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
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50 , ![Pharmacological inhibition of the RAF1 pathway inhibits HCMV infection. ( A, B ) Regorafenib (Reg) or ( C, D ) sorafenib (Sor) treated ( A, C ) MRC5 or ( B, D ) HFF fibroblasts were treated with the indicated concentration of drugs or DMSO control at the time of infection. ( A–D ) Cells were infected at an MOI of 0.1 with AD169-GFP for 5 days, then fixed. GFP-positive cells were counted per area of the well and plotted against drug concentration [avg ± standard error of the mean (SEM), n = 3]. IC50 values were calculated. The dotted line indicates the average maximal GFP in untreated cells. ( E–H ) Fibroblasts were infected at an MOI of 3.0 in the presence of DMSO or 2.17 µM regorafenib or sorafenib added at the time of viral adsorption. UN is an untreated control. Viral titers from ( E ) MRC5 or ( F ) HFF cells were assessed by TCID50 (avg ± SEM, n = 3) 120 hpi. The dotted line indicates the limit of detection. In ( G ) MRC5 or ( H ) HFF cells, at 24 h post-infection (hpi), phosphorylation of MEK1/2 and ERK1/2 (arrows indicate doublet) was assessed by western blot ( n = 1).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5854/pmc11915854/pmc11915854__jvi.01866-24.f003.jpg)
![Inhibition of the RAF1 pathway negatively impacts viral DNA and protein accumulation. ( A–C ) MRC5 fibroblasts were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with AD169 at an MOI of 3.0 for the indicated amount of time. ( A ) Viral DNA was quantified by RT-qPCR of the viral gene, IE1 [avg ± standard deviation (SD), n = 12]. ( B ) Samples in ( A ) were compared by area under the curve (AUC) to show the significance between treatments over the course of infection (avg ± SD, n = 12). Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( C ) Viral protein expression levels at 24, 72, and 96 h post-infection (hpi) were assessed by western blot ( n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( D–G ) Quantification of western blots in ( C ) (avg ± standard error of the mean, n = 2) normalized to GAPDH and DMSO, 24 hpi sample for viral proteins ( D ) IE1, ( E ) IE2, ( F ) UL44, and ( G ) pp28.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5854/pmc11915854/pmc11915854__jvi.01866-24.f005.jpg)
![RAF1 shRNA-mediated knockdown inhibits HCMV infection. ( A–H ) Indicated shRNA guides were delivered via lentiviral delivery in HFF fibroblast cells. ( A–D ) RAF1 shRNA (shRAF1) were assessed for ( A ) knockdown efficiency in uninfected fibroblasts by RT-qPCR [avg ± standard error of the mean (SEM), n = 3 or 4], ( B ) protein expression was assessed via western blot ( n = 1), and ( C–D ) viral titer was measured by TCID50 (avg ± SEM, n = 4) at ( C ) an MOI of 3.0 at 120 h post-infection, or ( D ) an MOI of 0.01 at 10 days post-infection. ( E–F ) ARAF shRNA (shARAF) were assessed for ( E ) RNA levels via RT-qPCR and ( F ) viral titers via the GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( G–H ) BRAF shRNA (shBRAF) were assessed for ( G ) RNA levels via RT-qPCR and ( H ) viral titers via GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( A, E, G ) RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and shRNA empty vector (shEV) control in all RT-qPCR experiments. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5854/pmc11915854/pmc11915854__jvi.01866-24.f006.jpg)
![CRISPR-mediated knockout of RAF1 attenuates cell-to-cell spread of HCMV. ( A ) Knockout (KO) scores for CRISPR RAF1 KO cell lines generated. ( B–D ) MRC5 parental (par) and RAF1 polyclonal CRISPR knockout (poly KO) cells were infected at an MOI of 3.0 for ( B ) 96 h, and protein expression was assessed by western blot during mock ( M ) or HCMV ( H ) infection ( n = 1). UL26 is a control for successful viral infection, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein loading control. ( C ) At 72, 96, and 120 h post infection (hpi), viral titers were assessed by TCID50 [avg ± standard error of the mean (SEM), n = 2]. Significance is based on Student’s t -test at 120 hpi. ( D ) MRC5 (avg ± SEM, n = 72) and ( E ) HFF (avg ± SEM, n = 48), parental, and RAF1 polyclonal CRISPR knockout cell lines were infected with AD169-GFP at an MOI of 0.05 for 10 days. Infected cells per area of the well were counted via GFP expression, and significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5854/pmc11915854/pmc11915854__jvi.01866-24.f007.jpg)
![Pharmacological inhibition of RAF1 attenuates HCMV TB40/E cell-to-cell spread in fibroblasts and epithelial cells. ( A, B ) MRC5 fibroblasts or ( C ) ARPE19 epithelial cells were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with ( A ) AD169-GFP or ( B, C ) TB40/E-mCherry at an MOI of 0.01. Viral spread was calculated by cells positive for ( A ) GFP or ( B ) mCherry on Day 12 in MRC5 cells or ( C ) mCherry on Days 15 and 30 in ARPE19 cells [avg ± standard error of the mean (SEM), n = 48]. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( D, E ) Mock-infected ARPE19 cells were treated with indicated concentrations of ( D ) regorafenib or ( E ) sorafenib at t = 0. Cells were fixed on Day 1, 5, or 11. Hoechst-stained nuclei were counted and plotted against time (avg ± SEM, n = 3).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5854/pmc11915854/pmc11915854__jvi.01866-24.f008.jpg)
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