Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: STOML2 expression is upregulated in HCC tissues. (A) Overall survival analysis using The Cancer Genome Atlas dataset showed that patients with HCC exhibiting high STOML2 expression had a significantly worse prognosis. P=0.017, log-rank test. (B and C) STOML2 expression levels across various cancer types determined using public datasets from the Gene Expression Omnibus. (B) GSE40367 dataset comparing normal liver tissue, non-metastatic HCC, HCC with lung metastasis and HCC with adrenal metastasis ( * P<0.05, ** P<0.01, **** P<0.0001, one-way ANOVA followed by Tukey's post hoc test, n=6, 10, 12 and 6, respectively). Data are presented as median with interquartile range. (C) GSE14520 dataset comparing HCC tissues and normal liver tissues ( **** P<0.0001, unpaired Student's t-test, n=65 and 50, respectively). Data are presented as median with interquartile range. (D) STOML2 protein expression in 72 paired HCC tissues (denoted as T) and adjacent normal tissues (denoted as N) was evaluated using IHC with tissue microarrays. (E) Semi-quantitative comparison of IHC staining (IHC score) for tumor tissues vs. adjacent normal tissues (n=72 pairs). Individual paired data points are shown as scatter plots, with lines connecting each tumor tissue to its matched adjacent normal tissue. The bars represent the median IHC score for each group, with error bars indicating the interquartile range. *** P<0.001, Wilcoxon signed-rank test. STOML2 expression was assessed in eight randomly selected paired HCC and normal tissues using (F) western blotting and (G) reverse transcription-quantitative polymerase chain reaction (n=8 pairs). (F) Representative images from three independent experiments are shown and (G) data were obtained from three independent experiments. **** P<0.0001, paired Student's t-test. Labels S1-S8 represent eight randomly selected paired HCC and adjacent normal tissue samples from individual patients. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; STOML2, stomatin-like protein 2.

Article Snippet: For MAPK pathway inhibition, Huh7 cells overexpressing STOML2 were pre-treated with sorafenib (20 μ M; cat. no. HY10201; MedChemExpress) for 24 h at 37°C prior to subsequent assays, including western blotting, flow cytometry and functional assays.

Techniques: Expressing, Gene Expression, Comparison, Immunohistochemistry, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: STOML2 promotes proliferation, migration, invasion and autophagy, and inhibits apoptosis in HCC cells. (A) STOML2 expression levels in five HCC cell lines and normal hepatocytes were determined using western blotting and RT-qPCR. Representative western blot images and quantitative RT-qPCR data (mean ± SD) from three independent experiments are shown ( **** P<0.0001, one-way ANOVA followed by Tukey's post-hoc tests). (B) Knockdown efficiency in HCCLM3 cells and overexpression efficiency in Huh7 cells were evaluated using western blotting and RT-qPCR. Representative western blot images and quantitative RT-qPCR data (mean ± SD) from three independent experiments are shown ( *** P<0.001, ** P<0.01, comparisons between two groups were analyzed by unpaired Student's t-test, whereas comparisons among three groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test). (C) Cell Counting Kit-8 assay showing the proliferation of STOML2-knockdown HCCLM3 cells, STOML2-overexpressing Huh7 cells and their respective controls. Data are presented as mean ± SD from three independent experiments ( **** P<0.0001, comparisons between two groups were analyzed by unpaired Student's t-test, whereas comparisons among three groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test). (D) Wound healing assay assessing the migratory ability of STOML2-overexpressing or -knockdown HCC cells. Representative images from three independent experiments are shown. (E) Cell cycle distribution analysis of STOML2-knockdown HCCLM3 cells, STOML2-overexpressing Huh7 cells and their respective controls. Representative images from three independent experiments are shown. (F) Flow cytometric analysis of apoptosis rates of STOML2-overexpressing or -knockdown HCC cells along with their respective controls ( *** P<0.001, comparisons between two groups were analyzed by unpaired Student's t-test, whereas comparisons among three groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test). Representative flow cytometry plots and quantitative data (mean ± SD) from three independent experiments are shown. (G) Transwell assay measuring the invasive capacity of HCC cells following STOML2 overexpression or knockdown ( *** P<0.001, ** P<0.01, comparisons between two groups were analyzed by unpaired Student's t-test, whereas comparisons among three groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test). Data are presented as mean ± SD. (H) Western blotting of autophagy markers p62 and Beclin1 in STOML2-overexpressing or -knockdown HCC cells. Representative western blot images from three independent experiments are shown. HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Scr, scramble; sh, short hairpin; STOML2, stomatin-like protein 2.

