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Characterization and expression of circSMEK1 in MASH and <t>HCC.</t> A) Analysis of circRNA expression profiles from MASH and HCC datasets. B) Heatmap of the top 8 differentially expressed circRNAs. C) Relative expression of circSMEK1 and circTFRC in paired peritumoral (Para‐HCC) and HCC tissues ( n = 12 pairs). D) Schematic of circSMEK1 derived from exons 3–4 of SMEK1 via back‐splicing. E) Sanger sequencing validation of the back‐splicing junction (BSJ). F) PCR product through gDNA and cDNA using convergent or divergent primers G) Stability of circSMEK1 and linear SMEK1 mRNA in actinomycin D‐treated cells ( n = 3). H) Nuclear/cytoplasmic distribution of circSMEK1 in HCC cells ( n = 3). U6 and GAPDH served as nuclear and cytoplasmic controls, respectively. I) RNA fluorescence in situ hybridization (FISH) for circSMEK1 (red) in normal hepatocytes and HCC cells. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. J) Serum circSMEK1 levels in Cohort 1: healthy controls ( n = 10), MASH ( n = 18), MASH‐HCC ( n = 5), HCC ( n = 20). K) Serum circSMEK1 levels in HCC patients with Vascular Invasion, (VI, n = 8) or without VI (Non‐VI, n = 12). L) Receiver operating characteristic (ROC) curve analysis of serum circSMEK1 for distinguishing HCC patients in Cohort 1 (AUC = 0.790, 95% CI [0.623 to 0.957]). M) Tissue circSMEK1 expression levels in Cohort 2: normal liver ( n = 8), MASH‐HCC ( n = 8), HCC ( n = 20). N) circSMEK1 expression in paired peritumoral and HCC tissues from Cohort 3 ( n = 55 pairs). O) ROC analysis of tissue circSMEK1 for discriminating HCC from peritumoral tissues in Cohort 3 (AUC = 0.742, 95% CI [0.646 to 0.839]). P) Kaplan–Meier survival analysis of HCC patients (Cohort 4) stratified by high vs low circSMEK1 expression (high vs low, total n = 206, p = 0.035). Q) Tissue circSMEK1 levels in HCC patients from Cohort 4 with (VI, n = 61) or without (Non‐VI, n = 145) vascular invasion. R) Representative Oil Red O staining images (left) and quantitative analysis of lipid content (right) in MASLD‐like and MASH‐like cell models following circSMEK1 knockdown ( n = 3). Scale bar, 100 µm. S,T) Relative mRNA expression levels of TNF‐α and TGF‐β upon circSMEK1 knockdown in MASLD‐like and MASH‐like cell models ( n = 3). U) circSMEK1 expression in normal hepatocyte cell lines (THLE‐2, THLE‐3), HCC cell lines <t>(Huh‐7,</t> <t>SNU‐398,</t> <t>Hep3B),</t> and hepatoblastoma cell line (HepG2) ( n = 3). Data are presented as mean ± SD. p ‐values were calculated using two‐tailed paired t‐test (C, N), two‐tailed unpaired t‐test (G, K, Q, R, S, T), one‐way ANOVA followed by Dunnett's multiple comparisons test (compared to the Normal group) (J, M), DeLong's test (L, O), Log‐rank test ( p ) * p < 0.05, ** p < 0.01, *** p < 0.001.

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1) Product Images from "CircSMEK1 Suppresses HCC via the hnRNPK‐IGF2‐AKT Axis: A Diagnostic Biomarker and Therapeutic Target"

Article Title: CircSMEK1 Suppresses HCC via the hnRNPK‐IGF2‐AKT Axis: A Diagnostic Biomarker and Therapeutic Target

