Journal: iScience

Article Title: Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

doi: 10.1016/j.isci.2023.108142

Figure Lengend Snippet: ALDH1A1 promoting tumor progress through the RA pathway (A and B) The protein expression of ALDH1A1 was increased with the treatment of gemcitabine and cisplatin in T24 and UMUC3 cells. The final concentration of gemcitabine and cisplatin were 0, 0.1 μg/mL, 0.3 μg/mL and 0.5 μg/mL respectively. (C) The cell proliferation and growth measured by the MTT assay in T24 cells. (D) The migration and cell invasive ability was reduced in T24 cell line by knocking down ALDH1A1. Scale bar = 200 μm. (E) Two stable cells (NC-shRNA and ALDH1A1-shRNA) were analyzed by sphere-forming assay. Scale bar = 100 μm. (F) Two stable cells (NC-shRNA and ALDH1A1-shRNA) were seeded into the 6-well plates to subject to colony formation analysis. (G and H) Western blotting analysis of ALDH1A1, RXRα, p -AKT, and β-catenin in T24 cells with different treatments. (I and J) shALDH1A1 cells were cultured with ATRA gained a stronger ability of proliferation, migration and invasion. Scale bar = 200 μm. (K and L) p -AKT and β-catenin were upregulated by added ATRA in shALDH1A1 cells by Western blotting. For cell experiments, each experiment was performed at least three times. Data are represented as mean ± SEM, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-sided Student’s t test.

Article Snippet: Mouse monoclonal anti-β-catenin , Cell Signaling , Cat #:2698; RRID:AB_1030945.

Techniques: Expressing, Concentration Assay, MTT Assay, Migration, shRNA, Western Blot, Cell Culture

Journal: iScience

Article Title: Modification of lysine-260 2-hydroxyisobutyrylation destabilizes ALDH1A1 expression to regulate bladder cancer progression

doi: 10.1016/j.isci.2023.108142

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-β-catenin , Cell Signaling , Cat #:2698; RRID:AB_1030945.

Techniques: Virus, Recombinant, Activity Assay, SYBR Green Assay, Software

Journal: bioRxiv

Article Title: Systems genetics analysis of human body fat distribution genes identifies Wnt signaling and mitochondrial activity in adipocytes

doi: 10.1101/2023.09.06.556534

Figure Lengend Snippet: Thirteen prioritized key driver genes may affect fat storage in adipocytes through the Wnt signaling pathway. (A) The Wnt signaling pathway consists of canonical β-catenin signaling and non-canonical pathways. Key driver genes interact with Wnt pathways in other cell types (gray and yellow). (B) Key driver gene expression in adipose tissue correlations with WHR adjBMI in STARNET. Pearson correlations are shown by color, p-values adjusted using FDR correction shown with * (*** = adj.P < 0.001, * = adj.P < 0.05). (C-F) Four selected key driver genes (red) regulate both WHR adjBMI downstream genes (yellow) and Wnt signaling downstream genes (blue, GO term “Wnt signaling pathway”) in GTEx and STARNET. (C) ANAPC2 in the GTEX Subcutaneous Female network, (D) PSME3 in the STARNET Visceral Female network, (E) RSPO1 in the GTEx Visceral Female network, and (F) TYRO3 in the GTEx Subcutaneous Male network.

Article Snippet: We labeled active and total β-catenin and β-actin control bands using primary antibodies (Cell Signaling Technologies, Danvers, Massachusetts, USA; Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Cat#8814, dilution 1:500; β-Catenin (15B8) Mouse mAb Cat#37447, dilution 1:1000).

Techniques: Expressing

Journal: bioRxiv

Article Title: Systems genetics analysis of human body fat distribution genes identifies Wnt signaling and mitochondrial activity in adipocytes