Article Snippet: For MAPK pathway inhibition, Huh7 cells overexpressing STOML2 were pre-treated with sorafenib (20 μ M; cat. no. HY10201; MedChemExpress) for 24 h at 37°C prior to subsequent assays, including western blotting, flow cytometry and functional assays.

Techniques: Migration, Expressing, Western Blot, Quantitative RT-PCR, Knockdown, Over Expression, Cell Counting, Wound Healing Assay, Flow Cytometry, Transwell Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: STOML2 promotes hepatocellular carcinoma growth and progression in vivo . (A-E) Subcutaneous implantation of STOML2-overexpressing Huh7 cells and STOML2-knockdown HCCLM3 cells along with their control cells in BALB/c nude mice. (A) Each mouse received 3×10 6 cells as a subcutaneous injection at the dorsal region of their neck. Tumors were harvested 25 days post-injection. (B) Tumor growth curves obtained at the indicated time points ( **** P<0.0001, comparisons between two groups were analyzed by unpaired Student's t-test, whereas comparisons among three or more groups were analyzed by two-way ANOVA followed by Tukey's post hoc test). Data are presented as mean ± SD. (C) Final tumor mass measured upon removal ( **** P<0.0001, unpaired Student's t-test or one-way ANOVA and Tukey's post hoc test for multiple comparisons). Data are presented as mean ± SD. (D) Representative immunohistochemistry images showing a positive association between STOML2 and Ki-67 expression in tumor tissues. Quantitative data are presented as mean ± SD from multiple fields of view ( **** P<0.0001, *** P<0.001, one-way ANOVA followed by Tukey's post hoc test for multiple comparisons). (E) Representative hematoxylin and eosin staining of tumor sections. Images are representative of tumors from each group. (F) Co-immunoprecipitation assay demonstrating interaction between STOML2 and PHB in vitro . Representative western blot images from three independent experiments are shown. (G) Immunofluorescence staining showing colocalization of STOML2 (green), PHB (red) and DAPI (blue) in HCCLM3, HepG2 and Huh7 cells. Representative images from three independent experiments are shown. PHB, prohibitin; Scr, scramble; sh, short hairpin; STOML2, stomatin-like protein 2.

Article Snippet: For MAPK pathway inhibition, Huh7 cells overexpressing STOML2 were pre-treated with sorafenib (20 μ M; cat. no. HY10201; MedChemExpress) for 24 h at 37°C prior to subsequent assays, including western blotting, flow cytometry and functional assays.

Techniques: In Vivo, Knockdown, Control, Injection, Immunohistochemistry, Expressing, Staining, Co-Immunoprecipitation Assay, In Vitro, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: PHB knockdown inhibits STOML2-induced proliferation and invasion while promoting pro-apoptotic autophagy in HCC cells. (A) Western blot analysis of STOML2, PHB and key proteins of the MAPK signaling pathway in Huh7-STOML2, HCCLM3-shSTOML2 and their respective control cells. Representative western blot images from three independent experiments are shown. (B) Wound healing assay (scale bar, 25 μ m), (C) cell cycle analysis and (D) Transwell assay ( **** P<0.0001, unpaired Student's t-test) demonstrating that PHB knockdown attenuated the cell cycle progression, migration and invasion of STOML2-overexpressing Huh7 cells. Representative images and quantitative analysis (mean ± SD) from three independent experiments are shown. (E) Huh7-STOML2-Scr control and Huh7-STOML2-shPHB cells were subcutaneously injected into BALB/c nude mice. Each mouse was injected with 3×10 6 cells in the dorsal region of their neck. (F) Flow cytometric analysis showing that PHB knockdown promoted apoptosis in STOML2-overexpressing Huh7 cells. Representative flow cytometry plots and quantitative data (mean ± SD) from three independent experiments are shown. (unpaired Student's t-test, *** P<0.001). (G) Tumor growth curves obtained at the indicated time points. Data are presented as mean ± SD ( **** P<0.0001, unpaired Student's t-test). (H) Western blotting of STOML2, PHB and MAPK signaling pathway proteins in Huh7-STOML2 cells with or without PHB knockdown. Representative western blot images from three independent experiments are shown. (I) Tumor mass measured at removal. **** P<0.0001, unpaired Student's t-test. Data are presented as mean ± SD. (J) Western blotting of p62 and Beclin1 in Huh7-STOML2 cells with or without PHB knockdown. Representative western blot images from three independent experiments are shown. p-, phosphorylated; PHB, prohibitin; Scr, scramble; sh, short hairpin; STOML2, stomatin-like protein 2.

