Journal: eLife

Article Title: Replication Study: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs

doi: 10.7554/eLife.56651

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , TaqMan probe PARD3 , Thermo Fisher Scientific , Hs00969077_m1 , .

Techniques: Recombinant, Plasmid Preparation, Sequencing, Software, Real-time Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Crumbs3 Is Essential for Proper Epithelial Development and Viability

doi: 10.1128/MCB.00999-13

Figure Lengend Snippet: Evaluation of epithelial polarity in Crumbs3 knockout lungs. (A) Frozen sections from E18.5 lungs were stained for Pals1, ZO1, and occludin (×600; zoom, 1.5). (B) Western blot of Crb complex proteins Pals1 and Patj in E18.5 lungs. β-Actin was used as a loading control. Samples were run on nonadjacent lanes of the same gel. (C) Frozen sections from E18.5 +/+ and −/− mouse lungs were stained for AQP5 (alveolar type I cells) (×600; zoom, 2), acetylated tubulin (cilia) (zoom, 1.5), Muc1 (apical surface) (zoom, 1.5), ezrin and phospho-ERM (cytoskeleton) (zoom, 3), E-cadherin and β-catenin (adherens junction/basolateral surface) (zoom, 1.5). (D) Frozen sections from E18.5 +/+ and −/− mouse lungs were stained for Par3 (×600) (zoom, 3) and aPKC (apical polarity complex) (zoom, 1.5).

Article Snippet: The primary antibodies used were acetylated tubulin (Sigma, St. Louis, MO; catalog no. T7451), aPKC (R&D, Minneapolis, MN; catalog no. AF4465), Aquaporin 1 (Aqp1) (Santa Cruz, Santa Cruz, CA; catalog no. SC-25287), Aqp2 (Santa Cruz; catalog no. SC-9882), Aqp5 (Abcam, Cambridge, MA; catalog no. AB78486), α-tubulin (Sigma; catalog no. T6199), β-actin (Sigma; catalog no. A2228), β-catenin (BD Bioscience, San Jose, CA; catalog no. 610154), CC10 (Santa Cruz; catalog no. SC-9772), chemokine C-C motif ligand 2 (CCL2) (R&D; catalog no. AF479-NA), claudin-4 (Santa Cruz; catalog no. SC-17664), cleaved caspase-3 (Cell Signaling, Danvers, MA; catalog no. 9661), Crumbs3a (Custom [ 24 ]), E-cadherin (R&D; catalog no. AF748), ERM (Cell Signaling; catalog no. 3142), ezrin (Sigma; catalog no. E8897), GFP-agarose (MBL, Woburn, MA; catalog no. D153-8), hemagglutinin (HA) (Santa Cruz; catalog no. SC-805), Hes1 (MBL; catalog no. D134-3), Ki67 (Abcam; catalog no. AB16667), mucin 1 (Muc1) (Novus Biologicals, Littleton, CO; catalog no. NB120-15481), Nkx2.1 (Epitomics, Burlingame, CA; catalog no. 2044-1), occludin (Invitrogen; catalog no. 71-1500), Pals1 for immunoblotting (IB) (Santa Cruz; catalog no. SC-365411), Pals1 for immunohistochemistry (IHC) (Custom [ 25 ]), Par3 (Novus Biologicals; catalog no. NBP1-88861), Patj (Abcam; catalog no. {"type":"entrez-nucleotide","attrs":{"text":"AB102113","term_id":"33353085","term_text":"AB102113"}} AB102113 ), phospho-ERM (Cell Signaling; catalog no. 3141), phospho-Yap1 (Cell Signaling; catalog no. 4911), surfactant protein B (SPB) (EMD Millipore, Billerica, MA; catalog no. 07-614), surfactant protein C (SPC) (Seven Hills Bioreagents, Cincinnati, OH; catalog no. WRAB-9337), Yap1 (Epitomics; catalog no. EP1674Y), and ZO1 (Invitrogen; catalog no. 33-9100).

