Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: CD133 interacts with DNMT1 depending on its C‐terminal cytoplasmic domain. A) Graphic representation of the proposed structural model of CD133. This protein is modeled as having an extracellular N terminus, a cytoplasmic C terminus, two small cytoplasmic loops, and two large extracellular loops. C‐terminal cytoplasmic domain of CD133 (residues 813–865) (indicated by square frame) is used as the bait for yeast two‐hybrid screen. B) In vitro interaction between CD133 and DNMT1. GST or GST‐CD133(813–865) proteins are incubated with purified His‐DNMT1 (112–235) protein. The GST pull‐down products are blotted with anti‐GST and anti‐His antibodies. C) CD133 interacts with DNMT1 in vivo. The lysates of CD133+ cells and CD133‐ cells isolated from glioblastoma samples are subjected to IP using anti‐CD133 (Clone W6B3C1) or anti‐DNMT1 antibodies, followed by immunoblotting (IB) with anti‐CD133 or anti‐DNMT1 antibodies. Whole‐cell lysates are analyzed by IB with anti‐CD133 or anti‐DNMT1 antibodies as input. D) The interaction between CD133 and the members of DNMT is examined by Co‐IP assay. Lysates of CD133+ cells are subjected to IP using anti‐CD133 antibody (Clone W6B3C1), followed by IB with anti‐CD133 (Clone W6B3C1), anti‐DNMT1, anti‐DNMT2, or anti‐DNMT3A antibodies. Whole‐cell lysates are analyzed by IB with anti‐CD133, anti‐DNMT1, anti‐DNMT2, or anti‐DNMT3A antibodies as input. E) Strep peptide or strep‐tagged CD133 c‐terminal deletion mutant are incubated with purified DNMT1 protein. The Strep pull‐down products are blotted with anti‐DNMT1 antibody. F) The lysates of CD133+ cells expressing FLAG or DNMT1‐FLAG or DNMT1(Del(155–163)) are subjected to IP using anti‐FLAG antibody, followed by IB with anti‐FLAG, or anti‐CD133 antibodies (Clone W6B3C1). Whole‐cell lysates are analyzed by IB with anti‐CD133 (Clone W6B3C1), anti‐FLAG, or anti‐GAPDH antibodies as input.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Two Hybrid Screening, In Vitro, Incubation, Purification, In Vivo, Isolation, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Expressing

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The CD133–DNMT1 interaction inhibits the nuclear translocation of DNMT1. A) Co‐localization of CD133 and DNMT1 is assessed by immunofluorescence staining of CD133 (red) and DNMT1 (green) in T12752 (upper panel) and T21286 (lower panel) CD133+ cells. Nuclei (blue) are counterstained with DAPI. Co‐localization of CD133 and DNMT1 is demonstrated by yellow fluorescence. The interaction between DNMT1 and cytoplasmic CD133 is indicated by dashed circle. Scale bars, 10 µM. B) Co‐localization of CD133 and EEA1 is assessed by immunofluorescence staining in CD133+ cells. Nuclei (blue) are counterstained with DAPI. Co‐localization of CD133 and EEA1 is demonstrated by yellow fluorescence. Scale bars, 10 µM. C) CD133 interacts with DNMT1 at endosome. Lysates of endosome protein isolated from CD133+ glioma cells are subjected to IP using anti‐CD133 antibody (Clone W6B3C1), followed by IB with anti‐CD133 (Clone W6B3C1) or anti‐DNMT1 antibodies. The efficiency of endosome isolation is checked by EEA1 (endosome marker). D) Nuclear distribution of DNMT1 in CD133+ cells expressing control shRNA, CD133 shRNA1, or CD133 shRNA2 is determined by IB. Histone H3 is used as the nuclear marker, and α ‐tubulin is used as the cytosolic marker. E,F) The level of nuclear DNMT1 in CD133+ cells and matched CD133‐ cells is determined by IB. Histone H3 is used as the nuclear marker, and α ‐tubulin is used as the cytosolic marker. E. The figures are presented out of three independent experiments. F) The relative densities of DNMT1 to Histone H3 are quantified using densitometry. Values are normalized to that of CD133+ cells. Results are expressed as mean ± SD from three independent experiments; t test, *** p < 0.001, Student's t ‐test. G) The level of nuclear DNMT1 in CD133+ cells expressing control shRNA, CD133 shRNA1, CD133 shRNA1 + shRNA‐resistant wild‐type CD133, or CD133 shRNA1 + shRNA‐resistant CD133(1–862) mutant is determined by IB. Histone H3 is used as the nuclear marker, and α ‐tubulin is used as the cytosolic marker. The relative densities of DNMT1 to Histone H3 are quantified using densitometry. Values are normalized to that of cells expressing control shRNA. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, Student's t ‐test. H) The level of nuclear DNMT1‐FLAG in CD133+ cells expressing FLAG‐DNMT1 or DNMT1(Del(155–163)) is determined by IB. Histone H3 is used as the nuclear marker, and α ‐tubulin is used as the cytosolic marker.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Translocation Assay, Immunofluorescence, Staining, Fluorescence, Isolation, Marker, Expressing, Control, shRNA, Mutagenesis

