (50 µM; ChQ) or MG132 (5 µM) for 1 d as indicated, followed by immunoblot (representative of three independent experiments). J EGFR-T790M CRISPR-barcoded PC9 cells were treated for 5 d as indicated with gefitinib (1 µM), sorafenib (5 µM), or a combination (iESM) of napabucasin (0.5 µM), S63845 (0.1 µM), and eFT-508 (1 µM). EGFR-T790M mean ± SEM is shown ( n = 5; representative of three independent experiments). K Osimertinib-resistant PC9 cells (EGFR-C797S) containing shMCL1, shSTAT3, and DN-MKNK1 inducible vectors were mixed with parental PC9 (1:100) and treated for 6 d as indicated with osimertinib (100 nM), sorafenib (5 µM), or doxycycline (2 µg/ml). EGFR-C797S mean ± SEM is shown ( n = 5, representative of three independent experiments). J , K p values were calculated by Mann–Whitney two-tailed test and “n” refers to biological replicates. Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Western Blot, Incubation, Recombinant, CRISPR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A Time-course effects of sorafenib (5 µM; Soraf) in PC9 cells. Immunoblot was performed using the indicated antibodies (representative of four independent experiments). B YUX-1024 and YU-1150 PDCs were treated with sorafenib (5 µM) for 5 d, followed by immunoblot (representative of three independent experiments). C PC9 cells were injected in the flanks of female SCID mice and, once the tumors reached a mean volume of about 200 mm 3 , the mice were randomized and treated with control vehicle or sorafenib (60 mg/kg) for 4 d. The mice were then sacrificed, and the tumors dissected. Immunoblot was performed using the indicated antibodies ( n = 5 mice). D PC9 cells were transduced with a lentiviral vector containing Flag-tagged EGFR-Ex19Del and the green fluorescent protein (GFP), separated by the T2A self-cleaving peptide and under the control of the PGK promoter. Two different clones were isolated and treated for 3 d in the presence or the absence of sorafenib (5 µM). The expression of EGFR and GFP was measured by immunoblot (upper panel) and FACS (lower panel), respectively. Representative blots and FACS analysis from three independent experiments. E PC9 cells were transduced with lentiviral vectors containing either Ex19Del-mutant or wt Flag-tagged EGFR, and two clones per condition were isolated. The clones were treated with or without sorafenib (5 µM) for 3 d, followed by immunoblot (representative of four independent experiments). F PC9 cells were treated with sorafenib (5 µM) alone or with different concentrations of chloroquine (ChQ) for 3 d, followed by immunoblot (representative of three independent experiments). G PC9 cells containing the EGFR-T790M CRISPR-barcode were treated with gefitinib (1 µM; Gef) alone or in combination with sorafenib (5 µM), with or without chloroquine (20 µM) for 4 d. The EGFR-T790M mean fraction ± SEM is shown ( n = 5 biological replicates; representative of three independent experiments). Statistics are Mann–Whitney test, two-tailed. Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Western Blot, Injection, Control, Transduction, Plasmid Preparation, Clone Assay, Isolation, Expressing, Mutagenesis, CRISPR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A Parental (Par) or osimertinib-resistant (OR) PC9 cells were treated for 5 d as indicated with osimertinib (0,1 µM; Osim), sorafenib (5 µM; Soraf), sunitinib (1 µM; Sun) or cabozantinib (5 µM; Cab). The cell viability mean ± SEM is shown ( n = 5 biological replicates; representative of four independent experiments). B Left panel: colony forming assay representative images of parental, gefitinib-resistant/osimertinib-sensitive (GR) or osimertinib-resistant PC9 cells treated for 5 d with osimertinib (1 µM) or sorafenib (5 µM), alone or in combination. Right panel: colony forming assay of H358 cells treated for 8 d with sotorasib (10 nM; Sotor) or sorafenib (5 µM), alone or in combination, or H3122 cells treated for 7 d with ceritinib (50 nM; Cerit) or sorafenib, alone or in combination ( n = 3 biological replicates; representative of three independent experiments). Osimertinib-sensitive and resistant PC9 cells were treated for 1 d ( C ) or 3 d ( D ) with osimertinib (1 µM), sorafenib (5 µM), or the combination, followed by immunoblot using the indicated antibodies (representative of three independent experiments). E Gene set enrichment analysis (GSEA) of genes up-regulated by EGFR-TKIs in NSCLC cells (KOBAYASHI_EGFR_SIGNALING_24HR_UP signature), performed on gene array data obtained from PC9 cells treated with osimertinib (1 µM) or sorafenib (5 µM), alone or in combination (Combo) for 2 d. Enrichment scores (ES) and p values are reported (two-sided permutation test). F The data described in ( E ) were analyzed using our osimertinib-sorafenib combination signature (COMBO_UP). Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Western Blot