Techniques: Knock-Out, Staining, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Crumbs3 Is Essential for Proper Epithelial Development and Viability

doi: 10.1128/MCB.00999-13

Figure Lengend Snippet: Evaluation of epithelial polarity and signaling pathways in Crumbs3 knockout kidneys. (A) Frozen sections from E17.5 kidneys were stained for Pals1 (green), ZO1 (red), and E-cadherin (blue) (×400 magnification). (B) Western blot of Crb complex protein Pals1 in E18.5 kidneys. β-Actin was used as a loading control. Samples were run on nonadjacent lanes of the same gel. (C) Frozen sections of kidneys from E17.5 to E18.5 mice were stained for ezrin (cytoskeleton), occludin and ZO1 (tight junction), E-cadherin (adherens junction/basolateral surface), claudin-4 (tight junction), Par3, and aPKC (apical polarity complex) (×600; zoom, 1.5). (D) Western blot of p-ERM and ERM protein levels among +/+ and −/− kidneys at E18.5. β-Actin and α-tubulin are shown as loading controls. Samples were run on nonadjacent lanes of the same gel. (E and F) Western blots of p-Yap1 (Hippo pathway activation) and Hes1 (Notch pathway activation) among +/+ and −/− kidneys at E18.5. α-Tubulin is shown as the loading control. Samples were run on nonadjacent lanes of the same gel.

Article Snippet: The primary antibodies used were acetylated tubulin (Sigma, St. Louis, MO; catalog no. T7451), aPKC (R&D, Minneapolis, MN; catalog no. AF4465), Aquaporin 1 (Aqp1) (Santa Cruz, Santa Cruz, CA; catalog no. SC-25287), Aqp2 (Santa Cruz; catalog no. SC-9882), Aqp5 (Abcam, Cambridge, MA; catalog no. AB78486), α-tubulin (Sigma; catalog no. T6199), β-actin (Sigma; catalog no. A2228), β-catenin (BD Bioscience, San Jose, CA; catalog no. 610154), CC10 (Santa Cruz; catalog no. SC-9772), chemokine C-C motif ligand 2 (CCL2) (R&D; catalog no. AF479-NA), claudin-4 (Santa Cruz; catalog no. SC-17664), cleaved caspase-3 (Cell Signaling, Danvers, MA; catalog no. 9661), Crumbs3a (Custom [ 24 ]), E-cadherin (R&D; catalog no. AF748), ERM (Cell Signaling; catalog no. 3142), ezrin (Sigma; catalog no. E8897), GFP-agarose (MBL, Woburn, MA; catalog no. D153-8), hemagglutinin (HA) (Santa Cruz; catalog no. SC-805), Hes1 (MBL; catalog no. D134-3), Ki67 (Abcam; catalog no. AB16667), mucin 1 (Muc1) (Novus Biologicals, Littleton, CO; catalog no. NB120-15481), Nkx2.1 (Epitomics, Burlingame, CA; catalog no. 2044-1), occludin (Invitrogen; catalog no. 71-1500), Pals1 for immunoblotting (IB) (Santa Cruz; catalog no. SC-365411), Pals1 for immunohistochemistry (IHC) (Custom [ 25 ]), Par3 (Novus Biologicals; catalog no. NBP1-88861), Patj (Abcam; catalog no. {"type":"entrez-nucleotide","attrs":{"text":"AB102113","term_id":"33353085","term_text":"AB102113"}} AB102113 ), phospho-ERM (Cell Signaling; catalog no. 3141), phospho-Yap1 (Cell Signaling; catalog no. 4911), surfactant protein B (SPB) (EMD Millipore, Billerica, MA; catalog no. 07-614), surfactant protein C (SPC) (Seven Hills Bioreagents, Cincinnati, OH; catalog no. WRAB-9337), Yap1 (Epitomics; catalog no. EP1674Y), and ZO1 (Invitrogen; catalog no. 33-9100).

