Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: NFATC2 is upregulated in CRC-SCs. ( A ) qRT-PCR analysis of NFATC2 in primary CRC spheres and re-adherent cells relative to adherent cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1, 2, 3, and 4). Spheres were obtained by suspension culture. ( B ) Western blot analysis of NFATC2 in primary CRC spheres and adherent and re-adherent cells. ( C ) qRT-PCR analysis of NFATC2 in sorted CD44 + (left) or CD133 + (right) primary CRC cells relative to negative cells. Primary CRC cells were isolated from the cancer tissues of colorectal cancer patients (No 1, 2, 3, and 4). CD44 + or CD133 + cells were obtained by flow cytometry. ( D ) The correlation between the transcription level of NFATC2 and CD44 (left) and CD133 (right) in pin primary CRC sphere-derived cells. The mRNA level of each gene was determined by qRT-PCR. Data were normalized to GAPDH as ∆CT and analyzed by Spearman’s correlation analysis. Data are represented as mean ± SD; * P <0.05, *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Quantitative RT-PCR, Isolation, Suspension, Western Blot, Flow Cytometry, Derivative Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Overexpression of NFATC2 promotes the stemness of CRC cells. ( A , B ) Sphere formation assay of NFATC2-overexpressing and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number and average diameters of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres isolated from the cancer tissues of CRC patients (No 1, 2, 3, and 4) was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (left) and CD133 (right) in NFATC2-overexpressing and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). NFATC2-overexpressing and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in NFATC2-overexpressing and control primary CRC cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Over Expression, Tube Formation Assay, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Knockdown of NFATC2 inhibits the stemness of CRC cells. ( A , B ) Sphere formation assay of NFATC2-knockdown and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number and average diameters of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (top) and CD133 (bottom) in NFATC2-knockdown and control primary CRC cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). NFATC2-knockdown and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in NFATC2-knockdown and control primary CRC cells. ( E ) Tumorigenesis of NFATC2-knockdown and control primary CRC cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Knockdown, Tube Formation Assay, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: YAP activity is necessary for NFATC2 for maintaining the stemness in CRC cells. ( A , B ) Sphere formation assay of YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). The 1st passaged spheres were obtained by suspension culture for 15 days and the number of the spheres were counted ( A ). The number of 1st, 2nd, and 3rd passaged spheres was also counted ( B ). ( C ) qRT-PCR analysis of CD44 (top) and CD133 (bottom) in YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Primary CRC cells were isolated from the cancer tissues of CRC patients (No 1 and 2). YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells were generated by lentivirus delivery system. ( D ) Western blot analysis of CD44 and CD133 in YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. ( E ) Tumorigenesis of YAP-knockdown NFATC2-overexpressing primary CRC cells and control cells. Data are represented as mean ± SD; *** P <0.001; two-tailed Student’s t -test. Abbreviations: CRC, colorectal cancer; NFATC2, nuclear factor of activated T-cells, cytoplasmic 2; CRC-SCs, colorectal cancer stem cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Activity Assay, Tube Formation Assay, Knockdown, Control, Isolation, Suspension, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques: Reverse Transcription

Journal: OncoTargets and therapy

Article Title: NFATC2 is a novel therapeutic target for colorectal cancer stem cells

doi: 10.2147/OTT.S169129

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: CD133 , IB , Mouse monoclonal , , Miltenyi Biotec , 130-092-395 , 100.

Techniques:

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (a) HepG2 with or without various treatments for 24 hours and stable HepG2 cells that expressed a control shRNA (sh-Ctrl) or the Atg5 shRNA (sh-Atg5) were subjected to flow cytometry analysis for CD133+ cells. Results represent the mean ± SEM of three independent experiments. None, no treatment. (B) HepG2 cells with the treatments shown in (A) were lysed for immunoblot analysis. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The β-actin protein was also analyzed to serve as the loading control. (C) Sphere-formation assay of CD133+ and CD133− HepG2 cells. The panels shown to the left are representative results of spheres formed by CD133+ and CD133− HepG2 cells with and without stable ATG5 knockdown. Scale bar=200 μm. The histogram shown to the right indicated the number of spheres larger than 100 μm in diameter when 500 CD133+ cells were seeded. The results represent the mean ± SEM of three independent experiments. (D) HepG2 cells with various treatments for 24 hours were incubated with MicroBeads (Miltenyi Biotec) for the isolation of CD133+ cells, which were then analyzed for their sphere-forming ability. 500 cells were seeded for the assay. Also see Figure S1.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Control, shRNA, Flow Cytometry, Western Blot, Tube Formation Assay, Knockdown, Incubation, Isolation

