a375p cells  (Korean Cell Line Bank)

Journal: Life

Article Title: Comparative Bioactivities and Fatty Acid Composition of Pinus koraiensis Leaf Oils Obtained Using Different Extraction Methods

doi: 10.3390/life16010049

Figure Lengend Snippet: Comparison of the cancer cell growth inhibitory effects of Pinus koraiensis essential oil (EPO) and supercritical CO 2 extract oil (SPO) on various human cancer cell lines. ( A , B ) Colon cancer (SW620, HCT116), ( C , D ) lung cancer (A549, H460), ( E , F ) prostate cancer (PC-3, DU145), ( G ) breast cancer (MDA-MB-231), and ( H ) melanoma (A375P) cells were treated with the indicated concentrations of EPO or SPO for 24 h. Cell viability was measured using the MTS assay, and the results are expressed as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control groups.

Article Snippet: HaCaT and A375P cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured in DMEM medium containing 10% fetal bovine serum (Cat: S101-07, WELGENE, Daegu, Republic of Korea) and 1% antibiotics (Cat: LS203-01, WELGENE, Daegu, Republic of Korea).

Techniques: Comparison, MTS Assay, Control

Journal: Life

Article Title: Comparative Bioactivities and Fatty Acid Composition of Pinus koraiensis Leaf Oils Obtained Using Different Extraction Methods

doi: 10.3390/life16010049

Figure Lengend Snippet: Comparison of the effects of Pinus koraiensis essential oil (EPO) and supercritical CO 2 extract oil (SPO) on iNOS mRNA expression and nitric oxide (NO) production. ( A ) THP-1 and ( B ) HaCaT cells were treated with or without LPS for 24 h, and LPS-stimulated cells were co-treated with EPO or SPO. ( C ) SW620 and ( D ) A375P cells were treated with EPO or SPO for 24 h without LPS stimulation. Relative mRNA expression levels of iNOS were analyzed via qRT-PCR. ( E , F ) Nitric oxide levels in LPS-stimulated THP-1 ( E ) and HaCaT ( F ) cells were measured by the Griess assay after co-treatment with EPO or SPO. Data are presented as the mean ± SD ( n = 3). Different letters indicate significant differences ( p < 0.05, one-way ANOVA).

Article Snippet: HaCaT and A375P cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured in DMEM medium containing 10% fetal bovine serum (Cat: S101-07, WELGENE, Daegu, Republic of Korea) and 1% antibiotics (Cat: LS203-01, WELGENE, Daegu, Republic of Korea).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Griess Assay

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: RT-qPCR analysis and the CUT&RUN peaks in indicated genes from dox-inducible A375P cells overexpressing either MITF-WT or MITF-sl of ( A ) endogenous mRNA MITF and mRNA MITF target genes: ( B ) NRP1, ( C ) CDH2, ( D ) PMEL, ( E ) TRIM63, ( F ) TYRP1, ( G ) MLANA, ( H ) TYR, and ( I ) DCT in the dox-inducible A375P overexpressing cells. The cells were treated with doxycycline for 6, 12, 24, and 36 h to induce MITF expression at the same level before harvesting for RNA isolation. Actin and hAPR was used as housekeeping genes. The fold change in target gene expression was assessed in cells overexpressing either MITF-WT or MITF-sl by comparing to those expressing EV-FLAG-HA followed by normalization to the proportion of MITF proteins retained in the nucleus. Error bars represent SEM of at least three independent experiments. Statistically significant differences (Student’s t test) are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant. ( J ) Venn Diagram showing the number of peaks shared and different between MITF-WT and the MITF-sl. ( K ) Peaks different between MITF-WT and MITF-sl mutant proteins shown in a Volcano plot ( P < 0.01). ( L ) Gene ontology analysis of the 10,636 ( P < 0.01) peaks that are different between MITF-WT and MITF-sl.