Article Snippet: For MAPK pathway inhibition, Huh7 cells overexpressing STOML2 were pre-treated with sorafenib (20 μ M; cat. no. HY10201; MedChemExpress) for 24 h at 37°C prior to subsequent assays, including western blotting, flow cytometry and functional assays.

Techniques: Knockdown, Western Blot, Control, Wound Healing Assay, Cell Cycle Assay, Transwell Assay, Migration, Injection, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: Sorafenib (an RAF1 inhibitor) significantly attenuated STOML2-induced cell cycle progression, migration and autophagy, while promoting apoptosis in HCC cells. (A) Cell cycle distribution analysis of Huh7-STOML2 and control cells following treatment with sorafenib (20 μ M, 24 h) or DMSO. Representative images from three independent experiments are shown. (B) Wound healing assay showing the migratory capacity of Huh7-STOML2 cells treated with sorafenib or DMSO. Representative images from three independent experiments are shown. (C) Flow cytometric analysis of apoptosis in STOML2-overexpressing Huh7 and control cells after treatment with sorafenib or DMSO (unpaired Student's t-test, **** P<0.0001). Representative flow cytometry plots and quantitative data (mean ± SD) from three independent experiments are shown. (D) Transwell assay showing the invasive capacity of Huh7-STOML2 and control cells following sorafenib treatment or DMSO. Data are presented as mean ± SD (unpaired Student's t-test, *** P<0.001). Western blot analysis of (E) MAPK signaling pathway proteins, and (F) p62 and Beclin1 in Huh7-STOML2 cells after treatment with sorafenib or DMSO. Representative images from three independent experiments are shown. p-, phosphorylated; STOML2, stomatin-like protein 2.

Article Snippet: For MAPK pathway inhibition, Huh7 cells overexpressing STOML2 were pre-treated with sorafenib (20 μ M; cat. no. HY10201; MedChemExpress) for 24 h at 37°C prior to subsequent assays, including western blotting, flow cytometry and functional assays.

Techniques: Migration, Control, Wound Healing Assay, Flow Cytometry, Transwell Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 localizes to the nucleolus. ( A ) Protein sequence of MED19. The predicted NoLS is highlighted in blue with lysine (K) residues included marked in red. ( B ) The NoLS prediction of MED19 protein using the NOD web tool. The x -axis indicates the amino acid position along the MED19 protein sequence, while the y -axis shows the nucleolar localization score assigned by the NOD for each amino acid. Each blue dot represents an individual amino acid. The pink region above 0.8 represents potential NoLSs. ( C ) Immunofluorescence staining of MED19 using specific antibodies in various cell lines. FBL serves as a commonly used nucleolar marker, and DAPI stains nuclear DNA. Scale bar: 10 μm. ( D ) Immunofluorescence images of the MED23, CDK8, MED4, and MED8 subunits of the Mediator complex in HuH7 cells, using respective antibodies. Scale bar: 10 μm. ( E ) Schematic representation of exogenously expressed full-length MED19 and its truncated or mutated variants. HA-MED19-FL represents full-length MED19. HA-MED19-NoLS represents MED19 with the NoLS. HA-MED19-ΔNoLS represents MED19 with the NoLS deleted. HA-MED19-K-A represents MED19 with all lysine residues (K) in the NoLS mutated to alanines (A). ( F ) Immunofluorescence images of exogenously expressed full-length MED19 and its truncated or mutated variants in HuH7 cells, detected using an anti-HA antibody. Scale bar: 10 μm. ( G ) Schematic illustration of the GFP localization experiment. GFP-WT, a plasmid expressing wild-type GFP protein. GFP-19NoLS, a plasmid expressing a GFP fusion protein with the NoLS of MED19 fused to its C-terminus. ( H ) Co-localization results of GFP-WT and GFP-19NoLS proteins. ( I ) Immunofluorescence was performed using an HA-tag antibody. Three representative cells were selected for display. The HA tag was knocked into the N-terminus of the MED19 protein via gene editing technology in the HuH7 hepatocellular carcinoma cell line.​

