human melanoma cell line a375  (ATCC)

Journal: bioRxiv

Article Title: Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8 + T cell fate to enhance anti-tumor functionality

doi: 10.64898/2026.01.21.700812

Figure Lengend Snippet: CRISPR/Cas9 engineered TIL (eTIL ® ) were manufactured to inactivate SOCS1 (KSQ-001EX), Regnase-1 (sgRegnase-1), or both SOCS1 and Regnase-1 (KSQ-004EX) with non-electroporated controls used as a benchmark (No EP). (A) Percent killing of A375-mOKT3 tumor spheroids by indicated eTIL. (B) Production of IFNγ from during eTIL co-culture with A375-mOKT3 tumor spheroids. (C) Repeat stimulation assay assessing the ability of eTIL manufactured from a treatment-refractory melanoma donor to kill A375-mOKT3 cells following each stimulation over time. (D) eTIL bulk RNA-Seq, with Principle Components (PC) PC1, PC2 and PC3 depicted. No EP contains 30 independent donors; KSQ-001EX contains 26 independent donors; sgRegnase-1 contains 13 independent donors, and KSQ-004EX contains 25 independent donors. (E) Select DEGs between pairwise eTIL comparisons are depicted, with log fold change depicted by heatmap. (F) The number of DEGs between the eTIL comparisons are depicted by Venn diagram. (G) Transcription factor activity analysis from eTIL bulk RNA-Seq (H) Heatmap of log fold changes of top DEGs between edited TILs versus their respective controls in both human and mouse. Statistical significance between treatment groups was determined using a two-way ANOVA in with ns = no significance, * = p value < 0.05, ** = p value < 0.01, and *** = p value < 0.001. For and , the * in the heatmap represent the adj-p value (Benjamini Hochberg) for pairwise differential expression analysis with DESeq2 where *** = adj-p value < 0.001; ** = 0.001- 0.01; * = 0.01-0.05 and ‘.’ = 0.05-0.1. For , the *s represent adj-p values (Benjamini Hochberg) for transcription factor activity analysis. is representative of n=16 independent donors, is representative of n=7 independent donors. See also Figure S10.

Article Snippet: The A375 human melanoma cell line was obtained from ATCC and engineered to express either a low-affinity or high-affinity membrane associated anti-CD3 binding domain from clone OKT3 (mOKT3) together with RFP as previously described (Schlabach et al. 2023), with the low-affinity A375-mOKT line used for in vitro spheroid cytotoxicity and IFN- release assays, and the high-affinity A375-mOKT3 line used for the chronic stimulation assay[ ].

Techniques: CRISPR, Co-Culture Assay, RNA Sequencing, Activity Assay, Quantitative Proteomics

Journal: iScience

Article Title: RPL18 promotes melanoma progression and drug resistance via BTF3/STAT3-dependent mechanisms and immune modulation

doi: 10.1016/j.isci.2025.114469

Figure Lengend Snippet: RPL18 promoted melanoma progression (A) Kaplan-Meier overall survival analysis was performed according to high and low expression of RPL5, RPL11, RPL18, RPL22, RPL23, and RPL26 genes in 570 melanoma patients from TCGA database. (Statistical analysis: Kaplan-Meier method; comparisons by log rank (Mantel-Cox) test; n = 570; exact p value as shown, p < 0.05 considered significant). (B) Immunostaining of RPL18 in melanoma tissues from patients, divided into Ki-67 negative ( n = 7) and positive ( n = 12) groups, metastatic ( n = 12) and non-metastatic ( n = 12) groups, and responding ( n = 15) and non-responding ( n = 9) groups. Scale bars: 100 μm. (Quantification: immunostaining signals quantified across indicated samples; group comparisons by two-tailed unpaired Student’s t test; exact n as shown; data represent mean ± SD; exact p value as shown, p < 0.05 considered significant). (C) Western blotting of RPL18 in vector and RPL18-overexpressed A375/WM793 cells. (D) Cell proliferation of vector and RPL18-overexpressed A375/WM793 cells (48 h). (Statistical test: two-tailed unpaired Student’s t test comparing groups; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (E) Cell migration of vector and RPL18-overexpressed A375/WM793 cells (24 h). (Statistical test: two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (F) Colony formation rates of vector and RPL18-overexpressed A375/WM793 cells in 3D Matrigel (5 days). (Statistical test: two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (G) Cell apoptosis of vector and RPL18-overexpressed A375/WM793 cells treated with TMZ (1 mg/mL, 48 h). (Statistical test: two-tailed unpaired Student’s t test or one-way ANOVA if multiple groups are compared; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). Data are presented as mean ± SD. Each experiment was repeated independently at least three times.