doi: 10.1101/2023.09.06.556534

Figure Lengend Snippet: RSPO1 and PSME3 activate canonical Wnt signaling while inhibiting the Ca 2+ non-canonical Wnt pathway. (A) Wnt transcriptional activity measured by luminescence of luciferase reporter (n = 3-6). (B) Representative images and (C) Quantification of active (non-phosphorylated) and total β-catenin by immunoblotting (n = 12). (D) Gene expression of AXIN2 measured by qPCR (n = 3). (E) Ratio of active (phosphorylated): total GSK3β measured by ELISA (n = 3). (F) Gene expression of IL6 measured by qPCR (n = 3). (G) Ratio of active (phosphorylated): total CAMK2A measured by ELISA (n = 2). (H) Ratio of active (phosphorylated): total JNK measured by ELISA (n = 3). All plots show mean ± standard error of the mean. Differences between groups determined using 1-way ANOVA by gene (Gene of Interest vs GFP controls), post-hoc tests were performed using pooled t-test with Dunnett’s adjustment. Adjusted p-values shown with * (*** = adj.P < 0.001, ** = adj.P < 0.01, * = adj.P < 0.05).

Article Snippet: We labeled active and total β-catenin and β-actin control bands using primary antibodies (Cell Signaling Technologies, Danvers, Massachusetts, USA; Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb Cat#8814, dilution 1:500; β-Catenin (15B8) Mouse mAb Cat#37447, dilution 1:1000).

Techniques: Activity Assay, Luciferase, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Secretory MPP3 reinforce myeloid differentiation trajectory and amplify myeloid cell production

doi: 10.1084/jem.20230088

Figure Lengend Snippet: Gal3 secretion contributes to myeloid amplification by leukemic MPP3. (A) HSPC and myeloid progenitor population size in age-matched Ctrl Gal3 +/+ and knockout Gal3 −/− mice (two independent experiments). (B) Changes in HSPC and myeloid progenitor population size in Ctrl and Gal3 −/− mice 2 d (D) after injection of anti-Ly6G depleting antibodies in one independent experiment. (C) Survival curve of BA tTA mice with Gal3 deletion. Results from Ctrl, Gal3 +/− , Gal3 −/− , BA tTA , BA tTA :Gal3 +/− , and BA tTA :Gal3 −/− mice from five independent cohorts are shown; induction, doxycycline withdrawal. Significance was assessed by a Mantel-Cox test. (D) Changes in population size for HSPCs, myeloid progenitors, and mature myeloid cells (My, Mac-1 + /Gr-1 + ) in 11- to 13-wk-old age-matched Ctrl, Gal3 −/− , BA tTA , and BA tTA :Gal3 −/− mice. Results are expressed as a percentage of Lin − /Sca-1 + /c-Kit + (LSK), Lin − /Sca-1 − /c-Kit + (MP), and BM cells, and are from five independent cohorts. (E) Quantification of nuclear β-catenin (βcat) positive HSC, MPP3, and MPP4 in a subset of age-matched Ctrl, Gal3 −/− , BA tTA , and BA tTA :Gal3 −/− mice shown in D. (F) Changes in the frequency of nuclearβcat-positive BA tTA :Gal3 −/− HSCs upon 18 h of in vitro treatment with the GSK3β inhibitor CHIR 99021 (CHIR, 30 µM) (two independent experiments). Data are means ± SD, and significance was assessed by a two-tailed unpaired Student’s t test except when indicated.

Article Snippet: Cells were incubated overnight at 4°C with a mouse monoclonal anti-KDEL (ab12223; Abcam) or a rabbit anti-mouse β-catenin (9582S; Cell Signaling) primary antibody, washed three times with PBS, and incubated for 1 h at RT with a goat anti-mouse IgG A488 (A11029; Invitrogen) or a donkey anti-rabbit-A555 (A31572; Invitrogen) secondary antibody.

Techniques: Amplification, Knock-Out, Injection, In Vitro, Two Tailed Test

Journal: The Prostate

Article Title: Inverse agonism at the Na/K-ATPase receptor reverses EMT in prostate cancer cells

