Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with Ad-ATF4 or Ad-GFP (50-MOI) for 24 hours and mRNA levels of Sesn2, Trib3 and Ddit4 were determined by qRT-PCR. Expression is reported as fold increase over untransduced neurons from the same culture (n=5; t-test, *p<0.05). B. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50µM) or 6-OHDA (10µM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels was determined by qRT-PCR. Expression is reported as fold increase over vehicle treated neurons from the same culture (n=3-4; t-test, *p<0.05). C. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for the indicated times and SESN2 and DDIT4 protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin or Cyclophilin B (n=3; 2-way-ANOVA *p<0.05). D. Mesencephalic neurons derived from ATF4+/+ and ATF4-/- mice were treated with MPP+ (25μM) or 6-OHDA (5μM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Representative images showing PD neurotoxin induced expression of Sesn2, Trib3 and Ddit4 mRNA fluorescence puncta in wildtype and ATF4-deficient dopaminergic (TH+) neurons (left scale bar=20uM, right scale bar=5uM). Data represent the mean ± SEM number of transcripts per DA neuron (n=3, 2-way-ANOVA, *p<0.05). E. Wildtype and ATF4-deficient mesencephalic neurons were seeded with α-Syn PFFs (5μg/ml) for 9 days and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Data represent the mean ± SEM number of transcripts per DA neuron (n=3, t-test, *p<0.05). F-H. RNA was isolated from N2 animals or atfs-1 LoF animals treated with MPP+ [100uM] or 6-OHDA [100uM] and a-syn or a- syn::atfs-1LoF animals at indicated time points. Heat maps represent fold change in gene expression over time as calculated by RT-qPCR. Values were normalized to act-1 housekeeping mRNA and fold change was calculated based on normalized mRNA levels in untreated or wildtype animals at respective time points (n=3, 2way-ANOVA, *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Control, Derivative Assay, Multiplex Assay, RNAscope, Fluorescence, In Situ Hybridization, Immunostaining, Isolation, Gene Expression

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with lenti-shRNA vectors targeting either Sesn2, Trib3, or Ddit4. After 5 days, neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for 24 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 9 days. The levels of p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p- FOXO3a (S253) and corresponding total protein was assessed by western blot and quantified by densitometry. The ratio of phosphorylated protein/total protein is reported relative to untreated control levels for each shRNA vector (n=5; 2-way-ANOVA, *p<0.05). B. Mesencephalic neuron cultures were transduced with lenti-shRNA and after 5 days treated with MPP+ (25μM) or 6-OHDA (5μM) for 48 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 12 days. The number of TH+ neurons was quantified and is reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by ANOVA (n=4; *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, shRNA, Western Blot, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical or neurons were transduced with recombinant adenoviral vectors (50-MOI) expressing GFP alone or co-expressing GFP and either SESN2, DDIT4 or Trib3 alone or indicated combinations for 24 hours and p-mTOR(S2448)/total mTOR was assessed by western blot and quantified by densitometry (n=3; ANOVA, *p<0.05). B. PUMA+/+ and PUMA-/- cortical neurons were co-transduced with Ad-Trib3/Ad- SESN2/Ad-DDIT4 or Ad-EGFP (3X) for 24 hours and p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p-Foxo3a (S253) levels were determined by western blot and quantified by densitometry (n=3; 2-way ANOVA *p<0.05). C. Mesencephalic neuron cultures were transduced with indicated vectors and dopamine neuron survival was assessed by TH+ counts at 48 hours. Survival is reported relative to untransduced neurons from the same culture (n=3; ANOVA *p<0.05). D. Midbrain neurons were treated with the dual mTOR inhibitor OSI-027 (5μM) for 24 hours and then co-immunostained for tyrosine hydroxylase (TH) and PUMA. Confocal images were acquired and the fluorescence intensity of PUMA was quantified in dopaminergic (TH+) neurons from 3 independent experiments (n=17-23 cells per group; t- test, *p<0.05). E. PUMA+/+ and PUMA-/- cortical neurons were treated with OSI-027 (5μM) and PUMA protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin (n=3; ANOVA *p<0.05). F. Midbrain neurons derived from PUMA+/+ and PUMA-/- mice were treated with OSI-027 for 48 hours and the number of TH+ cells was counted and reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by 2-way-ANOVA (n=4; *p<0.05). G. PUMA+/+ and PUMA-/- midbrain neuron cultures were transduced with Ad-Trib3/Ad-SESN2/Ad-DDIT4 or Ad-EGFP (3X) and DA neuron survival was assessed by TH+ cell counts after 48 hours (n=3; ANOVA *p<0.05).