Journal: Advanced Science

doi: 10.1002/advs.202505267

Characterization and expression of circSMEK1 in MASH and HCC. A) Analysis of circRNA expression profiles from MASH and HCC datasets. B) Heatmap of the top 8 differentially expressed circRNAs. C) Relative expression of circSMEK1 and circTFRC in paired peritumoral (Para‐HCC) and HCC tissues ( n = 12 pairs). D) Schematic of circSMEK1 derived from exons 3–4 of SMEK1 via back‐splicing. E) Sanger sequencing validation of the back‐splicing junction (BSJ). F) PCR product through gDNA and cDNA using convergent or divergent primers G) Stability of circSMEK1 and linear SMEK1 mRNA in actinomycin D‐treated cells ( n = 3). H) Nuclear/cytoplasmic distribution of circSMEK1 in HCC cells ( n = 3). U6 and GAPDH served as nuclear and cytoplasmic controls, respectively. I) RNA fluorescence in situ hybridization (FISH) for circSMEK1 (red) in normal hepatocytes and HCC cells. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. J) Serum circSMEK1 levels in Cohort 1: healthy controls ( n = 10), MASH ( n = 18), MASH‐HCC ( n = 5), HCC ( n = 20). K) Serum circSMEK1 levels in HCC patients with Vascular Invasion, (VI, n = 8) or without VI (Non‐VI, n = 12). L) Receiver operating characteristic (ROC) curve analysis of serum circSMEK1 for distinguishing HCC patients in Cohort 1 (AUC = 0.790, 95% CI [0.623 to 0.957]). M) Tissue circSMEK1 expression levels in Cohort 2: normal liver ( n = 8), MASH‐HCC ( n = 8), HCC ( n = 20). N) circSMEK1 expression in paired peritumoral and HCC tissues from Cohort 3 ( n = 55 pairs). O) ROC analysis of tissue circSMEK1 for discriminating HCC from peritumoral tissues in Cohort 3 (AUC = 0.742, 95% CI [0.646 to 0.839]). P) Kaplan–Meier survival analysis of HCC patients (Cohort 4) stratified by high vs low circSMEK1 expression (high vs low, total n = 206, p = 0.035). Q) Tissue circSMEK1 levels in HCC patients from Cohort 4 with (VI, n = 61) or without (Non‐VI, n = 145) vascular invasion. R) Representative Oil Red O staining images (left) and quantitative analysis of lipid content (right) in MASLD‐like and MASH‐like cell models following circSMEK1 knockdown ( n = 3). Scale bar, 100 µm. S,T) Relative mRNA expression levels of TNF‐α and TGF‐β upon circSMEK1 knockdown in MASLD‐like and MASH‐like cell models ( n = 3). U) circSMEK1 expression in normal hepatocyte cell lines (THLE‐2, THLE‐3), HCC cell lines (Huh‐7, SNU‐398, Hep3B), and hepatoblastoma cell line (HepG2) ( n = 3). Data are presented as mean ± SD. p ‐values were calculated using two‐tailed paired t‐test (C, N), two‐tailed unpaired t‐test (G, K, Q, R, S, T), one‐way ANOVA followed by Dunnett's multiple comparisons test (compared to the Normal group) (J, M), DeLong's test (L, O), Log‐rank test ( p ) * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure Legend Snippet: Characterization and expression of circSMEK1 in MASH and HCC. A) Analysis of circRNA expression profiles from MASH and HCC datasets. B) Heatmap of the top 8 differentially expressed circRNAs. C) Relative expression of circSMEK1 and circTFRC in paired peritumoral (Para‐HCC) and HCC tissues ( n = 12 pairs). D) Schematic of circSMEK1 derived from exons 3–4 of SMEK1 via back‐splicing. E) Sanger sequencing validation of the back‐splicing junction (BSJ). F) PCR product through gDNA and cDNA using convergent or divergent primers G) Stability of circSMEK1 and linear SMEK1 mRNA in actinomycin D‐treated cells ( n = 3). H) Nuclear/cytoplasmic distribution of circSMEK1 in HCC cells ( n = 3). U6 and GAPDH served as nuclear and cytoplasmic controls, respectively. I) RNA fluorescence in situ hybridization (FISH) for circSMEK1 (red) in normal hepatocytes and HCC cells. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. J) Serum circSMEK1 levels in Cohort 1: healthy controls ( n = 10), MASH ( n = 18), MASH‐HCC ( n = 5), HCC ( n = 20). K) Serum circSMEK1 levels in HCC patients with Vascular Invasion, (VI, n = 8) or without VI (Non‐VI, n = 12). L) Receiver operating characteristic (ROC) curve analysis of serum circSMEK1 for distinguishing HCC patients in Cohort 1 (AUC = 0.790, 95% CI [0.623 to 0.957]). M) Tissue circSMEK1 expression levels in Cohort 2: normal liver ( n = 8), MASH‐HCC ( n = 8), HCC ( n = 20). N) circSMEK1 expression in paired peritumoral and HCC tissues from Cohort 3 ( n = 55 pairs). O) ROC analysis of tissue circSMEK1 for discriminating HCC from peritumoral tissues in Cohort 3 (AUC = 0.742, 95% CI [0.646 to 0.839]). P) Kaplan–Meier survival analysis of HCC patients (Cohort 4) stratified by high vs low circSMEK1 expression (high vs low, total n = 206, p = 0.035). Q) Tissue circSMEK1 levels in HCC patients from Cohort 4 with (VI, n = 61) or without (Non‐VI, n = 145) vascular invasion. R) Representative Oil Red O staining images (left) and quantitative analysis of lipid content (right) in MASLD‐like and MASH‐like cell models following circSMEK1 knockdown ( n = 3). Scale bar, 100 µm. S,T) Relative mRNA expression levels of TNF‐α and TGF‐β upon circSMEK1 knockdown in MASLD‐like and MASH‐like cell models ( n = 3). U) circSMEK1 expression in normal hepatocyte cell lines (THLE‐2, THLE‐3), HCC cell lines (Huh‐7, SNU‐398, Hep3B), and hepatoblastoma cell line (HepG2) ( n = 3). Data are presented as mean ± SD. p ‐values were calculated using two‐tailed paired t‐test (C, N), two‐tailed unpaired t‐test (G, K, Q, R, S, T), one‐way ANOVA followed by Dunnett's multiple comparisons test (compared to the Normal group) (J, M), DeLong's test (L, O), Log‐rank test ( p ) * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: Expressing, Derivative Assay, Sequencing, Biomarker Discovery, Fluorescence, In Situ Hybridization, Staining, Knockdown, Two Tailed Test