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Targeted Gene Expression, Mutagenesis

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A ) Western blot analysis of the Mitf-Flag proteins upon cycloheximide treatment. The dox-inducible A375P cells expressing the MITF-WT, MITF-sp, and MITF-sl proteins were treated with doxycycline for 24 h to induce similar expression of the indicated mutant MITF proteins before treating them with 40 µg/ml cycloheximide (CHX) for 0, 1, 2, and 3 h. The blots were stained using Flag antibody and protein quantitated using the Odyssey imager and ImageJ. Actin was used as a loading control. ( B ) Half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment in A375P melanoma cells. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t-test. P values for the pS73-MITF form of WT compared to sp and sl were 0.9077 and 0.0037, respectively. P values for the S73-MITF form of WT compared to sp and sl were 0.7085 and 0.0007, respectively. ( C ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 h to overexpress different MITF mutant proteins. MITF-WT, MITF-Wh, MITF-sp, and MITF-sl protein in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. ( D ) Intensities of the indicated pS73- and S73-MITF protein bands in the cytoplasmic and nuclear fraction from the western blot analysis in ( C ) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT, Wh, sp, and sl were 0.3512, 0.040, 0.2150, and 0.0003, respectively. P values for the S73-MITF form of WT, Wh, sp, and sl were 0.3733, 0.3761, 0.8689, and 0.0004, respectively. ( E ) Western blot analysis of subcellular fractions isolated from A375P cells transiently co-overexpressing the MITF - sl protein with the MITF-Wh, MITF-mi, and MITF-ew mutant MITF proteins. MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. The MITF-sl protein migrates as a doublet at 50–55 kDa, whereas the other mutants migrate at 65–70 kDa. ( F ) The intensities of the pS73- and S73- MITF-sl protein in the cytoplasmic and nuclear fractions from western blot analysis ( E ) were quantified separately with ImageJ software and are depicted as percentages of the total protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73- and S73-MITF-sl co-expression with empty vector (-) Wh, mi, and ew were **** P < 0.0001. ( G ) Quantification of band intensities of the pS73- and S73-versions of the MITF-Wh, MITF-mi, and MITF-ew proteins as determined from western blots ( E ) in the nuclear fractions of A375P cells transiently co-overexpressing the MITF - sl protein with the indicated MITF mutant proteins. The intensities are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of Wh, mi, and ew with or without co-expressing with MITF-sl were 0.0021, 0.0002, and <0.0001, respectively. P values for the S73-MITF form of Wh, mi, and ew with or without co-expressing with MITF-sl were 0.0023, 0.0015, and 0.0072, respectively. ( H ) Western blot analysis of the degradation of the MITF-sl protein in the presence of non-DNA binding MITF mutations (MITF-Wh, MITF-mi, and MITF-ew). The A375P cells were transiently co-transfected with MITF-sl and either MITF-mi, MITF-ew, or MITF-Wh for 24 h before being treated with 55 µg/ml CHX. The amount of MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control and normalized to the expression of MITF protein expression. The band intensities were quantified using ImageJ software. ( I ) Half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The relative MITF protein levels to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF-sl form with or without co-expressing with Wh, mi, and ew were 0.0185, 0.0005, and 0.0015, respectively. P values for the S73-MITF-sl form with or without co-expressing with Wh, mi, and ew were 0.0005, <0.0001, and 0.0009, respectively. .

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Western Blot, Expressing, Mutagenesis, Staining, Control, Isolation, Software, Plasmid Preparation, Binding Assay, Transfection

