Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Representative immunofluorescence analysis of Acta2 (α-SMA), Col1, and Col3 (A). Analysis of fluorescence intensity for Acta2, Col1, and Col3 (B). Hepatic mRNA levels of Pdgfrb, Col1a1, and Acta2 (C). Immunoblot analysis of hepatic Acta2 and Pdgfrb (D). Representative immunofluorescence analysis of hepatic AFP, CK-19, Arg-1, Cd133, and Cd44 (E). Analysis of fluorescence intensity of hepatic AFP, CK-19, Argl, Cd133, and Cd44 (F). Data are expressed as mean ± SEM (n = 4 to 6 each group). #p < 0.05 and ##p < 0.01 for KO vs. WT. Immunofluorescence image analyses were performed using a fluorescence microscope (ZEISS, x200 magnification).

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: Immunofluorescence, Fluorescence, Western Blot, Microscopy

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Differentially expressed genes (DEGs) from the RNA-seq analysis of WT and Sens3-KO livers were presented by the volcano plot (A). A selected list of DEGs were presented in the table (B). Top 10 biological processes were over-represented in the significantly upregulated (C) and downregulated (D) genes. Hepatic Cd44 and Cd133 mRNA analysis by real-time qPCR (E). Immunoblot analysis of cancer signaling-related proteins in the liver of WT and Sesn3 KO mice (F). Co-IP analysis of Sesn3 and Gli2 interaction in HEK 293T cells (G). Data are expressed as mean ± SEM (n=4 for the immunoblot analysis, n=10 for ELISA, n=6 for qPCR analysis, respectively). #p < 0.05 and ##p < 0.01 for KO vs. WT.

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: RNA Sequencing, Western Blot, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Representative immunofluorescent images of Sesn3 and Gli2 after vector, Flag-Sesn3, and HA-Gli2 plasmid DNAs were transfected to Huh7 cells in the absence or presence of 100 nM SAG. Sesn3 and Gli2 were detected using anti-Sesn3 or anti-HA antibody followed by Alexa 594 and Alexa 488 secondary antibody, respectively (A, B). Vector or Flag-Sesn3 plus HA-Gli2 were transfected to Huh7 cells in the presence of 100 nM SAG. Sesn3, Gli2, and CD44 were detected using anti-Flag, anti-HA, and anti-CD44 antibody followed by Alexa 594, Alex 488, and Cy5 secondary antibody, respectively (C). Fluorescence images were captured using a Zeiss fluorescence microscope at x640 magnification. Real-time PCR analysis of endogenous GLI2 and CD44 mRNAs in vector or Sesn3 transfected Huh7 cells treated with vehicle or SAG (100 nM) for 24 hours (D, E). Data are expressed as mean ± SEM (n = 3). ###p < 0.001 for vehicle vs. SAG for vector or Sesn3 transfection, and ***p < 0.001 for vector vs. Sesn3 transfection after the SAG treatment.

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: Plasmid Preparation, Transfection, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2

doi: 10.3390/cells10061303

Figure Lengend Snippet: Immunoprofiling of the KAIMRC2 cell line. A strong panel of fibroblasts, epithelial, and stem cell biomarkers was used to characterize the cells by flow cytometry. Strongly positive CD47 and CD49c hints toward this cell line’s epithelial lineage, whereas strongly positive CD44, E-cadherin and ALDH-1A1 indicate CSCs potential of the KAIMRC2 cell line.

Article Snippet: To isolate population of human breast cancer stem cells we utilized R&D Systems MagCellect™ Human CD24 − CD44 + Breast Cancer Stem Cells Isolation Kit (Biotechne ® , Minneapolis, MN, USA).

Techniques: Flow Cytometry, Marker

Journal: Cells

Article Title: Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation

doi: 10.3390/cells13110912

Figure Lengend Snippet: Enumeration of TISC frequency in various murine passages following dietary salt modification. The frequency of CD44+CD24−TISCs cells in the isolated tumor cells among RS (grey) and HS (red) diet cohorts was enumerated by flow cytometry. ( A , B ) Representative flow cytometry plot of TISCs from passage 1 and passage 4 of Py230-C57Bl/6J HS diet cohort. ( C , D ) Changes in TISC frequency with each passage in Py230-C57Bl/6J ( C ) and 4T1-BALB/cJ ( D ) murine tumor models. ( E – N ) The mRNA expression of TISC markers Cadherin 1 ( E , J ), Snail2 ( F , K ), Aldh1A1 ( G , L ), Sox2 ( H , M ), and ITGA6 ( I , N ) in four passages of Py230-C57Bl/6J and 4T1-BALB/cJ tumor models (respectively). Data analyzed by one-way ANOVA for multiple comparisons and presented as mean ± SEM; n = 8 (biological replicates) per cohort; (*) p -value < 0.05.

Article Snippet: The TISCs were isolated from murine tumors by CD24 − CD44 + breast tumor stem cells using immunomagnetic beads as per the manufacturer’s specifications and reagents (catalog#MAGH111, R&D Systems, Minneapolis, MN, USA).

Techniques: Modification, Isolation, Flow Cytometry, Expressing

Journal: Cells

Article Title: Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation

doi: 10.3390/cells13110912

Figure Lengend Snippet: TGFβ signaling induced the expression of immune-inhibitory CD80 on TISCs. ( A ) Representative flow cytometry histogram of TGFβR2 expression in passages 1 (blue), 2 (pink), 3 (olive green), and 4 (purple) from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( B ) Comparison of the relative expression of TGFβR2 in each of the four passages on CD4+CD24-TISCs isolated from RS (grey) and HS (red) diet cohorts of the Py230-C57Bl/6J tumor model. ( C ) Representative flow cytometry histogram of pSMAD2/3 intracellular expression in each of the four passages from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( D ) Comparison of the relative expression of pSMAD2/3 in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( E ) Representative flow cytometry histogram of CD80 surface expression in each of the four passages from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( F ) Comparison of the relative expression of CD80 in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( G ) Representative flow cytometry plot to determine the TGFβR2/CD80 double-positive TISCs in passage 4 of the HS diet cohort from the Py230-C57Bl/6J tumor model. ( H ) Comparison of the relative expression of TGFβR2/CD80 double-positive TISCs in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( I ) Comparison of the relative expression of CD80 following TGFβ (80 ng/mL) stimulation for 5 days on CD4+CD24-TISCs isolated from each of the four passages of the RS and HS diet cohorts in the Py230-C57Bl/6J tumor model. ( J – M ) Comparison of the relative expression of TGFβR2 ( J ), pSMAd2/3 ( K ), CD80 ( L ), and TGFβR2/CD80 double-positive cells ( M ) in each of the four passages on CD4+CD24-TISCs isolated from the RS (grey) and HS (red) diet cohorts of the 4T1-BALB/cJ tumor model. ( N ) Comparison of the relative expression of CD80 following TGFβ (80 ng/mL) treatment for 5 days on CD4+CD24-TISCs isolated from each of the four passages of the RS and HS diet cohorts of the 4T1-BALB/cJ tumor model. Data analyzed by one-way ANOVA for multiple comparisons and presented as mean ± SEM; n = 8 (biological replicates) per cohort; (*) p -value < 0.05.

Article Snippet: The TISCs were isolated from murine tumors by CD24 − CD44 + breast tumor stem cells using immunomagnetic beads as per the manufacturer’s specifications and reagents (catalog#MAGH111, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Comparison, Isolation