doi: 10.1002/pros.24144

Figure Lengend Snippet: Antibodies and Reagents

Article Snippet: Antibody Property Supplier Catalogue number dilution α1 NKA antibody (α6F) Mouse monoclonal Developmental Studies Hybridoma Bank of University of Iowa (Iowa) a6f 1:1000 PhosphoSrc (Tyr419) antibody Rabbit polyclonal Invitrogen 44–660G 1:1000 c-Src B-12 antibody Mouse monoclonal Santacruz Biotechnology sc-8056 1:1000 Rat α1 NKA antibody Rabbit polyclonal Dr. T.A.Pressley (Texas Tech University, TX) Not applicable 1:1000 Anti-phosphotyrosine antibody, clone 4G10 Mouse monoclonal EMD Millipore 05–321 1:1000 c-Myc antibody Santacruz Biotechnology 1:1000 Anti-tubulin antibody Mouse monoclonal SIGMA T5168 1:2000 Cyclin D1 antibody Rabbit monoclonal Cell Signaling Technology 2978S 1:1000 Cyclin E1 antibody Rabbit monoclonal Cell Signaling Technology 20808S 1:1000 p53 antibody Mouse pantropic Calbiochem OP43 1:1000 p21 antibody Rabbit polyclonal Santacruz Biotechnology sc-397 1:1000 Phospho MAPK antibody Rabbit Cell Signaling Technology 9101 1:1000 ERK1/2 antibody Rabbit polyclonal Santacruz Biotechnology sc-94 1:1000 Phospho-FAK (Tyr576/7) antibody Rabbit polyclonal Cell Signaling Technology 3281S 1:1000 FAK antibody Rabbit polyclonal Cell Signaling Technology 3285S 1:1000 Phospho-FAK (Tyr 397) antibody Rabbit polyclonal Cell Signaling Technology 3283S 1:1000 Anti-Na+/K+-ATPase β1 antibody clone 464.8 Mouse monoclonal EMD Millipore 05–382 1:1000 E-cadherin (24E10) antibody Rabbit monoclonal Cell Signaling Technology 3195S 1:1000 Anti β-catenin antibody Mouse monoclonal BD Bioscience 610153 1:1000 ZO-1 antibody Rabbit polyclonal Thermo-Fisher Scientific 61–7300 1:1000 ZO-2 antibody Rabbit polyclonal Thermo-Fisher Scientific 38–9100 1:1000 Occludin antibody (OC-3F10) Mouse monoclonal Thermo-Fisher Scientific 33–1500 1:1000 SNAIL (C15D3) antibody Rabbit monoclonal Cell Signaling Technology 3879S 1:1000 TCF8/ZEB1 antibody Rabbit monoclonal Cell Signaling Technology 3396S 1:1000 Vimentin (D21H3) antibody Rabbit monoclonal Cell Signaling Technology 5741S 1:1000 MMP-2 antibody Rabbit monoclonal Cell Signaling Technology 87809S 1:1000 MMP-9 antibody Rabbit monoclonal Cell Signaling Technology 13667S 1:1000 PCNA antibody Mouse monoclonal Santacruz Biotechnology sc-56 1:2000 Lamin B antibody Goat polyclonal Santacruz Biotechnology sc-6216 1:1000 β actin antibody Mouse monoclonal Santacruz Biotechnology sc-47778 1:1000 SLUG (C19G7) antibody Rabbit monoclonal Cell Signaling Technology 9585S 1:1000 N cadherin (D4R1H) antibody Rabbit monoclonal Cell Signaling Technology 13116S 1:1000 Open in a separate window Antibodies and Reagents Rat α1 NKA- specific antibody (NASE) was a kind gift from Dr. T. A. Pressley (Texas Tech University, TX) All reagents were obtained from Sigma-Aldrich except FAK inhibitor (Cat. No. 324877; Millipore).

Techniques:

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: H. pylori infection increased the nuclear accumulation and transcriptional activity of YAP and β-catenin. (a) Western blots of total YAP and β-catenin expression in AGS cells following H. pylori ATCC43504 or 7.13 infection. (b) After H. pylori infection, cytoplasmic and nuclear fractions of AGS cells were separated. Western blots showing YAP and β-catenin expression. (c) Immunofluorescence staining showing the cellular localization of YAP and β-catenin in AGS cells infected with H. pylori at the indicated times. (d, e) AGS cells were infected with H. pylori at the indicated times. Relative luciferase activities of TOPFlash reporter (d) and 8×GITTC reporter (e) showing the transcriptional activation mediated by β-catenin and YAP, respectively. ***, P < 0.001; **, P < 0.01; *, P < 0.05. (f, g) Immunohistochemical staining showing the expression of β-catenin (f) and YAP (g) in the gastric mucosa of INS-GAS mice following infection with the H. pylori PMSS1 strain for 4 months (scale bars, 25 μm). *, P < 0.05.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Infection, Activity Assay, Western Blot, Expressing, Immunofluorescence, Staining, Luciferase, Activation Assay, Immunohistochemical staining