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Sequencing, Immunofluorescence, Staining, Marker, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 directly binds rRNA via its NoLS. ( A ) Schematic illustration of the silica enrichment assay. RNA-binding proteins can be enriched by silica material under UV cross-linking and denaturing conditions. ( B – F ) Western blot analysis of silica-enriched fractions probed with antibodies against FBL, NPM1, tubulin, actin, and MED19. Input: total cell lysates. UV: samples subjected to UV cross-linking. ( G ) Western blot analysis of silica-enriched fractions using an anti-HA antibody. HA-19-FL: full-length HA-tagged MED19. HA-ΔNoLS: HA-tagged MED19 lacking the NoLS. ( H ) Western blot analysis of silica-enriched fractions using an anti-GFP antibody. GFP-19-FL: full-length GFP-tagged MED19. GFP-NoLS: the NoLS of MED19 was fused to the C-terminus of GFP. ( I ) Western blot analysis of silica-enriched fractions using an anti-HA antibody. HA-19-K-A: HA-tagged MED19 with all lysine residues in the NoLS mutated to alanine. ( J ) Schematic diagram of identifying the RNA-binding sites on the corresponding protein by UV cross-linking mass spectrometry. ( K ) Distribution of RNA-binding sites across the MED19 protein. The x -axis represents the amino acid sequence positions; the y -axis represents the summed quantitative scores of the RNA-amino acid cross-linking sites. ( L ) Peaks distribution of MED19 CLIP-seq. IGS, Intergenic Spacer; 5′ETS, 5′ External Transcribed Spacer; 3′ETS, 3′ External Transcribed Spacer; ITS1, Internal Transcribed Spacer 1; ITS2, Internal Transcribed Spacer 2. ( M ) Reads frequency distribution of MED19-binding RNAs. ( N ) Reads frequency distribution of MED19-binding snoRNAs. ( O ) Denaturing UV-RIP-qPCR of MED19. Three kinds of denaturing wash buffer (10% guanidine hydrochloride, 8 M urea, 10% SDS) were used in the “wash” step of the UV-RIP-qPCR of HaloTag-MED19 and HaloTag-GFP. The Ct value of the target proteins was normalized to the input sample represented as the percentage of the corresponding RNA in the input. ( P ) EMSA blot of the purified MED19 proteins with rRNA probes. The rRNA probe without biotin was used as a competitive probe and was added at a molar amount 10 times that of the biotin-labeled probe. The signal on the membrane was detected using SA-HRP luminescence system.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: RNA Binding Assay, Western Blot, Structural Proteomics, Sequencing, Binding Assay, Purification, Labeling, Membrane