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A A PC9 mass population containing small pools (1:1000) of EGFR-C797S (expressing GFP), KRAS-G12D (expressing β-galactosidase), and PIK3CA-E545K (expressing mCherrry) osimertinib-resistant cells was subcutaneously injected in the flanks of female SCID mice. Once the tumors reached a mean volume of ~200 mm 3 , the mice were sacrificed (Ctrl) or treated for 4 weeks with osimertinib (5 mg/kg; Osim) alone or with sorafenib (60 mg/kg; Combo), followed by 3D-imaging. Images representative of two tumors per condition. The tumors from osimertinib-treated mice were cut in two. Scale bars: 1500 μm. B PC9 cells containing a small pool of EGFR-C797S cells were injected in the flanks of male SCID mice. Once the tumors were palpable (arrow), the mice were randomized and treated with or without osimertinib (5 mg/kg) and sorafenib (60 mg/kg; Soraf), alone or in combination, and tumor volume was measured by caliper. The mean tumor volumes ± SEM are represented ( n = 5 mice). Osim vs Combo p -value was calculated at day 91 using two-tailed unpaired t test. C YUX-1024 PDCs containing a 1:200 subpopulation of BRAF-V600E resistant cells expressing GFP were subcutaneously injected in female SCID mice. Once the tumors reached a mean volume of about 200 mm 3 , the mice were sacrificed (Ctrl) or treated for 4 weeks with osimertinib (10 mg/kg) alone or in combination with sorafenib (60 mg/kg), followed by 3D-imaging. Images representative of two tumors per condition. Scale bars: 1500 μm. D YUX-1024 PDCs containing a pool of BRAF-V600E cells (1:200) were injected in the right and left flanks of female SCID mice. When the tumors reached a mean volume of about 200 mm 3 , the mice were treated with vehicle, osimertinib (10 mg/kg), sorafenib (60 mg/kg), or the combination. The mean tumor volumes ± SEM are represented ( n = 6 mice for vehicle, n = 8 for the other groups); Osim vs Combo p value was calculated at day 83 using two-tailed unpaired t test. E Kaplan–Meier diagram of the experiment illustrated in ( D ). The mice were sacrificed when the volume of at least one of the tumors exceeded 800 mm 3 . Osim vs Combo p value was calculated (log-rank Mantel-Cox). Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Expressing, Injection, 3D Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A A PC9 mass population containing small pools of CRISPR-barcoded EGFR-C797S, KRAS-G12D, and PIK3CA-E545K cells or ERBB2-ex20ins overexpressing cells was injected in the flanks of male and female SCID mice. Once the tumors reached a mean volume of about 100 mm 3 , the mice were randomized and treated with vehicle, osimertinib (5 mg/kg; Osim) alone or with sorafenib (60 mg/kg; Combo). The mean tumor volumes ± SEM are shown ( n = 6 mice for vehicle, n = 10 mice for other groups). Osim vs Combo p value was calculated at day 47 using two-tailed unpaired t test. B Kaplan–Meier diagram of the experiment shown in ( A ). The mice were sacrificed when the volume of at least one of the tumors exceeded 800 mm 3 . Osim vs Combo p value was calculated (log-rank Mantel-Cox). C GSEA of the genes up-regulated by osimertinib, sorafenib, or the combination in PC9 cells based on an inflammatory response signature. D Mouse BEM-5 cells were injected in the flanks of syngeneic BALB/c mice. When the tumors reached a mean volume of about 50 mm 3 , the mice were treated for 10 days with vehicle, osimertinib (20 mg/kg), sorafenib (60 mg/kg; Soraf), or the combination, followed by IHC analysis. Images representative of three different tumors/mice per condition. E BEM-5 cells were injected in the right and left flanks of BALB/c mice. Once the tumors reached a mean volume of about 50 mm 3 , the mice were randomized and treated with vehicle, osimertinib (20 mg/kg), sorafenib (60 mg/kg), or the combination. The mean tumor volumes ± SEM are shown ( n = 5 mice for control, n = 6 mice for osimertinib and sorafenib, n = 16 mice for the combination). Osim vs Combo p value at day 57 was calculated using two-tailed unpaired t test. F Kaplan–Meier diagram of the experiment shown in ( E ). Osim vs Combo p value was calculated (log-rank Mantel-Cox). Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: CRISPR, Injection, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Prolonging lung cancer response to EGFR inhibition by targeting the selective advantage of resistant cells