Techniques: Protein-Protein interactions, Knock-Out, Staining, Western Blot, Control, Activation Assay

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A–B ) Pard3 associates with fusome as observed in E11.5-E13.5; Gonad stained for Pard3 (red), EMA (green) and DAPI (blue) ( A, A' ) and after rosette formation at E13.5 ( B, B' ). ( C-C' ) Ring canals (RACGAP, yellow) localize within the Pard3+ (red) apical domain in germ cells (GCNA, green). ( D ) A lineage-labeled E13.5 cyst (YFP, green); channels below show enrichment of Pard3 (red) with enriched fusome (EMA, gray). Graph: Quantification of Pard3 stained area colocalizing with large- ≥ 20 μm 2 and small <20 μm 2 fusome within lineage labeled cyst (Student’s t-test, N=13; *** p <0.001). ( E ) Xbp1 (green) enrichment in EMA (red) granule of E11.5 PGC. ( F–H ) scRNA-seq of E10.5-P5 gonad. UMAP of re-clustered germ cells at various stages ( F ), UMAP ( G ) UMI Feature Plot; NC = nurse cells. ( H ): UMAP with clusters labeled in ascending order of meiotic development. pre-meiotic (Pre-M), leptotene (Lp), zygotene (Zy), pachytene (Pa), diplotene (Dp), dictyate (Dc). ( I - I′ ) Bar plots: ( I ) Xbp1, Xbp1-target expression plots. ( I' ) Genes orthologous to fusome components. Scale bars: 10 μm ( A–C, E ), 20 μm ( D ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Labeling, Expressing

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) E13.5 ovaries stained for GCNA (blue), EMA (green) and PARD3 (red). ( B ) Lineage labeled E13.5 ovary stained for YFP (green), GCNA (blue), PARD3 (red), and EMA (gray). ( C ) Zoomed images of E13.5 gonad stained for RACGAP (green) and Pard3 (red). Scale bar = 10 μm ( A and B ), 20 μm ( C ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Labeling

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) Dnmt3a and EMA levels at E12.5. Dnmt3a levels are reduced in wild-type (WT) compared to Dazl -/- germ cells. Graph - Dnmt3a fluorescent levels within germ cells as normalized with somatic cells in WT versus Dazl mutant gonad. (N=10 tissues; **p<0.05). ( B ) Ring canals are smaller and defective in E13.5 Dazl -/- cysts compared to WT. (N=44; **p<0.05). ( C ) scRNA-seq of E11.5 and E12.5 WT and Dazl -/- gonad germ cells. UMAP. Germ cell clusters overlapped at E11.5 and segregated at E12 of WT and Dazl -/- . ( C’ ) Xbp1, Xbp1 targets, and fusome orthologs in WT vs Dazl -/- germ cells. ( D ) Validation of IRE1-Xbp1 assay: Ovarian cells visualized by fluorescent microscopy showing GCNA labeled bigger germ cells with higher Xbp1 fluorescence than smaller somatic cells ( D’-D”’ ) IRE1-Xbp1 assay comparing SSEA1+germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl -/- germ cells at E12.5. ( D’ ; 6 experiments: ~32 mice, ≥5 mice, and ≥20 ovaries per experiment, D”-D”’ ; 3 experiments: ~40 mice, ≥5 mice, and ≥25 ovaries per experiments, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) ( E - E″ ) Proteasome activity comparing SSEA1+germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl -/- germ cells at E12.5. (N=3 biological assays with ~35–60 E11.5 ovary per assay and ~25–28 E12.5 ovaries were used per assay. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) ( F ) Golgi fragmentation in E12.5 Dazl -/- germ cells stained with golgi marker Gs28 (red), EMA (green) and DAPI (blue). Graph: germ cell percent with fragmented Golgi in wild-type versus Dazl mutant mouse gonad (Student’s t-test: N=16, ***p<0.005) ( F’ ) Failure of E13.5 Dazl -/- germ cells to form EMA (gray) rosettes or enrich Pard3 (red). ( G ) Dazl -/- effects on fusome, Golgi and Pard3. ( H ) Proposed function of fusome-mediated regulation of ERAD-UPR proteostasis. Scale bar: 10 μm (except zoomed in 2 μm).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Mutagenesis, Biomarker Discovery, Microscopy, Labeling, Fluorescence, Activity Assay, Staining, Marker