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: Combined YAP and β-catenin silencing synergistically inhibited cell proliferation and expansion induced by H. pylori . (a, b) After knockdown of YAP and β-catenin alone or in combination, AGS cells were infected with H. pylori strain 7.13 (a) or ATCC43504 (b). Then, the CCK8 assay showed cell proliferation at different time points. *** , P < 0.001; ** , P < 0.01; * , P < 0.05. (c, d) AGS cells were transfected with YAP and β-catenin siRNA alone or a combination of YAP/β-catenin and then cocultured with H. pylori strain ATCC43504 or 7.13. An EdU cell proliferation assay was performed. C: Representative images (scale bars, 10 μm); D: the ratio of EdU-positive cells. *** , P < 0.001; * , P < 0.05. (e) Representative images showing spheroids derived from MKN45 cells with individual or combined knockdown of YAP and β-catenin following infection with H. pylori . Scare bars. (f) Quantification of spheroid size and number. *** , P < 0.001; ** , P < 0.01.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Infection, CCK-8 Assay, Transfection, Proliferation Assay, Derivative Assay

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: Common transcriptomic profiling of YAP and β-catenin in gastric cancer cells. (a) After transfection with Flag-YAP or HA-β-catenin plasmid, Western blots showing the expression of YAP and β-catenin, respectively. (b) Transcriptomic analysis using RNA-seq of AGS cells overexpressing YAP or β-catenin was performed in YAP-overexpressing cells. Volcano plot showing the DEGs in YAP-overexpressing or β-catenin-overexpressing cells compared with the control group. (c) Venn diagram showing the overlapping downstream genes of YAP and β-catenin. (d) KEGG pathway enrichment analysis for overlapping target genes. (e) Heatmap showing the significantly upregulated genes that were enriched in the cell cycle, apoptosis, MAPK and TNF signaling pathways. (f) AGS cells were transfected with Flag-YAP and β-catenin plasmids either alone or in combination treatment. RT‒PCR analysis showing the mRNA levels of CDX2, LGR5 and RUVBL1. *** , P < 0.001; ** , P < 0.01; * , P < 0.05. (G) RT‒PCR analysis showing the mRNA levels of MCM3, CUL1 and AXIN2. ** , P < 0.01; * , P < 0.05.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, RNA Sequencing Assay

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: YAP is required for H. pylori -induced β-catenin activity. (a) Following transient transfection with YAP siRNA and infection with H. pylori , cytoplasmic and nuclear fractions of AGS cells were prepared. Then, Western blotting was used to assess YAP and β-catenin expression. (b) Immunofluorescence staining for YAP and β-catenin cellular localization in AGS cells infected with the H. pylori PMSS1 strain alone or in combination with YAP siRNA (scale bars, 25 μm). (c) Immunoprecipitation assay for endogenous interaction between YAP and β-catenin. (d) Following H. pylori infection, immunoprecipitation analysis was performed to assess the YAP interaction with β-catenin. (e-g) After knockdown of individual or combined YAP and β-catenin with siRnas, AGS cells were infected with the H. pylori PMSS1 strain. RT‒PCR analysis showing the mRNA levels of CDX2 (e), LGR5 (f) and RUVBL1 (g). *** , P < 0.001; ** , P < 0.01; * , P < 0.05; NS, not significant.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Activity Assay, Transfection, Infection, Western Blot, Expressing, Immunofluorescence, Staining, Immunoprecipitation

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: YAP and β-catenin inhibitors ameliorated H. pylori infection-induced gastric pathology in mouse models. (a) Experimental protocol for panels. Mice were infected with H. pylori PMSS1 strains for 1 month, followed by intraperitoneal injection with 500 μg/kg Super-TDU for 9 weeks or 25 mg/kg KYA1797K for 7 weeks. (b) H&E staining of representative histological features of gastric mucosa of C57BL/6 mice in different groups (magnification 100×, scale bars: 25 μm). (c, d) the histopathological features of gastric mucosa for all mice were analyzed, including inflammation (c) and epithelial defects (d). (e) RT-PCR analysis showing the mRNA levels of inflammatory cytokines in gastric tissues for the indicated groups of mice, including IL-1β and IL-8. *, P < 0.05 ; NS, not significant . (f, g) Immunohistochemistry staining showing Ki67 expression from the indicated groups of mice (magnification 200×, scale bars: 25 μm). (f) Representative images. (g) the ratio of Ki67-positive cells. **, P < 0.01 ; *, P < 0.05, P < 0.05.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Infection, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Expressing