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Hep3B and Huh7 cells with various treatments for 24 hours were subjected to flow cytometry analysis for CD133+ cells. (B) HepG2 and Huh7 cells were transfected with the p53-expressing plasmid for two days, treated with 3-MA or rapamycin for another 24 hours and then subjected to flow cytometry analysis for CD133+ cells. (C) HepG2 cells transfected with the control siRNA (si-Ctrl) or the p53 siRNA (si-p53) for two days or treated with PFTα or DMSO for one day were analyzed for their CD133+ cells by flow cytometry (top panel) or sphere-forming ability of their CD133+ cells (bottom panel). The results shown in (A), (B) and (C) represent the mean ± SEM of three independent experiments. (D) HepG2 cells treated with DMSO or PFTα for one day or with siRNA for two days were lysed for immunoblot analysis. (E) Stable HepG2 cells that expressed control shRNA (sh-Ctrl), sh-Atg5, or both sh-Atg5 and sh-p53 were lysed for immunoblot analysis. (F) Cells mentioned in (E) were used for the sphere-formation assay. Also see Figure S2.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Control, Western Blot, shRNA, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) HepG2 cells with various treatments for 24 hours or stably expressing the control shRNA or the Atg5 shRNA were lysed for immunoblot analysis. (B) Immunoblot analysis of HepG2 cells transfected with either the control vector or the expression vector for various p53 proteins. Cells were lysed two days after DNA transfection for immunoblot analysis. None, control cells with no DNA transfection. (C) The experiments were conducted the same way as in (B), with the exception that Hep3B cells were used for the expression studies. (D) Hep3B or HepG2 cells were transfected with various p53-expressing plasmids or the control vector as indicated for two days followed by flow cytometry analysis for CD133+ cells. (E) HepG2 cells were transfected with the p53-expressing plasmids for two days, and CD133+ cells were then isolated for the sphere-formation assay. (F) The experiments were conducted the same way as in (E), with the exception that Hep3B cells were used for the studies. The results in (D–F) represented the mean ± SEM of three independent experiments. Also see Figure S3.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Isolation, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Top panel, HepG2 cells were treated with DMSO, Mdivi-1 or CCCP for one day and then subjected to flow cytometry analysis for CD133+ cells; bottom panel, CD133+ HepG2 cells were isolated and treated with Mdivi-1 or CCCP for two days and then analyzed for their sphere-forming ability. (B) HepG2 cells without treatment or with the treatment of DMSO, CCCP or Mdivi-1 for one day were lysed for immunoblot analysis. Cells were also subjected to subcellular fractionation for the isolation of mitochondria, cytosol, and nuclei for immunoblot analysis. Tom20, β-actin and lamin B1 were used as the loading controls for mitochondria, cytosol and nucleus, respectively, to ensure equal amount of proteins were loaded on the gel. (C) Confocal microscopy for the analysis of the subcellular localization of p53(pS392) in HepG2 cells treated with DMSO, Mdivi-1 or CCCP. TOM20 was used as the marker for mitochondria. The areas boxed are enlarged at the bottom. Scale bar, 10 μm. (D) The results shown in (C) were quantified with a Leica TCS SP8 fluorescent confocal microscope. The results indicated the percentages of p53(pS392) that colocalized with TOM20. The results represent the mean ± SEM of at least 30 cells that were analyzed. See also Figure S5.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Isolation, Western Blot, Fractionation, Confocal Microscopy, Marker, Microscopy