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A ) Schematic of MITF-sp truncation constructs. C-term truncations were generated by introducing stop codons at position Q326 or L378 or by deleting fragments 326–377 or 316–326. MITF-sp-326* introduces a stop-codon at residue 326 and, therefore, contains the SUMO-site at 316; MITF-sp-∆326–377 lacks the tentative activation domain AD3; MITF-sp-∆316–326 lacks the SUMO-site and adjacent amino acids; MITF-sp-378* lacks the series of phosphorylation sites at the carboxyl-end of the protein. ( B ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress the different MITF mutant proteins fused with Flag-tag at C terminus for 24 h. MITF-WT, MITF-sl, MITF-sp-326*, MITFmi-sp-378*, and MITF-sp-∆326–377 in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. ( C ) The intensities of the indicated pS73 MITF and S73 MITF proteins from the cytoplasmic and nuclear fractions of the western blot analysis in ( B ) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT, sl, 326*, ∆326–377, and 378* were 0.1455, <0.0001, 0.0033, 0.0054, and <0.0001. P values for the S73-MITF form of WT, sl, 326*, ∆326–377, and 378* were 0.1302, <0.0001, 0.0072, 0.3957, and 0.0021, respectively. ( D ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 h to overexpress the different MITF mutant proteins before treatment with TPA at 200 nM for 1 h. MITF-WT, MITF-sl, MITF-sp-326*, MITF-sp-∆326–377, MITF-sp-∆316–326, and MITF-sp-378* protein in cytoplasmic ( C ) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and γH2AX or H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. ( E ) Intensities of the indicated pS73-MITF proteins from the western blot analysis in ( D ) in the cytoplasmic and nuclear fractions from the cell treated with TPA were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT, sl, 326*, ∆326–377, 378*, and ∆316–326 were <0.0001, <0.0001, 0.0573, 0.0258, 0.0025, and 0.0050. P values for the S73-MITF form of sl, 326*, ∆326–377, 378*, and ∆316–326 compared to pS73-MITF-WT in the nuclear fraction were <0.0001, 0.0125, 0.5609, 0.0835, and 0.0019, respectively. ( F ) Western blot analysis of the MITF proteins from dox-induced A375P cells after treating them with 40 µg/ml CHX for 0, 1, 2, and 3 h. The MITF proteins were visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. ( G ) Half-life analysis of the indicated pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of sl, 326*, ∆326–377, 378*, and ∆316–326 compared with pS73-MITF-WT were <0.0001, 0.0093, 0.0008, 0.0004, and 0.0046, respectively. P values for the S73-MITF form of sl, 326*, ∆326–377, 378*, and ∆316–326 compared to S73-MITF-WT were <0.0001, 0.0042, 0.7240, 0.0064, and 0.8886, respectively. P values for the pS73-MITF form of 326*, ∆326–377, 378*, and ∆316–326 compared with pS73-MITF-sl were 0.0001, 0.1748, 0.4561, and 0.0002 respectively. P values for the S73-MITF form of 326*, ∆326–377, 378*, and ∆316–326 compared to S73-MITF-sl were 0.0077, <0.0001, <0.0001, <0.0001, respectively. .

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Construct, Generated, Residue, Activation Assay, Phospho-proteomics, Western Blot, Isolation, Mutagenesis, FLAG-tag, Software, Control

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A ) Graphical depiction of the MITF-WT, MITF-sl, MITF-sl-NES1, MITF-sl-NES2, and MITF-sl-NES1-NES2 proteins. The location of the NES1 and NES2 sequences in MITF-WT are also shown. ( B ) Western blot analysis of cytoplasmic (C) and nuclear (N) fractions from A375P melanoma cells induced for 24 h to overexpress the indicated MITF mutant proteins with or without treatment with 200 nM TPA for 1 h. MITF was visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. ( C , D ) MITF band intensities in the cytoplasmic and nuclear fractions from western blot analysis ( B ) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences (Student’s t test) are indicated by * P < 0.05 and ** P < 0.01.

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Western Blot, Mutagenesis, Software