doi: 10.1038/s41467-025-61788-w

Figure Lengend Snippet: A PC9 cells were treated for 1 d with osimertinib (1 µM; Osim) or sorafenib (5 µM; Soraf), alone or in combination (Combo), followed by immunoblot (representative of three independent experiments). B Drug-tolerant expanded persisters (DTEPs) labeled with a control lentivirus were mixed with parental PC9 cells (1:100) and treated for 15 d with osimertinib (0.1 µM) alone or with sorafenib (5 µM) or crizotinib (0.5 µM; Criz). DTEPs mean fraction ± SEM is shown ( n = 4 biological replicates; representative of three independent experiments). Mann–Whitney two-tailed test. C PC9 cells stably expression GFP were treated with osimertinib (0,1 µM) or sorafenib (5 µM), alone or in combination for 26 d. The fluorescence from each well was measured every day with an Incucyte imaging system. The mean ± SEM of n = 7 biological replicates is shown (representative of two independent experiments). D PC9 cells containing highly complex CRISPR-barcodes in the AAVS1 locus were treated for 2 weeks with osimertinib (1 µM) and sorafenib (5 µM), alone or in combination ( n = 4). Barcodes enriched at least 5-fold over the control in 4 or 3 biological replicates are shown. E Pearson correlation of the barcode distribution in control versus osimertinib or control versus combination. The coefficients of determination ( R 2 ) and the p value (Mann–Whitney, two-tailed) are indicated. F Mouse BEM4 cells were injected in the flanks of syngeneic BALB/c mice. Once the tumors reached a mean volume of about 100 mm 3 , the mice were randomized and treated 3 times a week with vehicle, osimertinib (20 mg/kg), sorafenib (60 mg/kg), or the combination. The mean tumor volumes ± SEM are shown ( n = 7 mice for the control, n = 8 mice for the other groups). Osim vs Combo p value at day 64 is shown (two-tailed unpaired t test). G Kaplan–Meier diagram of the experiment shown in ( F ). Osim vs Combo p value was calculated (log-rank Mantel-Cox). H The indicated blood parameters were measure from the mice in ( F ) at 10 d and 28 d after the beginning of the treatment. ns not significant (Kruskal–Wallis, two-tailed). Source data are provided as a file.

Article Snippet: Human embryonic kidney 293T cells were obtained from ATCC; PC9 cells (NSCLC, EGFR-Ex19Del) were obtained from ECACC (distributed by Sigma-Aldrich); HCC4006 (NSCLC, EGFR-Ex19Del) and H1975 cells (NSCLC, EGFR-L858R/T790M) were obtained from ATCC; H3122 cells (NSCLC, EML4-ALK) were obtained from Cytion; HCC827 cells (NSCLC, EGFR-Ex19Del) and H358 (NSCLC, KRAS-G12C) were a gift from Pr.

Techniques: Western Blot, Labeling, Control, MANN-WHITNEY, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Imaging, CRISPR, Injection