Article Snippet: Lentiviruses expressing shRNAs specifically targeting Trib3, SESN2 and DDIT4 were purchased from Vectorbuilder (shCTRL VB# VB010000-009mxc, shTrib3 VB# VB230127- 1198jed, shSESN2 VB# VB230127-1195msp, shDDIT4 VB# VB230127-1197gke).

Techniques: Transduction, Recombinant, Expressing, Western Blot, Fluorescence, Control, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: MG53 attenuates intestinal injury in mouse models. a Serum IL-6 and IL-18 levels of MG53-TG and their WT littermates at the indicated time points following DSS treatment. n = 3 for each group. Overexpression of MG53 attenuated DSS-induced IBD symptoms, including disease activity index (DAI; b ), body weight change ( c ), disruption of intestinal structure as demonstrated by H&E staining of the jejunum, ileum, and colon ( d ; scale bar, 100 μm), and shortening of the colon as evidenced by the representative images ( e ) and statistic results of colon length ( f ) in MG53-TG and WT mice. n = 7 for each group. g , h Depletion of MG53 exacerbated DSS-induced IBD symptoms. The DAI ( g ) and body weight change ( h ) of MG53-KO and the WT littermates at the indicated time points after DSS challenge. n = 5 for each group. Immunofluorescence staining of Ki-67 (red) to indicate the TA zone and the statistic results of its length in MG53-TG ( i , n = 20 for each group) or MG53-KO ( j , n = 13 for each group) as compared with their corresponding WT littermates. The nuclei were indicated by DAPI staining (blue); scale bar, 100 μm. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two tailed paired t test ( a – c , f – j ). All data were presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Over Expression, Activity Assay, Disruption, Staining, Immunofluorescence, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: MG53 overexpression promotes secretory lineage commitment. a Immunofluorescence staining of BrdU (red) and EdU (yellow) in the ileum of MG53-TG and their WT littermates subjected to DSS treatment and administered BrdU 48 h and EdU 24 h before euthanasia. n = 10 for each group. Scale bar, 100 μm. b UMAP of scRNA-seq of Epcam + CD45 − intestinal epithelial cells collected from MG53-TG and their WT littermates on Day 7 of DSS treatment, n = 5 for each group. c Proportions of stem, secretory, and absorptive cells in general and each particular cell clusters. d Representative images and statistic results of PAS staining of goblet cells of MG53-TG and WT mice in the colon. n = 10 for each group. Scale bar, 100 μm. e Representative images of intestinal organoids derived from MG53;Lgr5 mice and their Lgr5 littermates at the indicated time points. f Relative mRNA levels of the marker genes in the transcriptome analysis of the crypt organoids. n = 4 for each group. g Representative images of colonic organoids derived from MG53;Lgr5 mice and their Lgr5 littermates at the indicated time points. h Representative FACS profiles and statistic results of Lgr5 + cells in the colonic organoids derived from MG53;Lgr5 and the control Lgr5 mice. n = 6 for each group. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two tailed paired t test ( a , d , f , and h ), and were presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Over Expression, Immunofluorescence, Staining, Derivative Assay, Marker, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: PPAR signaling is enhanced by MG53 overexpression. a Trajectory analysis of Epcam + CD45 − intestinal epithelial cells Using Monocle 2. b KEGG pathway enrichment analysis of the differentially expressed genes between the secretory and absorptive branches. GSEA results ( c ) and the heatmap of differentially expressed genes ( d ) related to fatty acid oxidation and PPAR signaling derived from the bulk RNAseq data of the intestinal organoids from MG53-TG and WT