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 interacts with the BoxC/D-snoRNP. ( A ) Intersection analysis of MED19 interacting proteins identified by the MED19 IP-MS with nucleolar proteins annotated in the GO database. ( B ) Enrichment analysis of MED19 interactome based on the GO database at the molecular function level. ( C ) Enrichment analysis of annotated nucleolar proteins in the MED19 interactome by the GO database at the molecular function level. ( D ) Enrichment analysis of MED19 interactome by the Reactome database. ( E ) Enrichment analysis of annotated nucleolar proteins in the MED19 interactome by the Reactome database. ( F ) Identification of MED19 and four core Box C/D snoRNP subunit proteins by MED19 IP-MS. ( G ) Western blot analysis of MED19 immunoprecipitation (IP-WB) following RNase A treatment. ( H ) Western blot of FBL immunoprecipitation. ( I ) Western blot analysis of MED19 immunoprecipitation using exogenously expressed full-length and mutant forms of MED19. HA-MED19-FL: HA-tagged full-length MED19; HA-MED19-K-A: HA-tagged MED19 with all lysines in the NoLS mutated to alanines; HA-MED19-NoLS: HA-tagged NoLS domain of MED19. ( J ) Western blot of MED19 IP by exogenously expressed full-length and different truncation forms of MED19. HA-MED19-FL, HA-tagged MED19 full-length form; HA-MED19-ΔN, HA-tagged MED19 with the N-terminal region deleted (1–72); HA-MED19-ΔM, HA-tagged MED19 with the middle-region deleted (73–158); HA-MED19-ΔC, HA-tagged MED19 with the C-terminal region deleted (159–244). ( K ) IP experiments were performed using endogenously HA-tagged HA-MED23 and HA-MED19 by HA-magnetic beads. The results demonstrate that MED19 specifically associates with snoRNP proteins.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Protein-Protein interactions, Western Blot, Immunoprecipitation, Mutagenesis, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: Depletion of MED19 attenuates the 2′-O-methylation level of rRNA. ( A ) Mass spectrometry assay of 2′-O-methylation level of adenosine and guanosine in the total RNA. sh19, stable knockdown of MED19 using a retrovirus with shRNA sequence designed against the 3′UTR. Re-OE, re-expression of MED19 using its coding sequence (CDS). Statistical differences were calculated using a two-way unpaired Student’s t -test with experiments repeated three times. *Represents P < .05; **represents P < .01; ***represents P < .001. ns represents no significance. ( B ) Schematic diagram of the RTL-Q assay. RT, reverse transcription primer. R, reverse PCR primer located downstream of the 2′-O-Me site. Fu, forward PCR primer located upstream of the 2′-O-Me site. Fd, forward PCR primer located downstream of the 2′-O-Me site. ( C – E ) Changes of the 2′-O-methylation ratio of selected 2′-O-methylation sites after knockdown of MED19 (sh19) and re-expression (Re-OE) in 293T cells using RTL-Q method. ( F ) Schematic illustration of the Ribometh-seq principle. ( G – I ) Differentially changed 2′-O-methylated sites on rRNAs after MED19 knockdown in the HuH7 cell line. The x -axis represents the positions and corresponding nucleotides of the 2′-O-methylated sites on their respective rRNAs. The y -axis indicates the methylation modification ratio, where a value of 1 signifies 100% modification at that site. ( J ) Western blot of the MED19 and other BoxC/D-snRNP proteins after knockdown of MED19 in 293T cells. ( K, L ) CLIP-qPCR of FBL under MED19 knockdown (sh19) and re-overexpression (Re-OE). The pre-rRNA bound to FBL was measured by real-time qPCR both in 293T and HuH7 cell lines.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Methylation, Mass Spectrometry, Knockdown, shRNA, Sequencing, Expressing, Reverse Transcription, Modification, Western Blot, Over Expression

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: Phase separation of MED19 with FBL. ( A ) IDR profiling of FBL and MED19 proteins. The x -axis represents the amino acid sequence of the protein, while the y -axis represents the scores of disorder propensity calculated by the PONDA software . Scores >50 indicate potential IDR regions. ( B ) Coomassie blue staining of the purified mEGFP-MED19 and mCherry-FBL. ( C ) Droplet formation of mEGFP-MED19 protein (6 μM) under 1% PEG8000 condition. DIC, differential interference contrast microscopy. ( D ) Droplet formation of equimolar mixtures of mEGFP-MED19 and mCherry-FBL (6 μM) under 1% PEG8000 condition. ( E ) FRAP analysis mEGFP-MED19 in transfected HuH7 cell line. The MED19 plaque was bleached and gradually recovered within minutes. ( F ) The bleach and recovery profile of the bleached region.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Sequencing, Software, Staining, Purification, Microscopy, Transfection