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines present cysteine reliance on H 2 S production. ( A ) NSCLC cell lines showed similar basal levels of H 2 S production. To verify if H 2 S production was directly dependent on cysteine degradation, cells were exposed to AOAA/PAG treatment. ( B ) NSCLC cells cultured in the presence of the H 2 S donor NaHS alone and in combination with cysteine showed that H 2 S levels decreased when exposed to AOAA and PAG. Upon cysteine treatment, PC-9 showed a tendency to decrease the average levels of H 2 S in control conditions and compensated production of H 2 S upon exposure to inhibitors. ( C ) Upon cysteine supplementation, NSCLC cell lines tended to decrease H 2 S production at T = 0 h but compensated the CBS and CSE inhibition, maintaining (H292 and PC-9) or increasing (A549) H 2 S levels upon AOAA and PAG exposure. Analysis of H 2 S at T = 0 h indicated that A549 and H292 cell lines presented decreased levels of H 2 S in the presence of cysteine with and without glycolysis inhibition with BPA, while the PC-9 cell line presented no significant changes in all conditions. ( D ) Analysis of H 2 S levels showed that all cell lines presented a similar basal ability to produce H 2 S. Maximum score ( E ) or the average levels ( F ) of ATP production indicated that A549 cells increased the production of ATP upon cysteine supplementation independently of the presence of inhibitors, while H292 and PC-9 were able to maintain ATP levels in all experimental conditions. Maximum ATP score and average levels indicated that A549 cells increased the levels of ATP upon BPA and/or cysteine supplementation, and H292 and PC-9 cells maintained the ATP levels in all culture conditions independently of glycolysis inhibition. All data were normalized to the control condition and are represented as mean ± SD. * p < 0.5, ** p < 0.01, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Cell Culture, Inhibition

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cells express distinct expression patterns of enzymes and transporters involved in cysteine metabolism. ( A ) mRNA expression level analysis showed that H292 cells expressed noteworthy levels of CBS and SLC7A11 compared to A549 cells, while PC-9 cells showed low overall expression of CTH , MPST , SLC1A1, and SLC7A11 . ( B ) Immunofluorescence analysis showed that protein expression followed mRNA expression patterns, reporting similar differences. ( C ) mRNA expression level analysis further showed that H292 cells highly expressed GOT2 , while PC-9 showed high expression levels of GOT2 and CDO1 compared to A549 cells. All mRNA expression level data are relative to HPRT1 and represented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Expressing, Immunofluorescence

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: Distinct molecular backgrounds induce individual metabolic patterns in NSCLC cells. ( A – C ) 1 H-Nuclear magnetic resonance (NMR) of the NSCLC panel studied indicated alterations between A549, H292, and PC-9 regarding levels of intracellular amino acids and peptides, sugars and organic acids, and other metabolites. ( D – F ) NMR of the NSCLC panel studied further indicated alterations between A549, H292, and PC-9 regarding levels of extracellular amino acids and peptides and sugars and organic acids. Data are represented as mean ± SD. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( G , H ) PCA showed no differences in the endometabolome ( G ), but the exometabolome ( H ) indicated that A549 and PC-9 cells present distinct metabolic profiles, and H292 cells had common metabolic patterns with A549 and PC-9 cells. ( I ) PLS-DA analysis of the exometabolome allowed the discrimination of the three cell lines. From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate PC-9 from the other cell lines in the first component, while A549 could be discriminated in the second component (upper panel), Q 2 = 0.873. ( J ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. 2-Oxoisocaproate increased in A549, while aspartate, lactate, phosphocholine, and glycerophosphocholine increased in PC-9, making them important for discrimination between these cell lines. ( K ) From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate between A549 and PC-9 cell lines (upper panel), Q 2 = 0.975. ( L ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. ( M ) Metabolites significantly different between PC-9 and A549. Volcano plot (fold change >2 and p value < 0.05) of the intracellular metabolites between A549 and PC-9. Arginine, proline, and nicotinurate were significantly increased in A549 and guanosine in PC-9. ( N ) Nicotinurate was only present in A549 cells. Box plot of nicotinurate intracellular concentrations in the three cell lines (ANOVA analysis p value = 0.00028097 × 10 4 .

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Nuclear Magnetic Resonance

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines are chemoresistant, and SeChry@PURE G4 -FA induces decreased cell viability in NSCLC cells, with specificity toward tumor cells rather than nontumoral cells. ( A ) NSCLC cell lines exposed to the most commonly used therapy regimens showed overall resistance to these drugs, except for cisplatin. ( B ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in NSCLC indicated a higher sensitivity of A549 and H292 to the treatment than PC-9. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( C ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in noncancer cell lines (HaCaT and HK2) indicated no effect on cell viability. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( D ) Detection of FR-α by immunofluorescence indicated high protein levels in NSCLC but not in nontumoral cell lines. FR- α is labeled in green, and nuclei were counterstained with DAPI (blue). Magnification 400×, scale 20 µm. *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Immunofluorescence, Labeling