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Effects of PINK1 knockdown on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter using the Nanog-luc1 reporter (bottom panel). HepG2 cells transfected with either the control siRNA or the PINK1 siRNA for two days were analyzed. In the bottom panel, HepG2 cells were also transfected with the Nanog-luc1 reporter (see Figure 3B) for the analysis of luciferase activity. The luciferase activity of cells without the transfection of siRNA was arbitrarily defined as 1. The results represented the mean ± SEM of three independent experiments. (B) Effects of PINK1 over-expression on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter (bottom panel). The experiments were conducted the same way as in (A), except that instead of using siRNA, cells were transfected with either the control vector or the PINK1-expressing plasmid. (C) Immunoblot analysis of HepG2 cells with PINK1 knockdown (left panels) or PINK1 over-expression (right panels) were lysed for immunoblot analysis. Total cell lysates as well as the nuclear lysates (bottom two panels) were analyzed. (D) PINK1 in HepG2, Hep3B or Huh7 cells was immunoprecipitated with a control antibody (−) or the anti-PINK1 antibody (+) and then incubated with GST-p53 in the presence of ATP. The GST-p53 phosphorylated at S392 was analyzed using the anti-p53 antibody that recognized phosphoserine-392. GST-p53 added in the reaction and PINK1 immunoprecipitated were also analyzed by immunoblot (bottom two panels). Numbers to the left of the top panel indicate protein molecular weight markers. (E) GST-p53 was mixed with GST-PINK1 or GST and incubated in the presence of ATP. The phosphorylation of p53 at S392 was then analyzed with the antibody that recognized phosphoserine-392. GST-p53, GST-PINK1 and GST used for the reaction was also analyzed by anti-p53, anti-PINK1 and anti-GST antibodies, respectively (bottom three panels). Numbers to the left indicate protein molecular weight markers. (F) Co-immunoprecipitation of p53 and p53(pS392) with PINK1. HepG2 cells were lysed and immunoprecipitated using the anti-PINK1 antibody or the control antibody followed by immunoblot analysis for p53, p53(pS392) and PINK1. (G) Co-immunoprecipitation of p53 and p53(pS392) with PINK1 using the anti-PINK1 antibody in different subcellular fractions (top 3 panels). β-actin, lamin B1 and Tom20 were used as the markers for cytosolic (C), nuclear (N) and mitochondrial (M) fractions. Equal amounts of p53 were used for the co-immunoprecipitation experiment (bottom 2 panels). See also Figure S6.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Knockdown, Transfection, Control, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Incubation, Molecular Weight, Phospho-proteomics

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Virus, Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Mouse Assay, Mutagenesis, Software, Imaging, Extraction, Isolation, DNA Labeling

