target protein dna complexes  (Thermo Fisher)

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: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

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: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

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: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

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: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

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: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

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