Journal: British Journal of Cancer

Article Title: MYB is a novel regulator of pancreatic tumour growth and metastasis

doi: 10.1038/bjc.2015.400

Figure Lengend Snippet: MYB enhances growth and clonogenic potential of PC cells. Total protein was isolated from MYB ( A ) overexpressing (BxPC3-MYB) and ( B and C ) silenced MiaPaCa ( B ) and Panc1 ( C ) PC cells along with respective control cells, and the expression of MYB was examined by immunoblot assay. β -Actin served as an internal control. ( D ) Growth kinetics measurement of PC cell lines exhibiting forced expression (BxPC3-MYB) or silencing (MiaPaCa-shMYB and Panc-1-shMYB) of MYB relative to their respective controls (BxPC3-Neo, MiaPaCa-NT-Scr and Panc1-NT-Scr). ( E ) Plating efficiency of MYB-overexpressing and -silenced PC cells. Bars represent the mean of total number of colonies±s.d. ( n =3), * P <0.05. ( F ) Soft agar colony-forming assay. Bars represent the total number of colonies in 10 random view fields (mean±s.d.; n =3), * P <0.05.

Article Snippet: Luciferase-tagged paired MYB-knockdown and -overexpressing PC cell lines were injected into the pancreas of immunocompromised mice (Harlan Laboratories, Prattville, AL, USA) (10 mice/group).

Techniques: Isolation, Expressing, Western Blot

Journal: British Journal of Cancer

Article Title: MYB is a novel regulator of pancreatic tumour growth and metastasis

doi: 10.1038/bjc.2015.400

Figure Lengend Snippet: MYB promotes cell-cycle progression and imparts resistance to apoptosis in PC cells. ( A ) Synchronised cultures of MYB-overexpressing or -silenced PC cells along with their controls were grown in regular culture media for 24 h. Thereafter, cells were fixed, stained with propidium iodide and analysed using flow cytometry. ( B ) Cells were cultured under serum-free conditions for 72 h, and the extent of apoptotic cells were analysed by flow cytometry using PE Annexin V.

Article Snippet: Luciferase-tagged paired MYB-knockdown and -overexpressing PC cell lines were injected into the pancreas of immunocompromised mice (Harlan Laboratories, Prattville, AL, USA) (10 mice/group).

Techniques: Staining, Flow Cytometry, Cell Culture

Journal: British Journal of Cancer

Article Title: MYB is a novel regulator of pancreatic tumour growth and metastasis

doi: 10.1038/bjc.2015.400

Figure Lengend Snippet: MYB promotes malignant potential of PC cells. ( A and B ) Control and MYB-overexpressing/silenced cells were seeded on non-coated (for motility assay) or Matrigel-coated (for invasion assay) membranes. Media containing 10% FBS in the lower chamber served as a chemoattractant. ( A ) Migrated and ( B ) invaded cells were counted and presented as the average number of cells/field±s.d., * P <0.05. Photographs are representative of three independent experiments performed in triplicate. ( C ) Hanging drop assay showing cell–cell interaction.

Article Snippet: Luciferase-tagged paired MYB-knockdown and -overexpressing PC cell lines were injected into the pancreas of immunocompromised mice (Harlan Laboratories, Prattville, AL, USA) (10 mice/group).

Techniques: Motility Assay, Invasion Assay

Journal: British Journal of Cancer

Article Title: MYB is a novel regulator of pancreatic tumour growth and metastasis

doi: 10.1038/bjc.2015.400

Figure Lengend Snippet: MYB enhances tumour growth in nude mice. ( A ) Luciferase-tagged MYB-overexpressing (BxPC3-MYB) or -silenced (MiaPaCa-shMYB) cells along with their respective control sublines were implanted into the pancreas of athymic nude mice ( n =10 per group). Mice were imaged using IVIS in vivo imaging system to measure luminescence once a week after intraperitoneal injection of D-luciferin. Representative images of mice are from the day they were killed. ( B and C ) Upon killing the animals, their tumours were resected and measured for their weights and volumes. Graphs reflect the distribution of ( B ) weights and ( C ) volumes of tumours derived from MYB-overexpressing BxPC3 and -silenced MiaPaCa cells along with those originated from their respective controls.

Article Snippet: Luciferase-tagged paired MYB-knockdown and -overexpressing PC cell lines were injected into the pancreas of immunocompromised mice (Harlan Laboratories, Prattville, AL, USA) (10 mice/group).

Techniques: Luciferase, In Vivo Imaging, Injection, Derivative Assay