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , General morphology of livers 2 days after PHx. b , H&E staining of liver sections 2 days after PHx. c , d , Ki67 + percentages in HNF4α + hepatocytes ( c ) and HNF4α − NPCs ( d ) 2 days after PHx. Means ± SEM are shown, n=3 per group, **P < 0.01 (two-tailed unpaired t test). n.s., not significant. e , EpCAM was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). Stellate cells (GFAP) were not altered around the colony. f , CK19 was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). CD133 was highly expressed in the colony. g , Sox9 and CD44 were highly expressed in biliary epithelial cells (arrowhead), but was not upregulated in the colony (dashed line) compared to surrounding hepatocytes. h , AFP showed no difference between the colony (dashed line) and the surrounding tissue. i , Liver/body weight ratios after PHx. Means ± SEM from 3 or more mice analyzed for each time point and group are shown. j , k , Immunofluorescent staining of SKO liver sections 3 weeks after PHx. Colonies were at various sizes and locations as shown by arrows in j . Macroscopic colonies were found as shown by arrows and dashed lines in k . Immunofluorescent image in k corresponds to the magenta arrow in the liver image. PV, portal vein; CV, central vein. l , Vasculature shown by PECAM did not distinguish the colony (arrow). m , HGF expressed by NPCs was not concentrated in the colony (dashed line). Scale bars, 1 cm ( a ) 100 μm ( b , e - g , i - l ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence on liver tissue sections 2 days after PHx. HNF4α is a hepatocyte marker and Ki67 is a proliferation marker. Arrow in a points to an area enriched with proliferating hepatocytes. Dashed line in b shows an area with continuous CD133 expression. c , Quantification of proliferating rate in hepatocytes in CD133-positive and -negative areas in SKO livers 2 days after PHx. Each dot indicates one area. Data were collected from 3 mice. Means ± SEM are shown. ****P < 0.0001 (two-tailed unpaired t test). d , While WT hepatocytes proliferated at high frequency everywhere, proliferating hepatocytes in SKO liver were mostly located in patchy areas marked by CD133 expression. e , Immunofluorescence of CD133 on liver tissues at day 0 or 2, and 3 weeks (0d, 2d, 3 wk) after PHx. CD133 + hepatocyte clusters were only found in SKO livers after PHx (light green arrows). In WT livers, CD133 expression was only seen in bile duct epithelial cells (arrowheads). Scale bars, 100 μm ( a , e ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Marker, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Representative image of CD133 + colonies shown by immunofluorescence on SKO liver tissues 3 weeks after PHx. Dashed lines, vasculatures. b , H&E staining of SKO liver sections 3 weeks after PHx. Yellow dashed line: boundary between the colony and surrounding tissue; white dashed lines: vasculatures. c , Immunofluorescence on SKO liver sections at indicated time points after PHx. Arrowheads and dashed lines indicate the boundaries between the colonies and surrounding tissues, which was clear at 3 weeks but disappeared at 5 weeks. d , Unstained thick tissue section of SKO liver 3 weeks after PHx. e , Colonies ( C ) and non-colony control areas ( N ) were dissected out from thick tissue sections of SKO livers 3 weeks after PHx, and analyzed by immunoblotting. Random areas from WT livers 3 weeks after PHx, bile duct ( BD ) and E17.5 liver ( E ) were used for comparison. f , Illustration of epithelial lineages in the liver. Scale bars, 100 μm ( a - d ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Comparison

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Schematic illustration of mosaic rescue experiment of Shp2 in Shp2-deficient liver. b , Immunofluorescence showing Shp2-rescued hepatocytes also labeled with GFP. c , GFP + hepatocytes were proliferating (Ki67 + ; arrows), while other proliferating cells were not hepatocytes (arrowheads). Areas with colonies were not included here. d , Colonies were frequently constituted by GFP-negative hepatocytes (dashed line). Orange dots: non-specific stains of debris caused by HTVi. e , Some GFP + hepatocytes showed expression of CD133 (arrowheads). Dashed lines show CD133-positive areas. f , qRT-PCR analysis of WT and SKO liver lysates 2 days after PHx. n=3, means ± SEM are shown, *P < 0.05, **P < 0.01 (two-tailed unpaired t test). g , Immunofluorescence on SKO liver section 2 days after PHx. White dashed lines mark Porcupine + cells enriched in CD133 + colonies, rarely detected in non-colony area (arrowheads). Pink dashed lines: vasculature. h , Immunofluorescence on the colonies (arrows) in Shp2-deficient hepatocyte culture in vitro. Arrowhead, dying cells with high autofluorescence. i , Immunoblot analysis of WT and SKO liver lysates 2 days after PHx. j , qRT-PCR analysis of SKO liver tissue lysates without PHx (0d, 3 mice) and with PHx (2d, 3 mice). Means ± SEM are shown. **P < 0.01 (two-tailed unpaired t test). k , qRT-PCR analysis of hepatocyte and NPC fractions from SKO livers (3 mice) 2 days after PHx. HNF4α and CD45 were used as positive controls for the fractionation. β-actin was used for normalization, because GAPDH was highly expressed by hepatocytes. Means ± SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). l , qPCR analyses of PLC cells treated with Mek inhibitor and Porcn inhibitor. Means ± SD from 4 wells are shown. *P < 0.05, **P < 0.01, (two-tailed unpaired t test). n.s., not significant. Scale bars, 100 μm ( b - e , g , h ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Labeling, Expressing, Quantitative RT-PCR, Two Tailed Test, In Vitro, Western Blot, Fractionation