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: YAP and β-catenin inhibitors reduced DNA damage in the gastric mucosa of mice infected with H. pylori . (a) RT‒PCR analysis of RUVBL1 mRNA levels in stomach tissues from the indicated groups of mice. *, P < 0.05 . (b-c) Western blot analysis for γH2A× in stomach tissues from infected mice after treatment with Super-TDU (b) or KYA1797K (c). (d-e) Representative immunohistochemistry staining (magnification 200×, scale bars: 50 μm) (d) and quantification analysis (e) of γH2A× in stomach tissues from the indicated groups of mice. ***, P < 0.001 ; **, P < 0.01.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Infection, Western Blot, Immunohistochemistry, Staining

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: Elevated YAP was positively correlated with β-catenin expression in gastric cancer. (a, b) Representative immunohistochemical staining of YAP and β-catenin (b) in human gastric carcinoma and adjacent normal tissues. (Magnification 100× and 400×, Scale bars: 25 μm) (c, d) Immunohistochemistry staining scores for YAP (c) and β-catenin (d) ( n = 48). ***, P < 0.001 . (e) Kaplan‒Meier survival analysis for the low expression and high expression of YAP or β-catenin. (f) Spearman’s correlation between IHC staining scores of YAP and β-catenin in human gastric cancer tissues. (g) the expression of YAP and β-catenin in patients with stomach cancer from the GEPIA database. *, P < 0.05 . (h) the overall survival analysis for low and high expression of YAP or β-catenin based on the GEPIA database.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Gut Microbes

Article Title: YAP and β-catenin cooperate to drive H. pylori -induced gastric tumorigenesis

doi: 10.1080/19490976.2023.2192501

Figure Lengend Snippet: Working models of the crosstalk between the YAP and β-catenin pathways in H. pylori -induced gastric tumorigenesis. H. pylori infection invades the gastric epithelium and induces nuclear accumulation and transcriptional activation of YAP and β-catenin. Mechanistically, YAP interacts with β-catenin and promotes its nuclear activation. As a result, their common target genes, including CDX2, LGR5 and RUVBL1, are activated, which contributes to cell proliferation and expansion, ultimately leading to gastric carcinogenesis.

Article Snippet: Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology (Beverly, MA, USA); anti-CagA (sc -28,368) were from Santa Cruz (Dallas, TX, USA), anti-β-actin (#20536–1-AP) from proteintech (Wuhan, China).

Techniques: Infection, Activation Assay

Journal: Breast Cancer Research : BCR

Article Title: The WAVE3/β-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer

doi: 10.1186/s13058-023-01634-3

Figure Lengend Snippet: Loss of WAVE3 expression or phosphorylation in combination with chemotherapy induce β-catenin degradation. A & B Representative Western blots of protein lysates from parental and CIS-R ( A ) or DOX-R ( B ) MDA-MB-231 cells that were treated with or without cisplatin (30 µM) for 24 h, and subjected to immunoblotting with antibodies against β-Catenin. β-Actin was used as loading control. C – F Representative Western blots of protein lysates from MDA-MB-231 ( C & D ) or 4T1 cells ( E & F ) and their derivatives that were treated with or without cisplatin ( C & E ) or doxorubicin ( D & F ) for 24 h, and were subjected to immunoblotting with antibodies against β-Catenin. β-Actin was used as loading control. G Representative confocal microscopic images of sections of tumors derived from mice injected with parental (CTRL), cisplatin resistance (CIS-R) or WAVE3-deficient CIS-R (CIS-R-W3KO) MDA-MB-231, and stained for β-catenin (upper panels, green) or WAVE3 (lower panels, green). Cell nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. H and I Representative Western blots of nuclear ( N ) or cytoplasmic (C) fraction of protein lysates from parental and CIS-R ( H ) or DOX-R ( I ) MDA-MB-231 cells that were subjected to immunoblotting with antibodies against β-catenin or WAVE3. J & K Representative Western blots of protein lysates from parental MDA-MB-231 cells and their derivatives that were treated with or without cisplatin or doxorubicin for 24 h, and were subjected to immunoblotting with antibodies against β-catenin. The nuclear fraction is analyzed in ( J ), while the cytoplasmic fraction is analyzed in ( K ). α-Tubulin and Lamin B1 were used as loading controls for the nuclear and the cytoplasmic fraction, respectively. Data shown are representative of 3 replicates. The numbers under each WB band represent the fold change in signal intensity with respect to its respective control band in each panel after normalization to the loading control signal