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A ) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 h in A375P cells treated with 50 µg/ml CHX in the presence of either DMSO or 20 µg/ml MG132 or 0.2 µg/ml Baf-A1 for 3 h. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. ( B ) The indicated pS73- and S73-MITF protein band intensities from western blot analysis ( A ) were quantified separately with ImageJ software and are depicted relative to DMSO. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. Compared between DMSO and MG132 treated conditions in the presence of CHX, p values for the pS73-MITF form of WT, sp, and sl were 0.0008, 0.0443, and 0.0176, respectively. P values for the S73-MITF form of WT, sp, and sl were 0.0279, 0.3753, and 0.0035, respectively. Compared between DMSO and Baf-A1 treated conditions in the presence of CHX, P values for the pS73-MITF form of WT, sp, and sl were 0.5988, 0.6219, and 0.0003, respectively. P values for the S73-MITF form of WT, sp, and sl were 0.0005, 0.0028, and <0.0001, respectively. ( C ) Western blot analysis of the MITF-WT, MITF-sp, and MITF-sl proteins. Expression was induced for 24 h in A375P cells treated with either DMSO or 20 µg/ml MG132 or 0.2 µg/ml Baf-A1 for 3 h. The MITF protein was then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. ( D ) The indicated pS73- and S73-MITF protein band intensities from western blot analysis ( C ) were quantified separately with ImageJ software and are depicted relative to DMSO. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. Compared between DMSO and MG132 treated conditions, P values for the pS73-MITF form of WT, sp, and sl were 0.1532, 0.0007, and <0.0001, respectively. P values for the S73-MITF form of WT, sp, and sl were 0.0026, <0.0001, and 0.0011, respectively. Compared between DMSO and Baf-A1 treated conditions in the presence of CHX, P values for the pS73-MITF form of WT, sp, and sl were 0.0558, 0.0043, and 0.0372, respectively. P values for the S73-MITF form of WT, sp, and sl were 0.0427, 0.0005, and 0.0948, respectively. ( E ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced to overexpress MITF-WT protein before treating with either 200 nM TPA for 1 or 4 h or 40 µg/ml MG132 for 3 h or 200 nM TPA for 1 h and then adding 40 µg/ml MG132 for the next 3 h together with TPA. MITF-WT protein in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and γH2AX were loading controls for cytoplasmic and nuclear fractions, respectively. ( F ) Western blot analysis of the stability of the MITF-WT and MITF-sl mutant proteins after knocking down AKIRIN2 , a key regulator of the nuclear import of proteasomes, for 24 h and then inducing MITF expression using dox for 6 h. The inducible A375P cells were treated with 40 µg/ml CHX for 0, 1, 2, and 3 h. The MITF proteins were then visualized by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software. ( G ) The intensities of the indicated pS73- and S73-MITF protein bands were quantified from western blot analysis in ( F ) with ImageJ software and are depicted as relative protein expression to DMSO. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT and sl compared between siCTRL and siAKIRIN2 treated conditions were 0.8860 and 0.8731. P values for the S73-MITF form of WT and sl compared between siCTRL and siAKIRIN2 treated conditions were 0.0293 and 0.0395. .

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Western Blot, Expressing, Control, Software, Isolation, Mutagenesis