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) E17.5 ovary stained for WGA, GCNA, and TEX14. ( A’ ) E17.5 ovary stained for GCNA, RACGAP, and PARD3; Graph: Fusome volume and Pard3 Stained area versus ring canal number N=65 (Fusome volume; N=51 (Pard3); ANOVA, ***p<0.005, ****p<0.0001). ( B-B′ ) E18.5 ovary shows WGA-Fusome/PARD3 enrichment in large medullary oocytes vs smaller nurse cells; line: medulla/cortex boundary; dotted circle: large medullary oocytes; white dotted area: small nurse cells. The area marked as a white dotted rectangle is shown as a zoomed inset (white arrow). Black arrow in inset: WGA stained fusome; Graph compares fusome volume and Pard3 stained area versus Germ cell nucleus diameter (N=54 (WGA), N=37(PARD3); Student’s paired t-test, *p<0.05, ****p<0.001). ( C ) Single cell lineage labeled E18.5 ovary stained for YFP, DAPI, WGA, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary-Fusome volume difference according to germ cell nucleus size (N=10; ****p<0.0001). ( D ) Single cell lineage labeled E18.5 ovary stained for YFP, PARD3, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary- difference in PARD3 stained area according to germ cell nucleus size (N=10; ***p<0.005). ( G-G′ ) Dazl +/- E18.5 ovary- Fusome (WGA) and Pard3 enrichment failure in medullary oocytes (GCNA). Graph: Fusome volume in potential oocytes, i.e., bigger germ cells with nucleus diameter d ≥12 μm in wild-type versus Dazl +/- mutant F-F″ . Organelle enrichment analysis: E18.5 (WT- F - F’ , and Dazl +/- ovary F” ) stained for WGA, mitochondrial marker ATP5a and GCNA ( F and F” ). ( F’ ) - Electron microscopy (EM) image of Golgi-rich Fusome (arrow) surrounded by mitochondria. ( G-G′ ) Endoplasmic reticulum (ER)-mitochondria association in E18.5 WT ovary: G-EM image of ER tubules (arrow) wrapping mitochondria and G’ - E18.5 WT ovary- GCNA, ER, and Mitochondria tracker staining. Scale bars: 20 μm ( A-E , G-G′ , F,F” , G′ ), 5 μm ( B-, B′ - right most inset panel), 0.5 μm (EM images F′ , G ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Single Cell, Labeling, Mutagenesis, Marker, Electron Microscopy

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Pard3 is down-regulated in BC. (A) Representative IHC images showing Pard3 expression levels in BC tissues and paired paracancerous tissues. Magnification, × 200. Scale bars, 100 μm. Pard3 expression was observed in 67.1 % (43/64, IHC Score ≥3) of adjacent non-malignant tissues and 37.5 % (24/64, IHC Score≥ 3) of BC tissues. Normal, paracancerous tissue; Tumor, BC tissue, ***, P < 0.001. (B) Western blot showing that Pard3 levels were lower in BC tissues than in paracancerous tissues. Normal, paracancerous tissue; Tumor, BC tissue, ***, P < 0.001. (C) The expression of Pard3 in different subtypes of breast cancer. **, P < 0.01.

Article Snippet: Lentiviral vectors carrying Pard3 shRNA, Pard3 cDNA, as well as the USP28 pc-DNA, His-USP28 pc-DNA, His-USP28(DD) pc-DNA, Snail1 pc-DNA, HA-Snail1 pc-DNA, HA-Snail1 mutant plasmids, pGL3-USP28 promoter and pcDNA-p65 were purchased from Shanghai GeneChem Co., Ltd. (China).

Techniques: Expressing, Western Blot

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Clinicopathological analysis of Pard3 expression in BC.

Article Snippet: Lentiviral vectors carrying Pard3 shRNA, Pard3 cDNA, as well as the USP28 pc-DNA, His-USP28 pc-DNA, His-USP28(DD) pc-DNA, Snail1 pc-DNA, HA-Snail1 pc-DNA, HA-Snail1 mutant plasmids, pGL3-USP28 promoter and pcDNA-p65 were purchased from Shanghai GeneChem Co., Ltd. (China).

Techniques: Expressing

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Pard3 knockdown promotes BC progression in vitro and in vivo. (A) Western blot showing Pard3 levels in human BC cell lines and MCF10A cells. (B) Western blot and RT-PCR confirming Pard3 knockdown and overexpression in MDA-MB-231 and MCF7 cells. **, P < 0.01. ***, P < 0.001. (C) Scratch assay (Magnification, × 40) and transwell assay (Magnification, × 200) showing that Pard3 knockdown promoted BC cell migration and invasion, and Pard3 overexpression suppressed these processes. (D) Representative images of H&E-stained lung metastases and numbers of metastatic nodules. *, P < 0.05.