mice. n = 4 for each group. Representative images ( e ) and statistic results of signal intensity of the immunofluorescence staining of MG53 (green) and PPARα (red) determined by Aperio Scanscope scanner and Halo software ( f ), as well as the correlation of the levels of these two proteins ( g ) in the human small intestine of normal subjects ( n = 5) and patients with intestinal inflammation ( n = 8). Nuclei were stained with DAPI (blue); scale bars as indicated. Representative images ( h ) and statistic results of signal intensity of the immunofluorescence staining of MG53 (green) and PPARα (red) determined by Aperio Scanscope scanner and Halo software ( i ), as well as the correlation of the levels of these two proteins ( j ) in the human colon of normal subjects ( n = 4) and patients with intestinal inflammation ( n = 12). Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using Mann–Whitney U test ( f , i ) and Pearson correlation analysis ( g , j ). Data were presented as mean ± s.e.m. * p < 0.05 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Over Expression, Derivative Assay, Immunofluorescence, Staining, Software, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: PPARα mediates the effect of MG53 in promoting asymmetric division of Lgr5 + cells. Representative images ( a ) and statistic results ( b , n = 3 for each group) of asymmetric division of Lgr5 + cells with or without GW6471 (10 μM, 24 h) treatment. Lgr5 + cells were in green; all the cells were visualized by tubulin (red). Scale bar, 10 μm. c FACS analysis of Lgr5 + , Atoh1 + , and Notch1 + cells in the Lgr5 and MG53;Lgr5 organoids treated with GW6471 or vehicle. d Immunofluorescence staining of Ki-67 (red) and PPARα (green) and statistic results of PPARα positive cells ( n = 3 for each group) and the length of TA zone ( n = 18 for each group) in mice treated GW6471 or vehicle. Nuclei were stained with DAPI (blue); scale bar, 100 μm. Representative images and statistic results of PAS staining of goblet cells in the ileum ( e ; n = 10 for each group; TG, MG53-TG; scale bar, 50 μm) and colon ( f ; n = 10 for each group; scale bar, 100 μm). Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using one-way ANOVA with Tukey post hoc test ( b , d , e , and f ) and were presented as mean ± s.e.m. ns not significant and *** p < 0.001 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: Activation of PPARα signaling is required for MG53-induced alleviation of intestinal injury. Body weight change and DAI ( a ) as well as the representative images and statistic results of the intestinal length ( b ) of ISC-MG53-TG and the control MG53 tg-fl mice. n = 7 for each group. Body weight change and DAI ( c ) as well as the representative images and statistic results of the intestinal length ( d ) of the Lgr5 mice with ISC-specific inhibition of MG53 expression via infection with adeno-associated virus with inducible expression of shRNA targeting MG53 (AAV-shMG53) or the control mice infected with control shRNA (AAV-shCON). n = 6 for each group. Body weight change and DAI ( e ) as well as the intestinal length ( f ) of ISC-Ppara-KO mice with or without ISC-specific MG53 overexpression via injecting AAV-MG53 or AAV-CON. n = 8 for each group. Body weight change and DAI ( g ) as well as the intestinal length ( h ) of MG53-KO and control WT mice treated with PPARα agonist fenofibrate or vehicle following DSS treatment. n = 8 for each group. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two-tailed paired t test ( a – e , g ), Mann–Whitney U test ( f , h ), and were presented as mean ± s.e.m. ns not significant, * p < 0.05 and ** p < 0.01 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Activation Assay, Control, Inhibition, Expressing, Infection, Virus, shRNA, Over Expression, Two Tailed Test, MANN-WHITNEY