circSMEK1 Mediates hnRNPK Ubiquitination. A) Mass spectrometry analysis of proteins pull down by biotin‐labeled circSMEK1 probes. B) Top candidate proteins ranked by score. C) RNA immunoprecipitation assays test the binding of circSMEK1 to hnRNPK in HCC cells ( n = 3). D) Protein level of hnRNPK with or without OE‐circSMEK1 or sh‐circSMEK1. E) Quantification of hnRNPK levels in OE‐Ctrl vs OE‐circSMEK1 HCC cells ( n = 3). F) Protein level of hnRNPK in sh‐Ctrl and sh‐circSMEK1 cells treated with or without MG132 ( n = 3). G) Cycloheximide (CHX) chase assay of hnRNPK stability in OE‐Ctrl and OE‐circSMEK1 HCC cells ( n = 3). H,I) Immunoblot of hnRNPK ubiquitination in sh‐Ctrl and sh‐circSMEK1 cells with the treatment of MG132 to test exogenous H) and endogenous I) ubiquitination. J) AlphaFold‐3 prediction interaction sites with potential hydrogen bonds between the circSMEK1 sequence and KH1/KH3 domains of hnRNPK. K) Immunoblot for the FLAG‐tagged various domain‐truncated mutants of hnRNPK by RNA antisense purification by biotin‐labeled circSMEK1. Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired t ‐test (C, E, G). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure Legend Snippet: circSMEK1 Mediates hnRNPK Ubiquitination. A) Mass spectrometry analysis of proteins pull down by biotin‐labeled circSMEK1 probes. B) Top candidate proteins ranked by score. C) RNA immunoprecipitation assays test the binding of circSMEK1 to hnRNPK in HCC cells ( n = 3). D) Protein level of hnRNPK with or without OE‐circSMEK1 or sh‐circSMEK1. E) Quantification of hnRNPK levels in OE‐Ctrl vs OE‐circSMEK1 HCC cells ( n = 3). F) Protein level of hnRNPK in sh‐Ctrl and sh‐circSMEK1 cells treated with or without MG132 ( n = 3). G) Cycloheximide (CHX) chase assay of hnRNPK stability in OE‐Ctrl and OE‐circSMEK1 HCC cells ( n = 3). H,I) Immunoblot of hnRNPK ubiquitination in sh‐Ctrl and sh‐circSMEK1 cells with the treatment of MG132 to test exogenous H) and endogenous I) ubiquitination. J) AlphaFold‐3 prediction interaction sites with potential hydrogen bonds between the circSMEK1 sequence and KH1/KH3 domains of hnRNPK. K) Immunoblot for the FLAG‐tagged various domain‐truncated mutants of hnRNPK by RNA antisense purification by biotin‐labeled circSMEK1. Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired t ‐test (C, E, G). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Techniques Used: Ubiquitin Proteomics, Mass Spectrometry, Labeling, RNA Immunoprecipitation, Binding Assay, Western Blot, Sequencing, Purification, Two Tailed Test