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 promotes the IRES-dependent translation. ( A ) Schematic illustration of cap-dependent translation and IRES-dependent translation (cap-independent translation). ( B ) Schematic diagram of the bicistronic reporter system for measuring the IRES element translational activity. ( C ) Schematic diagram of the c-Myc mRNA composition with the IRES element located in the 5′UTR. ( D ) Predicted secondary structure of the c-Myc IRES sequence. ( E ) Measurement of the EMCV-IRES translation activity dynamics by the bicistronic reporter system. Left: overexpression of MED19 at increasing dosages. Right: knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). EMCV: Encephalomyocarditis virus. Statistical differences were calculated using a two-way unpaired Student’s t -test with experiments repeated three times. *Represents P < .05, ****represents P < .0001. ns represents no significance. ( F ) Measurement of the c-Myc-IRES translation activity by the bicistronic reporter system under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). Left: in 293T cells. Right: in HuH7 cells. ( G ) Western blot of the c-Myc and tubulin proteins expression levels under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). ( H ) RT-qPCR analysis of c-Myc mRNA expression levels under knockdown of MED19 (sh19) and re-overexpression of MED19 (Re-OE). Actin was used for normalization. Left: in 293T cells. Right: in HuH7 cells.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Activity Assay, Sequencing, Over Expression, Knockdown, Virus, Western Blot, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Nucleolar MED19 regulates 2′-O-methylation of rRNA in supporting cancer cell growth

doi: 10.1093/nar/gkaf1387

Figure Lengend Snippet: MED19 promotes cancer development and correlates with c-Myc at the protein level. ( A ) Expression profiling of MED19 in 33 types cancer clinical samples based on the TCGA database. The x -axis represents the sample names and quantities, while the y -axis represents the mRNA expression level of MED19. Red color indicates cancer samples, while blue color represents adjacent normal samples. ( B ) Cell proliferation disturbance profiling of various cancer cell lines under MED19 knockout. The x -axis values represent the cell proliferation changes compared to the control cell lines. A value >0 indicates that MED19 knockout promotes cell proliferation, while a value <0 indicates that MED19 knockout suppresses cell proliferation. The corresponding lineages of the cancer cell lines are plotted in the y -axis. The source data are from the DepMap database. ( C, D ) Effects of MED19 knockdown and re-expression on cell proliferation determined by CCK8 assay. ( E ) The mRNA expression correlation of MED19 and c-Myc across 1749 cancer cell lines. The source data are from the DepMap database. ( F ) The protein expression correlation of MED19 and c-Myc across 225 cancer cell lines. The source data are from the DepMap database. ( G ) The mRNA expression correlation of MED19 and c-Myc across 3850 tumor samples. The source data are from the TCGA database. ( H ) The protein expression correlation of MED19 and c-Myc across 203 tumor samples. The source data are from the CPTAC database . ( I, J ) Survival analysis of MED19 in multiple cancer types. LIHC: liver hepatocellular carcinoma. ACC: adrenocortical carcinoma. ( K ) Schematic model illustrating that MED19 enhances IRES-mediated translation of c-Myc and other genes through its role in promoting rRNA 2′-O-methylation in the nucleolus.

Article Snippet: The MED19 stable overexpression lentivirus plasmid pLVX-Puro-MED19 was constructed by inserting the MED19 CDS sequence into the pLVX-Puro plasmid (Clontech, 632164).

Techniques: Expressing, Knock-Out, Control, Knockdown, CCK-8 Assay, Methylation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Lactylation-driven KRT19 promotes non-small cell lung cancer progression by suppressing cellular senescence

doi: 10.1186/s13046-025-03602-5

Figure Lengend Snippet: KRT19 interacts with MYH9 and induces its expression A Schematic representation of experimental setup for identifying KRT19-interacting proteins B Representative SDS-PAGE separation and silver staining of protein lysates from Flag-KRT19-overexpressing A549 cells immunoprecipitated with anti-Flag or control IgG C List of potential KRT19-binding proteins identified by mass spectrometry D-E The interaction of endogenous KRT19 and MYH9 in A549 cells was determined by Co-IP F-I A549 (F-G) and HEK293T cells (H-I) were transfected with MYH9-overexpressing plasmid and transduced with Flag-KRT19 lentivirus (Flag-KRT19 group) or control empty vector (EV group). Representative immunofluorescence images of A549 (F) and HEK293T cells (H), and co-localization analysis of Flag and MYH9 in Flag-KRT19 A549 (G) and HEK293T cells (I) were shown. Scale bar, 10 μm J-K KRT19 and MYH9 protein levels in different groups of A549 cells (J) and PC-9 cells (K) were measured by immunoblot. Data are shown as mean ± S.E.M. and analyzed by Student’s t -test (J-K). * p < 0.05; ** p < 0.01. The experiments were repeated two (A-C) or three times (D-K)

