Journal: Current Issues in Molecular Biology

Article Title: Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness, and Epithelial–Mesenchymal Transition, Associated with u-PAR Downregulation Through JNK and ERK Pathways

doi: 10.3390/cimb48040353

Figure Lengend Snippet: HM inhibits colony formation, cell motility, and invasiveness in HT29 and SW620 cell lines. ( A ) In order to promote colony formation, HT29 and SW620 cells were incubated for 10–12 days at 37 °C alongside untreated HT29 and SW620 cells. Using a light microscope, the number of colonies was counted after applying crystal violet staining. An xCELLigence RTCA-DP system was used to monitor real-time CRC cell migration ( B ) and invasion ( C ). ( D ) In HT29 and SW620 cell lines, HM increases the expression of E-cadherin and decreases N-cadherin. Whole-cell lysates were subjected to immunoblotting using the designated antibodies directed against E-cadherin and N-cadherin. The bar graph shows the relative level of protein expression in relation to the loading control, β-actin. ( E ) A bar graph illustrating the relative levels of N-cadherin and E-cadherin mRNA expression in relation to GAPDH mRNA, the internal control. Results from three separate experiments ( n = 3) are displayed in bar graphs as mean ± SD. When reporting * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the control, the data were deemed significant.

Article Snippet: The human colorectal adenocarcinoma cell line HT29 (#HTB-38, harboring mutations, including BRAF-heterozygous-c.1799T>A; PIK3CA-heterozygous-c.1345C>A; TP53-homozygous-c.818G>A) and mCRC cell line SW620 (#CCL-227, harboring mutations, including KRAS-homozygous-c.35G>T; TP53-heterozygous-c.818G>A; and TP53-heterozygous-c.925C>T), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L L-glutamine added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Thermo Fisher Scientific).

Techniques: Incubation, Light Microscopy, Staining, Migration, Expressing, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness, and Epithelial–Mesenchymal Transition, Associated with u-PAR Downregulation Through JNK and ERK Pathways

doi: 10.3390/cimb48040353

Figure Lengend Snippet: (A) Analysis of human angiogenesis antibody array in HT29 and SW620 cell lysates after HM treatment. Whole HT29 and SW620 CRC cell lysates were subjected to immunoblotting against 23 antibodies. In the HT29 and SW620 cell lines, HM downregulated uPAR expression. Bar graphs showing the results expressed in a single experiment (single membrane). ( B ) HT29 and SW620 whole-cell lysates were subjected to immunoblotting against uPAR. The bar graph indicates the relative level of protein expression in relation to the loading control β-actin. ( C ) Bar graph illustrating the relative level of uPAR mRNA expression in relation to GAPDH mRNA, the internal control. Results from three independent experiments ( n = 3) are displayed in bar graphs as mean ± SD. When reporting * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the control, the p -values data were deemed significant.

Article Snippet: The human colorectal adenocarcinoma cell line HT29 (#HTB-38, harboring mutations, including BRAF-heterozygous-c.1799T>A; PIK3CA-heterozygous-c.1345C>A; TP53-homozygous-c.818G>A) and mCRC cell line SW620 (#CCL-227, harboring mutations, including KRAS-homozygous-c.35G>T; TP53-heterozygous-c.818G>A; and TP53-heterozygous-c.925C>T), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L L-glutamine added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Thermo Fisher Scientific).

Techniques: Ab Array, Western Blot, Expressing, Membrane, Control

Journal: Current Issues in Molecular Biology

Article Title: Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness, and Epithelial–Mesenchymal Transition, Associated with u-PAR Downregulation Through JNK and ERK Pathways

doi: 10.3390/cimb48040353

Figure Lengend Snippet: uPAR expression level in HT29 and SW620 cells after HM treatment following pretreatment with JNK inhibitor SP600125 and MEK inhibitor UO126. ( A ) HT29 and SW620 whole-cell lysates with/without 10 μM JNK kinase inhibitor (+/− SP600125) were subjected to immunoblotting against pJNK. ( B ) HT29 and SW620 whole-cell lysates with/without 10 μM MEK inhibitor (+/− UO126) were subjected to immunoblotting against pERK. The bar graph shows the relative level of protein expression in relation to the loading control β-actin. Results from three separate experiments ( n = 3) are displayed in bar graphs as mean ± SD. When reporting ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the control, the data were deemed significant.

Article Snippet: The human colorectal adenocarcinoma cell line HT29 (#HTB-38, harboring mutations, including BRAF-heterozygous-c.1799T>A; PIK3CA-heterozygous-c.1345C>A; TP53-homozygous-c.818G>A) and mCRC cell line SW620 (#CCL-227, harboring mutations, including KRAS-homozygous-c.35G>T; TP53-heterozygous-c.818G>A; and TP53-heterozygous-c.925C>T), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L L-glutamine added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness, and Epithelial–Mesenchymal Transition, Associated with u-PAR Downregulation Through JNK and ERK Pathways

doi: 10.3390/cimb48040353

Figure Lengend Snippet: uPAR expression levels in HT29 and SW620 cells after HM treatment following pretreatment with JNK inhibitor SP600125 and MEK inhibitor UO126. ( A ) HT29 and SW620 whole-cell lysates with/without 10 μM JNK kinase inhibitor (+/− SP600125) were subjected to immunoblotting against uPAR. ( B ) HT29 and SW620 whole-cell lysates with/without 10 μM MEK inhibitor (+/− UO126) were subjected to immunoblotting against uPAR. Bar graph showing the relative level of protein expression in relation to the loading control β-actin. Results from three independent experiments ( n = 3) are displayed in bar graphs as mean ± SD. When reporting ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the control, the data were deemed significant.

Article Snippet: The human colorectal adenocarcinoma cell line HT29 (#HTB-38, harboring mutations, including BRAF-heterozygous-c.1799T>A; PIK3CA-heterozygous-c.1345C>A; TP53-homozygous-c.818G>A) and mCRC cell line SW620 (#CCL-227, harboring mutations, including KRAS-homozygous-c.35G>T; TP53-heterozygous-c.818G>A; and TP53-heterozygous-c.925C>T), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L L-glutamine added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Herbal Melanin Inhibits Colorectal Cancer Cell Motility, Invasiveness, and Epithelial–Mesenchymal Transition, Associated with u-PAR Downregulation Through JNK and ERK Pathways

doi: 10.3390/cimb48040353

Figure Lengend Snippet: JNK inhibitor SP600125 (A) and MEK inhibitor UO126 (B) attenuated HM-inhibited real-time SW620 cell migration. Data are presented as mean ± SD from three separate experiments ( n = 3). When reporting * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the control, the data were deemed significant.

Article Snippet: The human colorectal adenocarcinoma cell line HT29 (#HTB-38, harboring mutations, including BRAF-heterozygous-c.1799T>A; PIK3CA-heterozygous-c.1345C>A; TP53-homozygous-c.818G>A) and mCRC cell line SW620 (#CCL-227, harboring mutations, including KRAS-homozygous-c.35G>T; TP53-heterozygous-c.818G>A; and TP53-heterozygous-c.925C>T), purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in 10% heat-inactivated fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L L-glutamine added to Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Thermo Fisher Scientific).

Techniques: Migration, Control