$Nucleic circSMEK1 interacts with hnRNPK to regulate IGF2/AKT demonstrates therapeutic potential for HCC. A) IGF2 ranked as the top protein‐coding gene in hnRNPK eCLIP‐seq analysis. B) Western blot analysis of hnRNPK and IGF2 protein levels in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. C) Dual‐luciferase reporter assay assessing hnRNPK activity on the 5′ UTR of IGF2 ( n = 3). D) Chromatin immunoprecipitation (ChIP) qPCR showing hnRNPK enrichment at the −2000 bp region upstream of the IGF2 transcription start site ( n = 3). E) Western blot of hnRNPK and IGF2 in nuclear and cytoplasmic fractions of HCC cells with or without circSMEK1 overexpression. F) ELISA measuring IGF2 levels in cell culture supernatant from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). G) Western blot analysis of secreted IGF2 by supernatant protein extraction from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). H) KEGG pathway analysis of differentially expressed genes from HepG2 cells with hnRNPK knockdown. I) Western blot of hnRNPK, IGF2, and phosphorylated AKT (p‐AKT) in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. J) Colony formation assay of HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). K) Western blot analysis of p‐AKT levels in HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). L) Schematic of the c‐MYC/AKT/NRAS PiggyBac transposon system for hydrodynamic tail vein injection in mice with or without circSMEK1 overexpression. M) Representative images of livers and lungs from transposon‐mediated mouse models ( n = 5 mice per group). N) Representative H&E staining of liver sections from transposon‐mediated mice with or without circSMEK1 overexpression ( n = 5 mice per group). O) Representative H&E staining (left) and quantification (right) of lung metastatic foci in transposon‐mediated mice with or without circSMEK1 overexpression (n = 5 mice per group). P) Western blot analysis of hnRNPK, IGF2, p‐AKT, p‐IGF1R, and VEGFA protein levels in liver tissues from the transposon‐mediated mouse model (n = 4 randomly chosen from 5 biological replicates). Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired (D, J, O) or One‐way ANOVA followed by Dunnett's multiple comparisons test (C, F). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.$
Figure Legend Snippet: Nucleic circSMEK1 interacts with hnRNPK to regulate IGF2/AKT demonstrates therapeutic potential for HCC. A) IGF2 ranked as the top protein‐coding gene in hnRNPK eCLIP‐seq analysis. B) Western blot analysis of hnRNPK and IGF2 protein levels in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. C) Dual‐luciferase reporter assay assessing hnRNPK activity on the 5′ UTR of IGF2 ( n = 3). D) Chromatin immunoprecipitation (ChIP) qPCR showing hnRNPK enrichment at the −2000 bp region upstream of the IGF2 transcription start site ( n = 3). E) Western blot of hnRNPK and IGF2 in nuclear and cytoplasmic fractions of HCC cells with or without circSMEK1 overexpression. F) ELISA measuring IGF2 levels in cell culture supernatant from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). G) Western blot analysis of secreted IGF2 by supernatant protein extraction from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). H) KEGG pathway analysis of differentially expressed genes from HepG2 cells with hnRNPK knockdown. I) Western blot of hnRNPK, IGF2, and phosphorylated AKT (p‐AKT) in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. J) Colony formation assay of HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). K) Western blot analysis of p‐AKT levels in HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). L) Schematic of the c‐MYC/AKT/NRAS PiggyBac transposon system for hydrodynamic tail vein injection in mice with or without circSMEK1 overexpression. M) Representative images of livers and lungs from transposon‐mediated mouse models ( n = 5 mice per group). N) Representative H&E staining of liver sections from transposon‐mediated mice with or without circSMEK1 overexpression ( n = 5 mice per group). O) Representative H&E staining (left) and quantification (right) of lung metastatic foci in transposon‐mediated mice with or without circSMEK1 overexpression (n = 5 mice per group). P) Western blot analysis of hnRNPK, IGF2, p‐AKT, p‐IGF1R, and VEGFA protein levels in liver tissues from the transposon‐mediated mouse model (n = 4 randomly chosen from 5 biological replicates). Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired (D, J, O) or One‐way ANOVA followed by Dunnett's multiple comparisons test (C, F). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Techniques Used: Western Blot, Over Expression, Knockdown, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Protein Extraction, Colony Assay, Injection, Staining, Two Tailed Test