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence of liver sections 2 days after CCl 4 injection. Proliferating hepatocytes were scattered in WT livers, whereas they were highly concentrated in CD133 + colonies (white dashed line) in SKO livers as shown in a . CD133 + /EpCAM + /HNF4α - bile duct epithelial cells (arrowheads) were not associated with the colonies. Pink dashed lines, injured areas. PV, portal vein. c , Immunofluorescence of Met hep-/- liver sections 2 days after PHx or CCl4 injection. White dashed lines, CD133 + colonies. Pink dashed line, injured area. d , qRT-PCR analysis of PLC cell lysates treated with Shp2 or MEK inhibitors (Shp2i and MEKi). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. e , Immunoblotting of PLC cell lysates treated with inhibitors or transfected with Shp2 targeting CRISPR vector. Guide RNA targeting the AAVS1 safe harbor site was used as a control (sgCtrl). f , qRT-PCR analysis of MCF10A cell lysates treated with the inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. g , Immunoblotting of MCF10A cell lysates treated with inhibitors. h , qRT-PCR analysis of lysates from various cell lines treated with Shp2 or MEK inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. Scale bars, 100 μm ( a - c ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Injection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transfection, CRISPR, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence on in vitro colonies of primary hepatocytes from SKO liver. CD133 was localized at filament-like structures in E-Cad + colonies as shown by arrowheads in a , which were connected between different hepatocytes as shown by arrows in b . c , 3D-reconstituted confocal image of immunofluorescence on PLC cells. Lower panel shows the Z-plane section of the orange box area. Arrowheads indicate the CD133 signal on continuous filament like structures bridged between neighboring cells. Pink dashed lines indicate the cell surface. d , Immunofluorescence of MCF10A cells treated with Shp2 inhibitor. e , f , Super-resolution STORM images of immunofluorescence on PLC cells without ( e ) or with ( f ) CD133 overexpression. Colocalization of CD133 and β-tubulin was analyzed as Pearson’s coefficient. Mismatched green and magenta channels from shuffled ROIs were measured as controls . Means ± SEM from 6 images are shown. **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). g , Immunofluorescence and Immuno-Gold EM images of cryo-ultramicrotome sections of SKO liver tissue after PHx. Cyan arrowheads and asterisk indicate apical lumens. White arrowheads indicate the CD133 signals aligned between apical lumens of neighboring cells. Light green arrow, CD133 staining (12 nm colloidal gold); Magenta arrows, α-tubulin staining (18 nm colloidal gold). h , Immunoblotting of CD133 + vesicles isolated from MEK inhibitor (MEKi) -treated PLC cells. Markers for different fractions were analyzed. CD133 antibody used for the vesicle isolation was IgG produced in mouse, which was detected by anti-mouse IgG antibody, showing efficient capture by the beads. Despite the efficient capture, the DMSO-treated PLC cells did not have much CD133+ vesicle to be bound with the antibody. Scale bars, 100 μm ( a ), 25 μm ( d ), 1 μm ( e , f , Fluorescence in g ), 100 nm (EM in g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, In Vitro, Over Expression, Two Tailed Test, Staining, Western Blot, Isolation, Produced, Fluorescence

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a - c , Immunofluorescence on PLC cells transfected with CD133-Myc-tag fusion protein expression construct. Cells with different expression levels are shown as representatives. In a and c , exposure times for the CD133 signals were adjusted separately to clearly demonstrate the patterns, rather than intensities. Note that cells with higher CD133 expression displayed bulky pattern of the filaments, with maintained colocalization with tubulin filaments. Arrowheads, CD133 + filaments. As shown in b , the overexpressed CD133 primarily localize to the filaments, without detectable membrane localization (arrowheads). With extreme overexpression, CD133 can also localize to the cellular membrane surface, altering the morphology of the surface (right panels). d , Immunofluorescence on HeLa and MC38 cells. e , Colocalization of CD133 and β-tubulin was analyzed as Pearson’s coefficient. Mismatched green and magenta channels from shuffled ROIs were measured as controls. f , Immunofluorescence on PLC cells transfected with cMet-GFP. cMet, an HGF receptor, shows the cell surface. CD133 did not colocalize with cMet-GFP on the cell surface, but instead localized to the filaments. g , Immuno-Gold EM images of cryo-ultramicrotome sections of SKO liver tissue after PHx. Light green arrowheads, CD133 staining (12nm colloidal gold); Magenta arrowheads, α-tubulin staining (18nm colloidal gold). Scale bars, 5 μm ( a , c ), 50μm ( b , d ), 25μm ( f ) and 50nm ( g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Transfection, Expressing, Construct, Membrane, Over Expression, Staining