Article Snippet: To identify the proteins interacted with KRT19, total extracts of A549 cells with stable Flag-KRT19 overexpression were immunoprecipitated with Flag tag antibody (66008-4-Ig, Proteintech) or control IgG as previously described, and subjected to SDS-PAGE.

Techniques: Expressing, SDS Page, Silver Staining, Immunoprecipitation, Control, Binding Assay, Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Transduction, Immunofluorescence, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Lactylation-driven KRT19 promotes non-small cell lung cancer progression by suppressing cellular senescence

doi: 10.1186/s13046-025-03602-5

Figure Lengend Snippet: KRT19 facilitates p21 ubiquitination at K16 via MYH9 A PC-9 cells were transfected with MYH9 siRNA (siMYH9) or control siRNA (si-NC). The protein levels of p21, p53 and MYH9 in PC-9 cells (si-NC, siMYH9) treated with CHX (15 µg/mL) for the indicated time were measured by immunoblot (Top), and quantitation of p21 protein levels based on band intensity was shown (bottom) B p21 and MYH9 protein expression in PC-9 cells (si-NC, siMYH9) treated with or without MG132 (20 µM) for 4 h was determined by immunoblot C Co-IP analysis of the interaction between p21 and ubiquitin (Ub) in PC-9 cells (si-NC, siMYH9) treated with MG132 (20 µM) for 4 h D HEK293T cells were co-transfected with Myc-p21, HA-Ub and MYH9-overexpressing (MYH9-OVE) plasmids or control plasmid. Co-IP analysis of the interaction between Myc-p21 and HA-Ub in different groups of HEK293T cells treated with MG132 (20 µM) for 4 h E Control or Flag-KRT19-overexpressing (Flag-KRT19) HEK293T cells were co-transfected with siMYH9 or si-NC, Myc-p21 and HA-Ub plasmids. F-G Prediction of ubiquitination sites of p21 protein using GPS-Uber H-I KRT19-Flag-OVE (H) or MYH9-OVE (I) HEK293T cells were co-transfected with HA-Ub and Myc-p21 (WT, K16R, K75R, K154R). The interaction between Myc-p21 and HA-Ub was determined by Co-IP after treatment with MG132 (20 µM) for 4 h J Sequence comparison around the K16 residue (red) of the p21 homologue in different species K-L PC-9 cells were transduced with shKRT19#2 or transfected with MYH9-OVE plasmid for 48 h. p21, KRT19, MYH9 protein expression levels (K) and SA-β-gal staining (L) of different groups of PC-9 cells were shown. Scale bar, 10 μm. Data are shown as mean ± S.E.M. and analyzed by one-way ANOVA (K-L). * p < 0.05; ** p < 0.01; *** p < 0.001. The experiments (A-E, H-I, K-L) were repeated three times

Article Snippet: To identify the proteins interacted with KRT19, total extracts of A549 cells with stable Flag-KRT19 overexpression were immunoprecipitated with Flag tag antibody (66008-4-Ig, Proteintech) or control IgG as previously described, and subjected to SDS-PAGE.