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Journal: Advanced Science

Article Title: CircSMEK1 Suppresses HCC via the hnRNPK‐IGF2‐AKT Axis: A Diagnostic Biomarker and Therapeutic Target

doi: 10.1002/advs.202505267

Figure Lengend Snippet: Characterization and expression of circSMEK1 in MASH and HCC. A) Analysis of circRNA expression profiles from MASH and HCC datasets. B) Heatmap of the top 8 differentially expressed circRNAs. C) Relative expression of circSMEK1 and circTFRC in paired peritumoral (Para‐HCC) and HCC tissues ( n = 12 pairs). D) Schematic of circSMEK1 derived from exons 3–4 of SMEK1 via back‐splicing. E) Sanger sequencing validation of the back‐splicing junction (BSJ). F) PCR product through gDNA and cDNA using convergent or divergent primers G) Stability of circSMEK1 and linear SMEK1 mRNA in actinomycin D‐treated cells ( n = 3). H) Nuclear/cytoplasmic distribution of circSMEK1 in HCC cells ( n = 3). U6 and GAPDH served as nuclear and cytoplasmic controls, respectively. I) RNA fluorescence in situ hybridization (FISH) for circSMEK1 (red) in normal hepatocytes and HCC cells. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. J) Serum circSMEK1 levels in Cohort 1: healthy controls ( n = 10), MASH ( n = 18), MASH‐HCC ( n = 5), HCC ( n = 20). K) Serum circSMEK1 levels in HCC patients with Vascular Invasion, (VI, n = 8) or without VI (Non‐VI, n = 12). L) Receiver operating characteristic (ROC) curve analysis of serum circSMEK1 for distinguishing HCC patients in Cohort 1 (AUC = 0.790, 95% CI [0.623 to 0.957]). M) Tissue circSMEK1 expression levels in Cohort 2: normal liver ( n = 8), MASH‐HCC ( n = 8), HCC ( n = 20). N) circSMEK1 expression in paired peritumoral and HCC tissues from Cohort 3 ( n = 55 pairs). O) ROC analysis of tissue circSMEK1 for discriminating HCC from peritumoral tissues in Cohort 3 (AUC = 0.742, 95% CI [0.646 to 0.839]). P) Kaplan–Meier survival analysis of HCC patients (Cohort 4) stratified by high vs low circSMEK1 expression (high vs low, total n = 206, p = 0.035). Q) Tissue circSMEK1 levels in HCC patients from Cohort 4 with (VI, n = 61) or without (Non‐VI, n = 145) vascular invasion. R) Representative Oil Red O staining images (left) and quantitative analysis of lipid content (right) in MASLD‐like and MASH‐like cell models following circSMEK1 knockdown ( n = 3). Scale bar, 100 µm. S,T) Relative mRNA expression levels of TNF‐α and TGF‐β upon circSMEK1 knockdown in MASLD‐like and MASH‐like cell models ( n = 3). U) circSMEK1 expression in normal hepatocyte cell lines (THLE‐2, THLE‐3), HCC cell lines (Huh‐7, SNU‐398, Hep3B), and hepatoblastoma cell line (HepG2) ( n = 3). Data are presented as mean ± SD. p ‐values were calculated using two‐tailed paired t‐test (C, N), two‐tailed unpaired t‐test (G, K, Q, R, S, T), one‐way ANOVA followed by Dunnett's multiple comparisons test (compared to the Normal group) (J, M), DeLong's test (L, O), Log‐rank test ( p ) * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HCC cell lines (SNU‐398, Huh‐7, Hep3B), liver cancer hepatoblastoma (HepG2), immortalized normal hepatocytes (THLE‐2, THLE‐3) were obtained from ATCC or the Chinese Academy of Sciences.