Article Snippet: Lentiviral vectors carrying Pard3 shRNA, Pard3 cDNA, as well as the USP28 pc-DNA, His-USP28 pc-DNA, His-USP28(DD) pc-DNA, Snail1 pc-DNA, HA-Snail1 pc-DNA, HA-Snail1 mutant plasmids, pGL3-USP28 promoter and pcDNA-p65 were purchased from Shanghai GeneChem Co., Ltd. (China).

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Wound Healing Assay, Transwell Assay, Migration, Staining

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Pard3 knockdown stabilizes the Snail1 protein by suppressing its ubiquitination. (A) Western blot data showing that Pard3 knockdown increased Snail1 and MMP9 protein levels. In contrast, Pard3 overexpression decreased the expression of Snail1 and MMP9 proteins. (B) Western blot showing that Pard3 regulated the expression of MMP9 via Snail1. (C) RT-PCR assay showing that neither Pard3 knockdown or overexpression altered the levels of Snail1 mRNA. ns, no significance. (D) The levels of Snail1 expression after CHX (100 μg/mL) treatment were detected by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001. (E) After transfection, lysates from BC cells were used for anti-Snail1 immunoprecipitation. Subsequently, western blots were performed with ubiquitin-specific antibodies. (F) Scratch assay data (Magnification, × 40) and transwell assay data (Magnification, × 200) showing that Pard3 regulated BC cell migration and invasion via Snail1. *P < 0.05, **P < 0.01.

Article Snippet: Lentiviral vectors carrying Pard3 shRNA, Pard3 cDNA, as well as the USP28 pc-DNA, His-USP28 pc-DNA, His-USP28(DD) pc-DNA, Snail1 pc-DNA, HA-Snail1 pc-DNA, HA-Snail1 mutant plasmids, pGL3-USP28 promoter and pcDNA-p65 were purchased from Shanghai GeneChem Co., Ltd. (China).

Techniques: Knockdown, Ubiquitin Proteomics, Western Blot, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Wound Healing Assay, Transwell Assay, Migration

Journal: Heliyon

Article Title: Inhibition of Pard3 promotes breast cancer metastasis via the USP28 mediated deubiquitination of Snail1

doi: 10.1016/j.heliyon.2023.e22599

Figure Lengend Snippet: Pard3 knockdown deubiquitinates Snail1 via the up-regulation of USP28. (A) Western blot assays were performed to measure the protein levels of USP28 and USP10, after Pard3 silencing or ever-expression. (B–C) Western blot assays and RT-PCR assays were performed to analyze the regulatory relationships among Pard3, USP28, and Snail1. (D) Western blot assays were performed to measure USP28, p -IκBα, total-IκBα, and p65 levels in Pard3-silenced or -overexpressing cells. (E) Western blot assays showing that PDTC inhibited Pard3 knockdown-induced NFκB pathway activation and USP28 up-regulation. In contrast, BetA rescued the NFκB pathway activation and USP28 expression that was inhibited due to Pard3 overexpression. (F) RT-PCR results showing that the activation of the NFκB pathway increases the mRNA levels of USP28. (G) Consensus sequence of the p-65 binding site, and schematic illustration of the potential p65 binding site on the USP28 promoter. ChIP assays with the p65 antibody and IgG were conducted to detect p65 binding on the USP28 promoter in BC cells. Then, luciferase activity was detected in HEK293T cells co-transfected with the wild-type (USP28-WT) promoter or mutant promoter (USP28-mut) containing the pGL3 reporter vector and pcDNA-p65 or the vector. After 48 h, the firefly luciferase activity was measured, and the Renilla luciferase activity was used as the standard control. *P < 0.05, **P < 0.01, ***P < 0.001. Ns, no significance.