Article Snippet: Human melanoma cell lines A375 and WM793 were obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling prior to use.

Techniques: Expressing, Immunostaining, Two Tailed Test, Western Blot, Plasmid Preparation, Migration

Journal: iScience

Article Title: RPL18 promotes melanoma progression and drug resistance via BTF3/STAT3-dependent mechanisms and immune modulation

doi: 10.1016/j.isci.2025.114469

Figure Lengend Snippet: RPL18 upregulated BTF3 in melanoma (A) Volcano Plot of differentially expressed genes in melanoma patients divided into RPL18 high and low groups ( n = 21). (Differential expression analysis performed with DESeq2; significance defined by adjusted p < 0.05). (B) Kaplan-Meier overall survival analysis was performed according to high and low expression of BTF3 in 570 melanoma patients from TCGA database. Kaplan-Meier method; comparisons by log rank (Mantel–Cox) test; n = 570; exact p value as shown, p < 0.05 considered significant). (C) Immunostaining of BTF3 in melanoma tissues from patients, divided into Ki-67 negative ( n = 7) and positive ( n = 12) groups, metastatic ( n = 12) and non-metastatic ( n = 12) groups, and responding ( n = 15) and non-responding ( n = 9) groups. Scale bars: 100 μm. (Quantification: groups compared by two-tailed unpaired Student’s t test; exact n as shown; mean ± SD; exact p value as shown, p < 0.05 considered significant). (D) Western blotting of BTF3 in vector and RPL18-overexpressed A375/WM793 cells. (E) mRNA expression of BTF3 in RPL18-overexpressed A375/WM793 cells, treated with scramble or BTF3 siRNAs. (Statistical test: one-way ANOVA followed by appropriate post-hoc test when >2 groups; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (F) Cell proliferation of vector and RPL18-overexpressed A375/WM793 cells, treated with scramble or BTF3 siRNAs (48 h). (One-way ANOVA or two-tailed unpaired Student’s t test as appropriate; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (G) Cell migration of vector and RPL18-overexpressed A375/WM793 cells, treated with scramble or BTF3 siRNAs (24 h). (One-way ANOVA or two-tailed unpaired Student’s t test as appropriate; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (H) Colony formation rate of vector and RPL18-overexpressed A375/WM793 cells, treated with scramble or BTF3 siRNAs in 3D Matrigel (5 days). (One-way ANOVA with post-hoc test when comparing multiple groups; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (I) Cell apoptosis of vector and RPL18-overexpressed A375/WM793 cells (pre-treated with scramble or BTF3 siRNAs) treated with TMZ (1 mg/mL, 48 h). (One-way ANOVA or two-tailed unpaired Student’s t test as appropriate; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). Data are presented as mean ± SD. Each experiment was repeated independently at least three times.

Article Snippet: Human melanoma cell lines A375 and WM793 were obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling prior to use.

Techniques: Quantitative Proteomics, Expressing, Immunostaining, Two Tailed Test, Western Blot, Plasmid Preparation, Migration

Journal: iScience

Article Title: RPL18 promotes melanoma progression and drug resistance via BTF3/STAT3-dependent mechanisms and immune modulation

doi: 10.1016/j.isci.2025.114469

Figure Lengend Snippet: RPL18/BTF3 axis mediated STAT3 signaling activation (A) Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes in melanoma patients divided into RPL18 high and low groups ( n = 21). (KEGG enrichment significance determined by adjusted p < 0.05; n = 21). (B) Heatmap of AKT3, WNT3A, STAT3, Nanog, Notch1 expression in vector and RPL18-overexpressed A375 determined by qPCR. (C) Western blotting of p-STAT3 and t-STAT3 in vector and RPL18-overexpressed A375/WM793 cells, treated with scramble or BTF3 siRNAs. (D) Cell proliferation of vector and RPL18-overexpressed A375/WM793 cells, treated with PBS or 20 nM STAT3-IN (48 h). (Two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (E) Cell migration of vector and RPL18-overexpressed A375/WM793 cells, treated with PBS or 20 nM STAT3-IN (24 h). (Two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant.). (F) Colony formation rate of vector and RPL18-overexpressed A375/WM793 cells, treated with PBS or 20 nM STAT3-IN in 3D Matrigel (5 days). (Two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (G) Cell apoptosis of vector and RPL18-overexpressed A375/WM793 cells (pre-treated with PBS or 20 nM STAT3-IN) treated with TMZ (1 mg/mL, 48 h). (Two-tailed unpaired Student’s t test; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). Data are presented as mean ± SD. Each experiment was repeated independently at least three times.