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunofluorescence for endosomal markers on PLC cells. b , Immunoblotting of CD133+ vesicles isolated from SKO liver after PHx. CHMP2B is a protein that is generally involved in vesicle formation. c , qRT-PCR analysis of housekeeping genes in RNAs extracted from WT (3 mice) and SKO tissues (3 mice) and from CD133 + vesicles and CD133-negative fractions (4 mice for both). Means ± SEM are shown. d , qRT-PCR analysis of RNAs extracted from CD133 + vesicles from PLC cells treated with MEK inhibitor. e , RNA-seq analysis showing enrichment scores of IEG contents in the two vesicle types compared to the whole cells. f , Immunofluorescence on SKO liver 2 days after PHx. Asterisks indicates vasculature. g , Immunofluorescence on MCF10A cells treated with inhibitors. h , qRT-PCR analysis of PLC cell lysates after treatment with CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. RNase and Triton X-100 were used to digest the RNA content of the vesicles. *P < 0.05, **P < 0.01 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 replicates are shown. Scale bars, 25μm ( a ) and 50μm ( f , g ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Western Blot, Isolation, Quantitative RT-PCR, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Agarose gel electrophoresis of total RNAs extracted from WT and SKO livers and CD133-positive vesicle and negative fractions from SKO liver after PHx. Arrowheads show rRNAs and arrow shows microRNAs. b , qRT-PCR analysis of RNAs extracted form WT (3 mice) and SKO tissues (3 mice) and from CD133 + vesicles and CD133 − fractions (4 mice for both). Means ± SEM are shown. *P < 0.05, **P < 0.01 (uncorrected Dunn’s multiple comparison test, performed after Kruscal-Wallis test). n.s., not significant. c , RNA-seq analysis of the different cell types and the whole cells. The bars indicate proportions between numbers of deficient and enriched gene transcripts in each RNA types. d , Comparison of IEG contents between the different vesicle types with RNA-seq. e , RNA-FISH for MYC mRNA and immunostaining for CD133. f , Quantitative colocalization analysis of CD133 and MYC mRNA in PLC cells shown in e . Means ± SEM from 14 images are shown. g , Experimental design to detect the traffic of CD133+ intercellsome between neighbor cells. h , Immunostaining of Myc-tag and CD133 on GFP+ and mCherry+ PLC cells mixed as shown in g . Note that Myc-tag only indicates exogenous CD133, while CD133 indicates both endogenous and exogenous CD133. Myc-tag was primarily detected in the GFP+ cells, but also detected on the bridges (arrowheads) and in the mCherry+ cells (arrows). GFP was not detected at the same locations (arrowheads and arrows), indicating the specific traffic of CD133-Myc-tag. Scale bars, 10 μm (e) and 50 μm ( h )

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Agarose Gel Electrophoresis, Quantitative RT-PCR, Comparison, RNA Sequencing, Immunostaining