Journal: Signal Transduction and Targeted Therapy

Article Title: Mitsugumin 53 drives stem cell differentiation easing intestinal injury and inflammation

doi: 10.1038/s41392-025-02268-x

Figure Lengend Snippet: Enhanced PPARα activation by palmitoleic acid contributes to MG53-mediated amelioration of intestinal injury. a Heatmap of free fatty acids (FFA) in MG53-TG and WT intestinal tissues at the indicated time points during DSS treatment. n = 3 for each group. b Changes in the intestinal content of FFA in the MG53-TG and their WT littermates at day 7 after DSS treatment. n = 3 for each group. c Relative change in POA concentrations in the serum of IBD patients in remission ( n = 11) or flare ( n = 26). d Luciferase reporter activity driven by the PPARα-responsive element in the presence of different FFA in HEK293 cells. n = 3 for each group. Body weight change and DAI ( e ) as well as the colon length ( f ) of MG53-TG (TG) and their WT littermates challenged with DSS with or without POA treatment. n = 12 for each group. g Immunofluorescence staining of Ki-67 (red) and statistic results of the length of TA zone of MG53-TG and their WT littermates with or without POA treatment. n = 15 for each group. The nuclei were indicated by DAPI staining (blue); scale bar, 100 μm. h Representative images and statistic results of PAS staining of goblet cells in the colon after POA treatment. n = 10 for each group. Scale bar, 100 μm. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using one-way ANOVA with Tukey post hoc test ( c , e ), two tailed t test ( d , f – h ) and were presented as mean ± s.e.m. ns not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls

Article Snippet: To knockdown MG53 expression in HCT116, cells were infected with lentivirus (Suzhou GenePharma) expressing shRNA specifically targeting MG53 (sequence of shRNA: 5’-GACTGAGTTCCTCATGAAATA-3’), as previously described.

Techniques: Activation Assay, Luciferase, Activity Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: iScience

Article Title: ANKRD17 induces pro-survival signaling pathways that enhance cellular invasion and migration during hepatocellular carcinoma tumorigenesis

doi: 10.1016/j.isci.2025.112463

Figure Lengend Snippet: ANKRD17 enhances tumorigenic and pro-metastatic characteristics during HCC disease progression (A) ANKRD17 -overexpression in a CTNNB1 S33Y and shp53 predisposed background ( n = 3), resulted in larger liver to body weight percentage ratios and higher tumor burden in Fah /SB11 mouse injected livers at around 356-day PHI compared to control cohorts (GFP only, n = 2; GFP in a CTNNB1 S33Y and shp53 predisposed background, n = 3). Scale bars, 0.5cm. (B) Quantitative PCR (qPCR) results showing upregulated ANKRD17 and CTNNB1 S33Y expression levels in liver samples from experimental and control injected Fah /SB11 mice. (C) qPCR results show significant upregulation of EMT markers in liver tumors of experimental Fah /SB11 injected mice compared with control cohorts. Arbitrary value relative to Actb mRNA levels expressed as mean ± SD. GFP, GFP only control; GFP/CP, GFP , CTNNB1 S33Y , and shp53 control; ANK/CP, ANKRD17 , CTNNB1 S33Y , and shp53 experimental; N, peripheral normal liver; T, tumor nodule. p , unpaired Student’s t test: ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05.

Article Snippet: A set of short hairpin RNA (shRNA) constructs, including four gene-specific shRNA expression vectors targeting ANKRD17 and a scrambled negative control, was purchased from Origene.

Techniques: Biomarker Discovery, Over Expression, Injection, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: iScience

Article Title: ANKRD17 induces pro-survival signaling pathways that enhance cellular invasion and migration during hepatocellular carcinoma tumorigenesis

doi: 10.1016/j.isci.2025.112463

Figure Lengend Snippet: ANKRD17 -overexpression enhanced cellular migration and invasion abilities in human liver cancer cell lines ANKRD17 -overexpressing MHCC97L (top) and C3A (bottom) cells both exhibited significant increases in cellular motility and invasion abilities compared with control orange fluorescent protein ( OFP )-transfected cells. Representative graphs of stained migrated or invaded cell intensity expressed as mean ± SD; P , unpaired Student’s t test: ∗∗∗, p < 0.001; ∗∗, p < 0.01.

Article Snippet: A set of short hairpin RNA (shRNA) constructs, including four gene-specific shRNA expression vectors targeting ANKRD17 and a scrambled negative control, was purchased from Origene.