Article Snippet: Lentiviral vectors carrying Pard3 shRNA, Pard3 cDNA, as well as the USP28 pc-DNA, His-USP28 pc-DNA, His-USP28(DD) pc-DNA, Snail1 pc-DNA, HA-Snail1 pc-DNA, HA-Snail1 mutant plasmids, pGL3-USP28 promoter and pcDNA-p65 were purchased from Shanghai GeneChem Co., Ltd. (China).

Techniques: Knockdown, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Over Expression, Sequencing, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Association of LKB1 with SIK1−3 and PARD3 and their roles in GC. ( A ) The gene association network of LKB1 , SIK1 − 3 , and PARD3 was constructed using the GeneMANIA data portal. ( B ) Relationship between SIK2 expression and the survival curve of GC patients. ( C ) Relationship between SIK3 expression and the survival curve of GC patients. ( D ) The relationship between PARD3 and the survival curve of GC patients. ( E ) IHC staining of SIK2, SIK3, and PARD3 in paracancerous and GC tissues of GC patients (400×). ( F , G ) Differences in gene and protein expression levels of SIK2 , SIK3 , and PARD3 in paracancerous and GC tissues of GC patients ( n = 3). ( H , I ) Differential protein expression of LKB1, SIKs, and PARD3 in four GC cell lines and GES-1. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the normal group ( F ) or GES-1 group ( I ).