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 h to overexpress the indicated MITF mutant proteins. The MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. Actin or GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. ( B ) The intensities of the indicated pS73- and S73-MITF proteins in the cytoplasmic and nuclear fractions from western blot analysis in ( A ) were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT, WT-K316R, WT-E318K, sp-K316R, sp-E318K, sp-K316R-S409A, sp-E318K-S409A and sl were 0.1774, 0.2128, 0.1697, 0.1261, 0.4814, 0.0157, 0.0271 and <0.0001, respectively. P values for the S73-MITF form of WT, WT-K316R, WT-E318K, sp-K316R, sp-E318K, sp-K316R-S409A, sp-E318K-S409A and sl were 0.7878, 0.2038, 0.0958, 0.2712, 0.1098, 0.0222, 0.0101, and <0.0001, respectively. ( C ) Western blot analysis of subcellular fractions isolated from A375P melanoma cells induced for 24 h to overexpress the indicated MITF mutant proteins before treatment with 200 nM TPA for 1 h leading to phosphorylation of S73 of MITF. The mutant MITF proteins in cytoplasmic (C) and nuclear (N) fractions were visualized using FLAG antibody. GAPDH and H3K27me3 were loading controls for cytoplasmic and nuclear fractions, respectively. ( D ) The intensities of the indicated pS73-MITF proteins bands in the cytoplasmic and nuclear fractions of the western blot analysis in ( C ), respectively, were quantified separately with ImageJ software and are depicted as percentages of the total amount of protein present in the two fractions. Error bars represent SEM of three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT, WT-K316R, WT-E318K, sp-K316R, sp-E318K, sp-K316R-S409A, sp-E318K-S409A and sl were <0.0001, <0.0001, 0.0007, 0.0028, <0.0001, 0.6584, 0.0060, and <0.0001, respectively. ( E , F ) Half-life analysis of the pS73- and S73-MITF proteins over time after CHX treatment. The MITF protein levels relative to T0 were calculated, and non-linear regression analysis was performed. Error bars represent SEM of at least three independent experiments. Statistically significant differences were calculated using unpaired Student’s t test. P values for the pS73-MITF form of WT-K316R, sp-K316R, sp-K316R-S409A, sp-326*-K316R, sp-378*-K316R, and sl compared to WT were <0.0001, 0.0026, 04614, 0.0007, <0.0001, and <0.0001, respectively. P values for the pS73-MITF form of sp-326*-K316R and sp-378*-K316R compared to sl were 0.1869 and 0.1159, respectively. P values for the S73-MITF form of WT-K316R, sp-K316R, sp-K316R-S409A, sp-326*-K316R, sp-378*-K316R, and sl compared to WT were 0.0169, 0.0452, 0.0118, 0.0072, 0.0050, and 0.0005, respectively. P values for the S73-MITF form of sp-326*-K316R and sp-378*-K316R compared to sl were 0.0093 and 0.1561, respectively. P values for the pS73-MITF form of sl, sp-E318K, sp-E318K-S409A, sp-326*, sp-326*-E318K, sp-378*, and sp-378*-E318K compared to WT were 0.0002, 0.0033, 0.0022, 0.0004, 0.0010, 0.0008, and 0.0001, respectively. P values for the pS73-MITF form of sp-E318K, sp-E318K-S409A, sp-326*, sp-326*-E318K, sp-378*, and sp-378*-E318K compared to sl were 0.0379, 0.1455, 0.0017, 0.0031, 0.1045, and 0.2853, respectively. P values for the S73-MITF form of sl, sp-E318K, sp-E318K-S409A, sp-326*, sp-326*-E318K, sp-378*, and sp-378*-E318K compared to WT were 0.0003, 0.0288, 0.0026, 0.0238, 0.1406, 0.0100, and 0.0024, respectively. P values for the S73-MITF form of sp-E318K, sp-E318K-S409A, sp-326*, sp-326*-E318K, sp-378*, and sp-378*-E318K compared to sl were 0.0453, 0.1788, 0.0004, <0.0001, 0.0010, and 0.0616, respectively. .

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Western Blot, Isolation, Mutagenesis, Software, Phospho-proteomics

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: ( A , B ) Western blot analysis of the stability of the MITF proteins. The inducible A375P cells were treated with doxycycline for 24 h to express the indicated mutant MITF proteins before treating them with 40 µg/ml CHX for 0, 1, 2, and 3 h. The MITF protein was then compared by western blot using FLAG antibody. Actin was used as a loading control. The band intensities were quantified using ImageJ software.

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Western Blot, Mutagenesis, Control, Software

Journal: EMBO Reports

Article Title: Novel mechanisms of MITF regulation identified in a mouse suppressor screen

doi: 10.1038/s44319-024-00225-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The cell lines A375P (CRL-3224), and SkMel28 (HTB-72) were purchased from ATCC.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Labeling, Magnetic Beads, Saline, Software, Cloning, Mutagenesis, Reverse Transcription

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: IL-33 stimulates the anticancer activities of eosinophils through extracellular vesicle-driven reprogramming of tumor cells