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunoblotting of HuR in the CD133 + vesicles from SKO liver after PHx. GAPDH and HNF4α were used as controls for cytoplasmic and nuclear fractions, respectively. b , Immunofluorescence on PLC cells. Arrows, peri-nuclear areas enriched with HuR in the cytoplasm; Arrowheads show colocalization of HuR on the CD133 + filament bridging two cells. c , Immunofluorescence on PLC cells treated with the Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. d , Immunofluorescence on PLC cells transfected with CD133 expression vector treated with a Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. e , Immunoblotting of CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. f , qRT-PCR analysis of PLC cell lysates after treatment with CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. RNase and Triton X-100 were used to digest the RNA content of the vesicles. *P < 0.05 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 replicates are shown. g , A model and predictions for single cell RNA-seq data analysis. h , Total IEG expression levels. Cyclin D1-positive and -negative cells were analyzed separately to evaluate the influence of cell cycle on the IEG analysis. See Methods section for what the bars and dots represent. i , j , Box plots (Tukey’s) of IEG diversity within each cell (calculated as entropy) and IEG variations among cells. Analyses were focused on cyclin D1 + cells in all groups for fair comparison. k , l , Plot of intracellular IEG diversity against total IEG expression levels in SKO hepatocytes 2 days after PHx. Blue color gradient indicates cyclin D1 expression levels. For the simulation in l , the parameters used were: Group of 5 cells, X=1/12, model 3 (see also and Methods). Green and Black arrows show typical profiles of CD133-positive and -negative cells, respectively. m , Box plot (Tukey’s) of intracellular IEG diversity after simulation. The analysis was not limited to cyclin D1-positive or -negative cells. Note the simulation of the IEG exchange attracted the cells from cyclin D1-low profile to cyclin D1-high profile ( k - m ). Statistics was performed by Wilcoxon rank sum test adjusted by FDR in i , j and m . Scale bars, 50 μm ( b , c ), 5 μm ( d ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Western Blot, Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Isolation, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a, Heatmap analysis of single hepatocytes. b , Detection of CD133 expression in the single cell RNA-seq data. c , Expression levels of indicated hepatocyte subtype markers and stem cell-like markers. CD133- and CD133+ hepatocytes in the SKO liver 2 days after PHx were compared. d , Principal component analysis with the IEGs. e , tSNE analysis. Clusters likely reflect spatial locations within the tissue.

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence ( a ) on liver sections of WT and Prom1 KO mice 2 days after PHx with or without Shp2 deletion using AAV-Cre and quantification of Ki67 + ratio in hepatocytes in the indicated genotypes ( b ). Separate analyses of pericentral and periportal hepatocytes showed insignificance of zonal difference. **P < 0.01, (two-tailed unpaired t test). Means ± SEM are shown. n = 3, 3, 4 and 5 mice, respectively. c , d , Immunofluorescence ( c ) on primary hepatocytes isolated from SKO and Shp2/Prom1 double KO (DKO) mouse livers and quantification of Ki67+ ratio ( d ). Images of representative colonies are shown. ***P < 0.001, (two-tailed unpaired t test). Means ± SD from 4 wells are shown. e , Experimental design with primary hepatocytes isolated from GFP labeled SKO liver and unlabeled DKO liver. E-Cad+ colonies were analyzed. f , Immunofluorescence images of E-Cad+ colonies in SKO, DKO and mixed culture as shown in e . The arrows show a GFP+ SKO cell forming part of the colony with the surrounding DKO cells. g , Quantification of the Ki67 ratio in E-Cad+ colony-forming cells shown in f . ***P < 0.001 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 wells are shown. h - j , scRNA-seq analyses of DKO liver compared to SKO liver 2 days after PHx. Porcn was used as a marker to represent the Prom1 promoter active hepatocyte populations in both SKO and DKO ( h ). A proliferation marker CyclinD1 was reduced in Porcn+ DKO hepatocytes compared to Porcn+ SKO hepatocytes ( i ). The IEG variations among cells were analyzed ( j ). See also a model in , regarding the predicted relationship between the intercellsome exchange and IEG variations among cells. Scale bars, 100 μm ( a , c , f ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Two Tailed Test, Isolation, Labeling, Marker

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunofluorescence on WT and Prom1 KO mouse intestinal tissue sections and intestinal organoids. b , c , Immunofluorescence ( b ) on WT and Prom1 KO mouse intestinal organoids treated with MEK inhibitor (MEKi) and quantification of Ki67+ ratio in the crypt cells ( c ). Dashed lines indicate the crypt buds. *P < 0.05, (two-tailed unpaired t test). Means ± SD from 3 wells are shown. Each dot represents each crypt buds, and symbols indicate each well. Scale bars, 20 μm ( a ) and 50 μm ( b ).

