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: 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