doi: 10.1186/s13046-024-03129-1

Figure Lengend Snippet: IL-33 stimulates EV secretion by human eosinophils that reprogram human melanoma cells. A Western blot analysis of CD81 and Calnexin expression in human eosinophils stimulated with IL-5 (hEo5) or IL-33 (hEo33) and their derived EV (hEo5-EV and hEo33-EV). B Flow cytometry quantification of fluorescent EV generated by Bodipy FL-C16 labelling of hEo5 and hEo33. Data are expressed as number of EV released per cell. Mean (SD) of three experiments is shown. C Incorporation of fluorescent eosinophil-derived EV into A375P melanoma cells following co-culture with C16-labelled hEo5 or hEo33 in 0.4 Transwell system for the indicated times. Mean (SD) of three replicates is shown. D Tumor spheroid formation of A375P human melanoma cells cultured alone (CTR), with hEo5-EV or hEo33-EV. Left, representative micrographs at the indicated times. Bars: 1000 μm. Right, quantitative analysis of tumor spheroid area. Mean (SD) of several spheroids is shown. *** P < 0.001. E Gene expression analysis of CDKI in A375P cells following 24 h exposure to hEo5-EV or hEo33-EV. Mean (SD) of three replicates is shown. ** P < 0.01; *** P <0.001; **** P < 0.0001. E Cell cycle analysis in A375P cells after 24 h exposure to hEo5-EV or hEo33-EV. Mean (SD) of three replicates is shown. ** P < 0.01; *** P < 0.001. ( F ) Expression of CDH1 and CDH2 genes in A375P cells exposed to hEo5-EV or hEo33-EV (24 h). Mean (SD) of three replicates is shown. * P < 0.05; *** P < 0.001. **** P < 0.0001

Article Snippet: Murine B16.F10 metastatic melanoma cells (ATCC, CRL-6475), TC1 lung adenocarcinoma cells (provided by Dr. Guido Kroemer, Gustave Roussy Cancer Institute, France), MCA205 fibrosarcoma cells (Merk Millipore, SCC173) and human metastatic melanoma A375P cells (ATCC, CRL-3224) were used in this study.

Techniques: Western Blot, Expressing, Derivative Assay, Flow Cytometry, Generated, Co-Culture Assay, Cell Culture, Gene Expression, Cell Cycle Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells

doi: 10.3390/ijms25052956

Figure Lengend Snippet: BCR-ABL tyrosine kinase inhibitors reduce proliferation and migration of human melanoma A375P cells. ( A ) Cell viability. A375P cells were inoculated into 96-well plates and treated with different concentrations of AT-9283 (0.25 μ M, 0.5 μ M, 0.75 μ M, 1 μ M, and 1.5 μ M), imatinib (5 μ M, 10 μ M, 15 μ M, 20 μ M, and 25 μ M), nilotinib (2.5 μ M, 5 μ M, 7.5 μ M, 10 μ M, and 15 μ M), or ZM-306416 (10 μ M, 20 μ M, 30 μ M, 40 μ M, and 50 μ M) for 24 h. Cell viability was determined by CCK-8 assay (n = 4). ( B ) BrdU cell proliferation. A375P cells were seeded into 96-well plates, then treated with 2 μ M palbociclib, 2.5 μ M nilotinib, 0.25 μ M AT-9283, 10 μ M Imatinib, or 20 μ M ZM-306416 for 24 h. Cell proliferation was examined by the BrdU assay kit (n = 4). ( C ) Wound-healing assay. A375P cells were incubated with 2 μ M palbociclib, 0.25 μ M AT-9283, 2.5 μ M nilotinib, 10 μ M imatinib, or 20 μ M ZM-306416 for 24 h and 48 h. Cell migration was captured by using bright-field microscopy. Bar, 250 μ M. ( D ) Quantification of cell migration. The migration area was quantified using ImageJ software (version 1.53 e) and is represented as a graph at 0 h, 24 h, and 48 h (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not a significant p value.

Article Snippet: The A375P human melanoma cell line (CRL-3224TM) was purchased from the American Type Culture Collection (ATCC).

Techniques: Migration, CCK-8 Assay, BrdU Staining, Wound Healing Assay, Incubation, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells

doi: 10.3390/ijms25052956

Figure Lengend Snippet: Cell cycle stage distribution in response to drug treatments ( A ) Cell cycle analysis by flow cytometry. A375P cells were exposed to 2 μ M palbociclib, 0.25 μ M AT-9283, 2.5 μ M nilotinib, 10 μ M imatinib, and 20 μ M ZM-306416 for 24 h. Subsequently, the harvested cells were stained with propidium iodide (PI) and subjected to flow cytometric analysis. The obtained data on cell cycle distribution were further processed using Flowjo software. ( B ) A graphical representation of the percentage of cells residing in the G0/G1, S, and G2/M phases (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, ns; not a significant p value.