Article Snippet: Human melanoma cell lines A375 and WM793 were obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling prior to use.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Western Blot, Two Tailed Test, Migration

Journal: iScience

Article Title: RPL18 promotes melanoma progression and drug resistance via BTF3/STAT3-dependent mechanisms and immune modulation

doi: 10.1016/j.isci.2025.114469

Figure Lengend Snippet: RPL18/STAT3 signaling promoted M2 macrophages polarization (A) Correlation analysis between RPL18 and the infiltration of different immune cell subtypes in melanoma patients ( n = 452) was performed using the Sangerbox online tool. (Correlation assessed by Spearman’s rank correlation; n = 452; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, p < 0.05 considered significant). (B) Schematic diagram of macrophages and tumor cells co-culture system. (C) mRNA expression of IL-10, CD206, Arg1 in THP-1 cells co-cultured with vector or RPL18-overexpressed A375 cells. (Two-tailed unpaired Student’s t test for pairwise comparisons or one-way ANOVA for multiple groups; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (D) Heatmap of IL-6, TGF-β, IL-10, CCL2, CCL5, and VEGF expression in vector and RPL18-overexpressed A375/WM793 cells, treated with PBS or 20 nM STAT3-IN, determined by qPCR. (E) 20 nM STAT3-IN, 100 ng TGF-β, or IL-10-neutralizing antibodies were added into macrophage and A375 co-culture system. After co-cultured with RPL18-overexpressed A375 for 3 days, the expression of IL-10, CD206, Arg1 in THP-1 cells were determined by q-PCR. (Comparisons performed by one-way ANOVA followed by post-hoc testing; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). Data are presented as mean ± SD. Each experiment was repeated independently at least three times.

Article Snippet: Human melanoma cell lines A375 and WM793 were obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling prior to use.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: RPL18 promotes melanoma progression and drug resistance via BTF3/STAT3-dependent mechanisms and immune modulation

doi: 10.1016/j.isci.2025.114469

Figure Lengend Snippet: Blockade STAT3 signaling improved the outcome of TMZ in melanoma patients (A) Immunostaining of CEA in tumor organoids from melanoma patients. Scale bars, 100 μm. (Quantification of staining intensity performed using two-tailed unpaired Student’s t test or one-way ANOVA where appropriate; organoid biological replicates n ≥ 3 unless otherwise indicated; mean ± SD; exact p value as shown, p < 0.05 considered significant). (B) Cell apoptosis of organoids treated with TMZ (1 mg/mL, 48 h) for 48 h (Statistical test: two-tailed unpaired Student’s t test or one-way ANOVA as appropriate; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (C) Cell apoptosis of organoids treated with Dabrafenib (left, 300 nM, 48 h) or Binimetinib (right, 300 nM, 48 h). (Two-tailed unpaired Student’s t test for pairwise comparisons; biological replicates n ≥ 3; mean ± SD; exact p value as shown, p < 0.05 considered significant). (D) Vector and RPL18-overexpressed A375 cells were subcutaneously injected into immunodeficient mice on day 0 and the tumor volume was recorded ( n = 5 in each group). On day 25, the mice were treated with intratumoral injection of TMZ (0.1 mg). On day 28, the tumor cells were isolated from the mice and apoptosis was determined. (Tumor volumes compared by two-way ANOVA for repeated measures with post-hoc tests; apoptosis quantified and analyzed by two-tailed unpaired Student’s t test; n = 5 mice per group and n = 3 in apoptosis analysis as indicated; mean ± SD; exact p value as shown, p < 0.05 considered significant). (E) RPL18-overexpressed A375 bearing mice were treated with PBS, TMZ (5 mg/kg), STAT-IN (5 mg/kg), or combination on day 10 and 15 ( n = 5 in each group). Tumor volume and survival information were analyzed. (Tumor growth analyzed by two-way ANOVA with appropriate post-hoc tests; survival analyzed by Kaplan–Meier method and compared by log rank (Mantel–Cox) test; n = 5 per group; mean ± SD; exact p value as shown, p < 0.05 considered significant). Data are presented as mean ± SD. Each experiment was repeated independently at least three times.

Article Snippet: Human melanoma cell lines A375 and WM793 were obtained from the American Type Culture Collection (ATCC) and authenticated by short tandem repeat (STR) profiling prior to use.

Techniques: Immunostaining, Staining, Two Tailed Test, Plasmid Preparation, Injection, Isolation