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Construct, Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin inhibits the expression of LKB1, SIK2/3 and PARD3, but not SIK1, in GC cells. ( A ) Molecular docking conformation of Erianin and LKB1 (PDB ID: 2WTK). Arrows indicated the locations of LKB1 and Erianin. The black box magnifies the 3D view of the docking cavity of Erianin and LKB1. ( B , C ) Distribution of amino acid residues around the binding pocket of Erianin and LKB1 and the chemical bonds formed. The oxygen atom on Erianin formed hydrogen bonds with Ser148, Ser199, Ser154, Thr147, Met150 and Thr98. The oxygen atom on Erianin formed a weak hydrogen bond with Ser199, and the carbon atom formed a weak hydrogen bond with Asp157. The carbon atom on the benzene ring of Erianin formed hydrophobic interactions with Leu202 and Ile75. The carbon atom on the C=C connecting the two benzene rings in Erianin formed a hydrophobic interaction with Phe415. ( D , E ) Erianin inhibited the fluorescence intensity of LKB1 in MGC803 and MKN45 cells (scale bar = 50 μm). ( F ) Effects of treating MGC803 cells with an Erianin concentration gradient on the expression of LKB1 , SIK1 − 3 , and PARD3 genes. ( G , H ) Effects of treating MGC803 cells with an Erianin concentration gradient on the expression of LKB1, SIK1−3, and PARD3 proteins. ( I ) Effects of treating MKN45 cells with an Erianin concentration gradient on the expression of LKB1 , SIK1 − 3 , and PARD3 genes. ( J , K ) Effects of treating MKN45 cells with an Erianin concentration gradient on the protein expression of LKB1, SIK1−3, and PARD3. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the control group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Binding Assay, Fluorescence, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Silencing the expression of LKB1, SIKs, and PARD3 and their interaction. ( A , B ) Effects of silencing LKB1 expression on the expression of SIK1−3 and PARD3 in GC cells. ( C , D ) Effects of silencing SIK2 expression on the expression of LKB1 and PARD3 in GC cells. ( E , F ) Effects of silencing SIK3 expression on the expression of LKB1 and PARD3 in GC cells. ( G , H ) Effects of silencing PARD3 expression on the expression of LKB1 and SIK1−3 in GC cells. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the NC group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin inhibits tumor growth and the expression of LKB1, SIK2, SIK3, and PARD3 in the CDX mouse model. ( A ) Schematic diagram of the CDX model construction and drug administration treatment strategy. ( B ) Axillary tumor growth of mice in the five groups. ( C ) The size of isolated tumors from mice in the five groups. ( D ) Body weight changes of mice in the five groups during the administration period. ( E ) Changes in tumor volume during the administration of mice in the five groups. ( F ) Tumor mass in the five groups of mice. ( G ) Liver, spleen, and kidney index of mice in each group. ( H ) Differences in liver MDA and SOD levels of mice in the five groups. ( I ) Representative photomicrographs of pathological staining of tumors, liver, spleen, and kidney tissues of mice in the five groups (200×). ( J ) IHC staining results of LKB1, SIK2, SIK3, and PARD3 in mouse tumor tissues in the five groups (400×). ( K ) Changes in protein expression of LKB1, SIK2, SIK3, and PARD3 in tumor tissues of mice in the five groups. ( L ) Changes in the expression of EMT marker proteins in tumor tissues of mice in the five groups. ** p < 0.01 indicates statistical difference compared to the vehicle control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the 5-FU 50 mg/kg group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Isolation, Staining, Immunohistochemistry, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: 5-FU inhibits the expression of LKB1, SIK2, SIK3, and PARD3 in GC cells. ( A ) CCK8 assay showing the inhibitory effect of different concentrations of 5-FU on MGC803 cells. ( B ) CCK8 detected the inhibitory effect of different concentrations of 5-FU on MKN45 cells. ( C , D ) 5-FU inhibited the protein expression of LKB1, SIK2, SIK3 and PARD3 in MGC803 cells in a dose-dependent manner. ( E , F ) 5-FU inhibited the proteins expression of LKB1, SIK2, SIK3 and PARD3 in MKN45 cells in a dose-dependent manner. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the control group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, CCK-8 Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: 5−FU combined with Erianin inhibits the expression of LKB1, SIK2/3, and PARD3 in GC cells and the migration and invasion ability of cells. ( A ) CCK8 examined the inhibitory effect of Erianin 25 nM or Erianin 50 nM combined with different concentrations of 5-FU on the cell viability of MGC803 or MKN45 cells. ( B ) Inhibitory effects of Erianin at 25 nM combined with 2.5 μg/mL 5-FU, and Erianin at 50 nM combined with 2.5 μg/mL 5-FU on the cell viability of MGC803 or MKN45. ( C ) Erianin at 25 nM combined with 5-FU at 2.5 μg/mL inhibited the fluorescent expression of LKB1 in MGC803 cells (scale bar = 50 μm). ( D ) 50 nM Erianin combined with 2.5 μg/mL 5-FU inhibited the fluorescent expression of LKB1 in MKN45 cells (scale bar = 50 μm). ( E ) Erianin combined with 5-FU inhibited the protein expression of LKB1, SIK1−3, and PARD3 in MGC803 cells. ( F ) Erianin combined with 5-FU inhibited the protein expression of LKB1, SIK1−3, and PARD3 in MKN45 cells. ( G ) Erianin combined with 5-FU inhibited the migration and invasion of MGC803 cells (scale bar = 100 μm). ( H ) Erianin combined with 5-FU inhibited the migration and invasion of MKN45 cells (scale bar =100 μm). ( I ) Erianin combined with 5-FU inhibited wound healing of MGC803 cells (scale bar = 100 μm). ( J ) Erianin combined with 5-FU inhibited wound healing of MKN45 cells (scale bar = 200 μm). * p < 0.05, ** p <0.01 indicates statistical difference compared to the control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the Erianin+5FU group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin combined with 5-FU inhibits tumor growth in CDX mouse model. ( A ) Schematic diagram of the CDX mouse model construction and the administration strategy for Erianin combined with 5-FU. ( B ) Growth of subcutaneous tumors in mice. ( C ) Photographs of subcutaneous tumors in mice. ( D ) Body weight change curve of mice in each group during the administration period. ( E ) Tumor volume change curves of mice in the four groups during the administration period. ( F ) Tumor weight of mice in the four groups. ( G ) Liver, spleen, and kidney indices of mice in the four groups. ( H ) Differences in liver MDA and SOD contents among the four groups of mice. ( I ) H&E staining of tumors, liver, spleen, and kidneys of mice in the four groups (200×). ( J ) IHC staining of LKB1, SIK2, SIK3, and PARD3 in tumor tissues from mice in the four groups (400×). ( K ) Protein expression of LKB1, SIK2, SIK3, and PARD3 in tumor tissues from mice in the four groups. ( L ) Expression of EMT marker proteins in tumor tissues from mice in the four groups. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the Vehicle control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the Erianin+5-FU group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Staining, Immunohistochemistry, Expressing, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Gene sequences (human).

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Sequencing