Techniques: Ubiquitin Proteomics, Transfection, Control, Western Blot, Quantitation Assay, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Sequencing, Comparison, Residue, Transduction, Staining

Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: Effects of HG and C3a on podocyte damage and mitophagy. Normal glucose refers to 5.5 mM glucose, while the mannitol high osmotic control group was subjected to 24.5 mM mannitol + 5.5 mM glucose, and HG represents the intervention group (30 mM glucose). (A) Representative images (left) and quantification (right) of podocyte cytoskeleton, with F-actin (green) stained using phalloidin (n=6). Scale bar, 20 μ m. (B) Protein levels of synaptopodin and podocin in podocytes (n=6). Corresponding histograms are shown on the bottom panel of representative protein bands. (C) ELISA detection of C3a levels in podocytes at different time points (n=6). (D) Protein levels of C3 and C3aR in podocytes (n=6). Corresponding histograms are shown on the right panel of representative protein bands. (E) Protein levels of LC3B I/II, parkin and PINK1 in podocytes (n=8). Corresponding histograms are shown on the bottom panel of representative protein bands. (F) Protein levels of parkin and PINK1 in podocytes (n=5). Corresponding histograms are shown on the right panel of representative protein bands. (G) Representative images and quantification (bottom) of podocyte cytoskeleton induced by different times and concentrations of C3a (10 −7 M for 12, 24 and 48 h; or 10 −8 , 10 −7 and 10 −6 M for 24 h) (n=6). Scale bar, 20 μ m. * P<0.05, ** P<0.01 and *** P<0.001. ns, no statistically significant difference; HG, high glucose.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: C3a downregulates mitophagy levels through the PI3K/AKT/FoxO1 signaling pathway in a HG environment, leading to podocyte damage. (A) Immunofluorescence images of F-actin-stained podocyte cytoskeleton. Scale bar, 20 μ m. Flow cytometric analysis of podocyte apoptosis under different intervention conditions using annexin V-FITC PI. (B) Expression of podocyte functional and mitophagy-related proteins after PINK1 inhibition (n=5). Corresponding histograms are shown on the right panel of representative protein bands. (C) Expression of downstream signaling molecules after direct stimulation of podocytes by C3a (n=6). (D) Expression of downstream signaling molecules after inhibiting C3aR in a HG environment (n=5). Corresponding histograms are shown on the bottom panel of the representative protein bands. (E) Confocal microscopy images capturing fluorescence of immortalized human podocytes transfected with adenovirus GFP-LC3B (green) and Mito-DsRed (red). Scale bar, 20 μ m. The right panel represents the statistical analysis of the number of GFP-LC3B-positive spots per cell and the proportion of LC3B spots on mitochondria (Mito) to total LC3B. Quantification of GFP-LC3B-associated Mito-DsRed staining intensity normalized by GFP-LC3B area (n=5). (F) Effects of PI3K inhibition on downstream pathway proteins and mitophagy proteins under HG conditions with C3a overexpression (n=5). Corresponding histograms are shown on the bottom panel of representative protein bands. (G) Immunofluorescence micrographs demonstrating the nuclear/cytoplasmic distribution of FoxO1 in podocytes, with dual-color staining of phosphoryalted-FoxO1 (red) and FoxO1 (green). Scale bar, 20 μ m. * P<0.05, ** P<0.01 and *** P<0.001. ns, no statistically significant difference; HG, high-glucose.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Immunofluorescence, Staining, Expressing, Functional Assay, Inhibition, Confocal Microscopy, Fluorescence, Transfection, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: C3a/C3aR axis is involved in diabetic kidney injury by regulating podocyte mitophagy in diabetic nephropathy

doi: 10.3892/ijmm.2025.5664

Figure Lengend Snippet: Role and mechanism of C3a/C3aR in a DN model. In a high-glucose environment, the complement component C3 is activated. The C3a/C3aR axis modulates the PI3K-AKT signaling pathway, resulting in an enhanced phosphorylation level of FoxO1, leading to the loss of its transcriptional activity. Consequently, there is inhibition of PINK1/parkin-mediated mitophagy, contributing to podocyte injury and DN progression. DN, diabetic nephropathy; C3aRA, C3aR antagonist.

Article Snippet: Under HG conditions with C3a overexpression, treatment with LY294002 (a PI3K inhibitor; cat. no. HY-10108; MedChemExpress), which was dissolved in DMSO and applied to podocytes at a final concentration of 20 μ M for 24 h prior to protein extraction, reduced PI3K/AKT phosphorylation, restored FoxO1 expression and increased the number of mitochondrial LC3 + puncta (by 2.3-fold, P<0.001) with elevated mitophagic flux ( ).

Techniques: Phospho-proteomics, Activity Assay, Inhibition