Techniques: Expressing, Derivative Assay, Sequencing, Biomarker Discovery, Fluorescence, In Situ Hybridization, Staining, Knockdown, Two Tailed Test

Journal: Advanced Science

Article Title: CircSMEK1 Suppresses HCC via the hnRNPK‐IGF2‐AKT Axis: A Diagnostic Biomarker and Therapeutic Target

doi: 10.1002/advs.202505267

Figure Lengend Snippet: circSMEK1 Mediates hnRNPK Ubiquitination. A) Mass spectrometry analysis of proteins pull down by biotin‐labeled circSMEK1 probes. B) Top candidate proteins ranked by score. C) RNA immunoprecipitation assays test the binding of circSMEK1 to hnRNPK in HCC cells ( n = 3). D) Protein level of hnRNPK with or without OE‐circSMEK1 or sh‐circSMEK1. E) Quantification of hnRNPK levels in OE‐Ctrl vs OE‐circSMEK1 HCC cells ( n = 3). F) Protein level of hnRNPK in sh‐Ctrl and sh‐circSMEK1 cells treated with or without MG132 ( n = 3). G) Cycloheximide (CHX) chase assay of hnRNPK stability in OE‐Ctrl and OE‐circSMEK1 HCC cells ( n = 3). H,I) Immunoblot of hnRNPK ubiquitination in sh‐Ctrl and sh‐circSMEK1 cells with the treatment of MG132 to test exogenous H) and endogenous I) ubiquitination. J) AlphaFold‐3 prediction interaction sites with potential hydrogen bonds between the circSMEK1 sequence and KH1/KH3 domains of hnRNPK. K) Immunoblot for the FLAG‐tagged various domain‐truncated mutants of hnRNPK by RNA antisense purification by biotin‐labeled circSMEK1. Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired t ‐test (C, E, G). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Labeling, RNA Immunoprecipitation, Binding Assay, Western Blot, Sequencing, Purification, Two Tailed Test

Journal: Advanced Science

Article Title: CircSMEK1 Suppresses HCC via the hnRNPK‐IGF2‐AKT Axis: A Diagnostic Biomarker and Therapeutic Target