Article Snippet: The A375P human melanoma cell line (CRL-3224TM) was purchased from the American Type Culture Collection (ATCC).

Techniques: Cell Cycle Assay, Flow Cytometry, Staining, Software

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells

doi: 10.3390/ijms25052956

Figure Lengend Snippet: Cell cycle stage distribution in response to different doses of drugs. ( A – D ) Cell cycle distribution flow cytometric analysis. A375P cells were incubated with different concentrations of each drug for 24 h as follows: AT-9283 (0.25 μ M, 0.5 μ M, 0.75 μ M, and 0.1 μ M), nilotinib (2.5 μ M, 5 μ M, 6 μ M, and 7.5 μ M), imatinib (10 μ M, 15 μ M, 20 μ M, and 25 μ M), ZM-306416 (10 μ M, 20 μ M, 30 μ M, and 40 μ M). Cell cycle distribution was quantified by Flowjo software. ( E ) A graphical representation of the percentage of cells residing in the G0/G1, S, and G2/M phases (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not a significant p value.

Article Snippet: The A375P human melanoma cell line (CRL-3224TM) was purchased from the American Type Culture Collection (ATCC).

Techniques: Incubation, Software

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells

doi: 10.3390/ijms25052956

Figure Lengend Snippet: AT-9283 and nilotinib regulate the gene or protein levels associated with the cell cycle ( A ) RT-qPCR assay. A375P cells were incubated with 2 μ M palbociclib, 0.25 μ M AT-9283, and 2.5 μ M nilotinib for 24 h. The relative mRNA levels of CCNE1, CCNA2, and CDK2 genes were examined by qPCR analysis and quantified using GAPDH control (n = 3). ( B ) Western blot assay. Cell extracts of A375P cells treated with 2 μ M palbociclib, 0.25 μ M AT-9283, and 2.5 μ M nilotinib for 24 h were subjected to Western blot analysis. Cyclin A, cyclin E, and CDK2 proteins were detected by their specific primary antibodies and subsequently incubated with secondary antibodies. β actin was used as an internal normalization control. ( C ) A graphical representation of the relative intensity of cyclin A, cyclin E, and CDK2 normalized by β actin (n = 3). Data represent the mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The A375P human melanoma cell line (CRL-3224TM) was purchased from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Incubation, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells

doi: 10.3390/ijms25052956

Figure Lengend Snippet: AT-9283 and nilotinib control RB1 phosphorylation and E2F1-dependent transcriptional activity. ( A ) Western blot assay. A375P cells were treated with 2 μ M palbociclib, 0.25 μ M AT-9283, and 2.5 μ M nilotinib for 24 h. Cell lysates were analyzed by Western blot assay using antibodies against RB1, RB1 phosphorylation (S807/811), E2F1, α tubulin, and lamin A/C. α tubulin was used as a normalization of nilotinib for 24 h. Cell lysates were analyzed by Western blot assay using antibodies against RB1, RB1 phosphorylation (S807/811), E2F1, α tubulin, and lamin A/C. α tubulin was used as a normalization control for cytoplasm, and lamin A/C was used as a normalization control for nuclear fractions. ( B ) Quantification of the relative intensity of RB1 phosphorylation (S807/811) and E2F1 normalized by α tubulin and lamin A/C (n = 3). ( C ) RT-qPCR assay. A375P cells were incubated with 2 μ M palbociclib, 0.25 μ M AT-9283, and 2.5 μ M nilotinib for 24 h. The mRNA levels of CCNE1, CCNA2, POLA1, and TK-1 were determined by qPCR analysis using a pair of primers for each gene . GAPDH was used as an internal normalization control (n = 3). Data represent the mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The A375P human melanoma cell line (CRL-3224TM) was purchased from the American Type Culture Collection (ATCC).

Techniques: Control, Phospho-proteomics, Activity Assay, Western Blot, Quantitative RT-PCR, Incubation