Techniques: Over Expression, Migration, Control, Transfection, Staining

Journal: iScience

Article Title: ANKRD17 induces pro-survival signaling pathways that enhance cellular invasion and migration during hepatocellular carcinoma tumorigenesis

doi: 10.1016/j.isci.2025.112463

Figure Lengend Snippet: ANKRD17 -overexpression induces the dysregulation of several important signaling pathways (A) Liver tumors from experimental mice demonstrating dysregulated HIPPO signaling pathway with significantly higher expression of Yap1 than control cohorts. Quantitative PCR results of Yap1 relative to Actb levels expressed as mean ± SD. GFP, GFP only control; GFP/CP, GFP , CTNNB1 S33Y , and shp53 control; ANK/CP, ANKRD17 , CTNNB1 S33Y , and shp53 experimental; N, peripheral normal liver; T, tumor nodule. (B) Representative Western blot demonstrating dysregulated AKT/PTEN signaling pathway in tumors (T) and their peripheral normal (N) tissues. Semi-quantitative analyses of AKT activity (pAKT/total AKT) relative to ACTB levels expressed as mean ± SD. (C) Representative Western blot demonstrating dysregulated AKT/PTEN signaling pathway in ANKRD17 -overexpressing MHCC97L cells. Semi-quantitative analyses of AKT activity (pAKT/total AKT) relative to ACTB levels expressed as mean ± SD. (D) Representative Western blot demonstrating dysregulated STAT3 signaling pathway in ANKRD17 -overexpressing MHCC97L cells. Control, OFP -overexpressing cells. Semi-quantitative analyses of STAT3 activity (pSTAT3/total STAT3) relative to ACTB levels expressed as mean ± SD. (E) Liver tumors from experimental mice demonstrated significantly higher expression of Ddr1 than control cohorts at both the transcriptional (top) and translational (bottom) levels. Western blot for DDR1 as shown in (B). Semi-quantitative analyses of Ddr1 and DDR1, relative to Actb and ACTB levels, respectively, expressed as mean ± SD. (F) Induced expression of DDR1 at both the transcriptional (top) and translational (bottom) levels in ANKRD17 -overexpressing MHCC97L cells. Western blot for DDR1 as shown in (C). Semi-quantitative analyses of DDR1 expression at both the transcriptional (top) and translational (bottom) levels relative to ACTB and ACTB levels, respectively, expressed as mean ± SD. P , unpaired Student’s t test: ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05.

Article Snippet: A set of short hairpin RNA (shRNA) constructs, including four gene-specific shRNA expression vectors targeting ANKRD17 and a scrambled negative control, was purchased from Origene.

Techniques: Over Expression, Protein-Protein interactions, Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: iScience

Article Title: ANKRD17 induces pro-survival signaling pathways that enhance cellular invasion and migration during hepatocellular carcinoma tumorigenesis

doi: 10.1016/j.isci.2025.112463

Figure Lengend Snippet: Inhibition of DDR1 suppressed ANKRD17 -induced cellular migration ability in HCC cancer cell line (A) Cell survival assay of DDR1 inhibitor 7rh at different concentrations to determine the sub-cytotoxic conditions for subsequent experiments. (B) Cell motility assessed by transwell migration assay using DDR1 inhibitor 7rh at 0, 0.25, 0.5, and 0.75 μM in ANKRD17 -overexpressing MHCC97L cells. (C) Semi-quantitative analyses of the cellular migration results from (B) expressed as mean ± SD. (D) Representative Western blot demonstrates reduction in DDR1 levels but no alterations in AKT phosphorylation at Ser473 were detected with DDR1 inhibitor 7rh at all tested concentrations. Control, OFP -overexpressing cells. Semi-quantitative analyses of DDR1 and AKT activity (pAKT/total AKT) relative to ACTB levels expressed as mean ± SD. P , unpaired Student’s t test : ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05.

Article Snippet: A set of short hairpin RNA (shRNA) constructs, including four gene-specific shRNA expression vectors targeting ANKRD17 and a scrambled negative control, was purchased from Origene.

Techniques: Inhibition, Migration, Clonogenic Cell Survival Assay, Transwell Migration Assay, Western Blot, Phospho-proteomics, Control, Activity Assay