Article Snippet: Rabbit anti-WAVE3 (1:1000) and mouse anti- β-catenin (1:1000) were from Cell Signaling Technologies (Danvers, MA).

Techniques: Expressing, Western Blot, Derivative Assay, Injection, Staining

Journal: Breast Cancer Research : BCR

Article Title: The WAVE3/β-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer

doi: 10.1186/s13058-023-01634-3

Figure Lengend Snippet: Inhibition of the proteasome-induced protein degradation stabilizes β-catenin in chemotherapy-treated WAVE3-deficient cells. A – D Representative Western blots of protein lysates from WAVE3-deficient MDA-MB-231 (MDA-MB-231-W3KO) ( A ), WAVE3-deficient 231 cells overexpressing phospho-mutant WAVE3 (231-W3KO-W3Y4) ( B ), WAVE3-deficient 4T1 (4T1-W3KO) or WAVE3-deficient and ( C ) or WAVE3-deficient 4T1 cells overexpressing phospho-mutant WAVE3 (4T1-W3KO-W3Y4) cells ( D ) that were treated for 24 h with 0.1% DMSO, 10 µM cisplatin (Cis), 1 µM GM6001 (GM), or both GM6001 and cisplatin (GM + Cis), and were subjected to immunoblotting with antibodies against β-Catenin. β-Actin was used as loading control. E & F Representative Western blots of nuclear ( E ) or cytoplasmic ( F ) fraction of protein lysates from MDA-MB-231-W3KO cells that were treated for 24 h with 0.1% DMSO, 10 µM cisplatin (Cis), 1 µM GM6001 (GM), or both GM6001 and cisplatin (GM + Cis), were subjected to immunoblotting with antibodies against β-Catenin. α-Tubulin and Lamin B1 were used as loading controls for the nuclear and the cytoplasmic fraction, respectively. The numbers under each WB band represent the fold change in signal intensity with respect to its respective control band in each panel after normalization to the loading control signal. Data shown are representative of 3 replicates

Article Snippet: Rabbit anti-WAVE3 (1:1000) and mouse anti- β-catenin (1:1000) were from Cell Signaling Technologies (Danvers, MA).

Techniques: Inhibition, Western Blot, Mutagenesis

Journal: Breast Cancer Research : BCR

Article Title: The WAVE3/β-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer

doi: 10.1186/s13058-023-01634-3

Figure Lengend Snippet: Both β-catenin and WAVE3 expression levels correlate with the aggressiveness of breast cancer and with poor outcome in BC patients. A Representative Western blots of protein lysates from the indicated breast cancer cell lines that were subjected to immunoblotting with antibodies against WAVE3 (upper panel) or β-catenin (middle panel). β-Actin (lower panel) was used as loading control. Data shown are representative of 3 replicates. B , C Breast Cancer Kaplan–Meier plotter (KM, http://kmplot.com/analysis/ ) correlating survival of triple negative breast cancer patients with expression levels of WASF3/WAVE3 ( B ) and CTNNB1/β-catenin ( C ). High WAVE3 and β-catenin expression levels correlate with poor survival probability in BC patients. D Interrogation of the TCGA breast cancer PanCancer Atlas cohort ( n = 803 BC patients) showed a significant ( p < 2.2e-16) positive correlation between WAVE3 and β-catenin mRNA expression levels

Article Snippet: Rabbit anti-WAVE3 (1:1000) and mouse anti- β-catenin (1:1000) were from Cell Signaling Technologies (Danvers, MA).

Techniques: Expressing, Western Blot