Article Snippet: Antibodies for Ki67 (14-5698-80; eBioscience), HNF4α (sc-8987; Santa Cruz Biotechnology), mouse CD133 (14-1331-82; eBioscience), human CD133 (86781; CST), E-cadherin (sc-7870; Santa Cruz Biotechnology), GFP (04404-84; Nacalai Tesque), Shp2 (sc-280; Santa Cruz Biotechnology), Porcupine (ab105543; abcam), β-catenin (sc-7199; Santa Cruz Biotechnology), CHMP2B (ab157208; abcam), HuR (ab200342; abcam, and 66549-1-Ig; proteintech), VE-Cad (AF1002; R&D SYSTEMS), BrdU (555627; BD Biosciences), PECAM (13-0311-81; eBioscience), HGF (ab83760; abcam), GFAP (Z0334; Dako), EpCAM (552370; BD Biosciences), Sox9 (AB5535: Millipore), CD44v6 (GTX75661; GeneTex) and AFP (AF5369; R&D SYSTEMS) were used as primary antibodies.

Techniques: Immunofluorescence, Two Tailed Test

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Primers used for real-time PCR

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques:

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: CIH promoted CSC-like properties in NSCLC cells. a Microscopic observation of NSCLC cells. A549 and SPCA1 spheroids were obtained and then cultured under Nor or CIH conditions for 24 h. b qRCP analyses in triplicate of CD44, Sox2, Nanog, Oct4, and CD133 in NSCLC cells. Adherent A549 and SPCA1 were treated with 24 h-Nor or CIH exposure. GADPH expression served as an internal control. c Flow cytometry analyses of the percentage of CD44 + CD133 + cells in A549 or SPCA1 populations. Error bars represent the mean ± SEM of at least triplicate experiments. * P < 0.05, ** P < 0.01. CIH chronic intermittent hypoxia, CSC cancer stem cell

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Knockdown of Bach1 decreased the stemness and mitochondrial ROS accumulation in CIH-treated NSCLCs. Bach1 shRNA or parental negative control (NC) was transfected into A549 or SPCA1. Then the cells were cultured under CIH conditions for 48hrs. Flow cytometry analyses of CD44 + CD133 + cells in Nor or CIH-treated A549 ( a ) or SPCA1 ( b ). c The percentage of CD44 + CD133 + cells in Nor or CIH-treated cells was measured and illustrated. Fluorescence microscopy analysis for localization of mtROS production in A549 ( d ) and SPCA1 ( e ). Nuclei was stained with Hoechst (blue), mitochondria ROS were stained with MitoSOX-red. The merged panels showed the MitoSOX-red positive cells in total cells. f The percentage of MitoSOX positive cells was measured and illustrated. All experiments were performed in triplicate, and data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control group

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: shRNA, Negative Control, Transfection, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Staining

Journal: International Journal of Oncology

Article Title: RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling

doi: 10.3892/ijo.2023.5541

Figure Lengend Snippet: RPL27 silencing suppresses CRC stemness. (A) Separation of CD133 + HT29 CSC population from CD133 − cell population. (B) Relative levels of CD133 mRNA. Decreased CD133 and PLK1 (C) mRNA and (D) protein levels in CD133 + HT29 CSCs treated with NC or RPL27-1 siRNA. Inhibition of (E) proliferation and (F) sphere formation of CD133 + HT29 CSCs following RPL27 silencing. * P<0.05, ** P<0.01, *** P<0.001 vs. NC. si, small interfering; NC, negative control; RPL27, ribosomal protein L27; PLK1, polo-like kinase 1; CRC, colorectal cancer.

Article Snippet: CD133 MicroBead kit (cat. no. 130-100-857) and AutoMACS Pro (both Miltenyi Biotec, Inc.) were used to isolate CD133 + HT29 CSC populations from HT29 parental cell culture according to the manufacturer's instructions.

Techniques: Inhibition, Negative Control