doi: 10.1002/advs.202505267

Figure Lengend Snippet: Nucleic circSMEK1 interacts with hnRNPK to regulate IGF2/AKT demonstrates therapeutic potential for HCC. A) IGF2 ranked as the top protein‐coding gene in hnRNPK eCLIP‐seq analysis. B) Western blot analysis of hnRNPK and IGF2 protein levels in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. C) Dual‐luciferase reporter assay assessing hnRNPK activity on the 5′ UTR of IGF2 ( n = 3). D) Chromatin immunoprecipitation (ChIP) qPCR showing hnRNPK enrichment at the −2000 bp region upstream of the IGF2 transcription start site ( n = 3). E) Western blot of hnRNPK and IGF2 in nuclear and cytoplasmic fractions of HCC cells with or without circSMEK1 overexpression. F) ELISA measuring IGF2 levels in cell culture supernatant from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). G) Western blot analysis of secreted IGF2 by supernatant protein extraction from cells with or without sh hnRNPK or sh circSMEK1 ( n = 3). H) KEGG pathway analysis of differentially expressed genes from HepG2 cells with hnRNPK knockdown. I) Western blot of hnRNPK, IGF2, and phosphorylated AKT (p‐AKT) in HCC cells with or without hnRNPK overexpression or circSMEK1 knockdown. J) Colony formation assay of HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). K) Western blot analysis of p‐AKT levels in HCC cells treated with sh‐circSMEK1 and/or Xentuzumab (0.5 µM) ( n = 3). L) Schematic of the c‐MYC/AKT/NRAS PiggyBac transposon system for hydrodynamic tail vein injection in mice with or without circSMEK1 overexpression. M) Representative images of livers and lungs from transposon‐mediated mouse models ( n = 5 mice per group). N) Representative H&E staining of liver sections from transposon‐mediated mice with or without circSMEK1 overexpression ( n = 5 mice per group). O) Representative H&E staining (left) and quantification (right) of lung metastatic foci in transposon‐mediated mice with or without circSMEK1 overexpression (n = 5 mice per group). P) Western blot analysis of hnRNPK, IGF2, p‐AKT, p‐IGF1R, and VEGFA protein levels in liver tissues from the transposon‐mediated mouse model (n = 4 randomly chosen from 5 biological replicates). Data are presented as mean ± SD. p ‐values were calculated by two‐tailed unpaired (D, J, O) or One‐way ANOVA followed by Dunnett's multiple comparisons test (C, F). Significant results are presented as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Techniques: Western Blot, Over Expression, Knockdown, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Protein Extraction, Colony Assay, Injection, Staining, Two Tailed Test

Relative mRNA expression of SLC41A1 in HCC cell lines (CCLE).

Journal: International Journal of Medical Sciences

Article Title: SLC41A1 overexpression correlates with immune cell infiltration in HCC and promotes its malignant progression

doi: 10.7150/ijms.100155

Figure Lengend Snippet: Relative mRNA expression of SLC41A1 in HCC cell lines (CCLE).

Article Snippet: The HCC cell line SNU398 was purchased from the American Type Culture Collection (Manassas, USA), while Huh7 was obtained from Japanese Cancer Research Bank (Tokyo, Japan), and the two cell lines were preserved in our institute.

Techniques: Expressing

SLC41A1 promotes HCC cell proliferation, migration and invasion. (A) Transfection efficiencies of lentiviruses in SNU398 and Huh7 were measured by qRT-PCR. (B-C) The CCK-8 (B, n=6) and EdU (C, n=3) assays were conducted to assess cell proliferation. (D) The Transwell assays were performed to measure cell migration and invasion (n=3). (E)The EdU positive cell percent was shown. (F)The number of migrated and invaded cells was shown. ** P <0.01, *** P <0.001.

Journal: International Journal of Medical Sciences

Article Title: SLC41A1 overexpression correlates with immune cell infiltration in HCC and promotes its malignant progression

doi: 10.7150/ijms.100155

Figure Lengend Snippet: SLC41A1 promotes HCC cell proliferation, migration and invasion. (A) Transfection efficiencies of lentiviruses in SNU398 and Huh7 were measured by qRT-PCR. (B-C) The CCK-8 (B, n=6) and EdU (C, n=3) assays were conducted to assess cell proliferation. (D) The Transwell assays were performed to measure cell migration and invasion (n=3). (E)The EdU positive cell percent was shown. (F)The number of migrated and invaded cells was shown. ** P <0.01, *** P <0.001.

Techniques: Migration, Transfection, Quantitative RT-PCR, CCK-8 Assay

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