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The CD133–DNMT1 interaction upregulates p21 and p27. A) Western blot analysis of FLAG‐DNMT1 in CD133+ cells expressing FLAG or FLAG‐DNMT1(Del(155–163)). GAPDH is used as a loading control. B) By Infinium MethylationEPIC BeadChip arrays, the methylation of 680 annotated genes is increased in CD133+ cells expressing DNMT1 (Del(155–163)) compared to control cells ( p < 0.001, Δ β ≥ 0.15). Gene ontology results (top five, according to p value) for 680 genes in which methylation is upregulated in CD133+ cells expressing FLAG‐DNMT1(Del(155–163)) are shown. C) qRT‐PCR quantification of the indicated gene mRNA levels in T21286 CD133+ cells expressing FLAG or FLAG‐DNMT1(Del(155–163)). Data is shown as mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01, * p < 0.05, # p > 0.05, Student's t‐test. D‐E) The methylation rate of p21 and p27 promoters in CD133+ cells and matched CD133‐ cells from T21286 (D) and T12752 (E) are analyzed by bisulfite sequencing. Methylation levels are determined by the ratio of converted C nucleotides to total C nucleotides following bisulfite treatment under CpG island. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, Student's t ‐test. F) Western blot analysis of p21 and p27 expression in T21286 CD133+ cells and CD133‐ cells. GAPDH is used as a loading control. G) Western blot analysis of p21 and p27 expression in T21286 CD133+ cells treated with 2% FBS for 7 days. GAPDH is used as a loading control. H) Chromatin immunoprecipitation (ChIP) assay is performed in CD133+ cells and CD133‐ cells using a DNMT1 specific antibody, followed by PCR amplification of p21 and p27 promoter regions between +250 to position −100. Chromatin (defined as input) and GAPDH products immunoprecipitated by DNMT1 Ab are used as positive and negative control.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Western Blot, Expressing, Control, Methylation, Quantitative RT-PCR, Methylation Sequencing, Chromatin Immunoprecipitation, Amplification, Immunoprecipitation, Negative Control

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: Nuclear localization of DNMT1 inhibits the self‐renewal ability and the tumorigenesis of GSCs. A) Western blot analysis of FLAG‐DNMT1, p21, p27 expression in CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)), or its C1229S mutant. GAPDH is used as a loading control. B) The level of total 5‐methylcytosine in CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)) or its C1229S mutant is examined by ELISA kit. Values are normalized to that of cells expressing FLAG. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01, Student's t ‐test. C) Analysis of 5‐ethynyl‐2′‐deoxyuridine (EdU)‐labeled CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)) or its C1229S mutant. The percentage of EdU‐positive cells is measured. Results are expressed as mean ± SD from six independent experiments; *** p < 0.001, ** p < 0.01, Student's t ‐test. D,E) The number of spheres derived from 100 CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)), or its C1229S mutant at passages 1 and 2. D) Representative images are shown. E) Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01, Student's t ‐test. Scale bar represents 10 µM. F,G) Limiting dilution assay shows overexpression of DNMT1(Del(155–163)) reduced stem cell frequency in T12752 (F) or T21286 (G) CD133+ cells. n = 10, *** p < 0.001 by ELDA analysis. H) The tumor‐initiating capacity of CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)) or its C1229S mutant. An intracranial limiting dilution tumor formation assay (employing 10000, 5000, 1000, and 500 cells per mouse) is performed using CD133+ cells infected with the indicated lentivirus. The table displays the number of mice developing tumors. I–K) T21286 (I) or T12752 (J) CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)) or its C1229S mutant are implanted into immunocompromised mice brain (5000 cells per mouse). Mice are sacrificed when they are moribund or 120 days after implantation. I,J) Survival of mice ( n = 6) is evaluated by Kaplan‐Meier analysis (** p < 0.01, log rank test). K) H&E staining of mouse brain shows tumors formation by CD133+ cells expressing FLAG, FLAG‐DNMT1(Del(155–163)) or its C1229S mutant. Scale bar, 1 cm. C1229 (Cys at position 1229 ) in the FLAG‐DNMT1 Del(155‐163) protein corresponds to Cys at position 1226 in wild type human DNMT1 protein.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Western Blot, Expressing, Mutagenesis, Control, Enzyme-linked Immunosorbent Assay, Labeling, Derivative Assay, Limiting Dilution Assay, Over Expression, Tube Formation Assay, Infection, Staining

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The CD133–DNMT1 interaction maintains the self‐renewal capacity and tumorigenesis of GSC. A,B) Single cell neurosphere formation assay of CD133+ cells expressing control shRNA, CD133 shRNA1, CD133 shRNA1+shRNA‐resistant wild type CD133, or CD133 shRNA1+shRNA‐resistant CD133(1–862) at passages 1–3. A) Representative images of neurosphere are shown. B) The number of neurospheres is shown. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01, Student's t ‐test. Scale bar represents 10 µM. C) Wild type CD133, but not CD133(1–862) mutant, rescues the effect of CD133 knockdown on the tumor‐initiating capacity of CD133+ cells. An intracranial limiting dilution tumor formation assay (employing 10 000, 5000, 1000, and 500 cells per mouse) is performed using CD133+ cells infected with the indicated lentivirus. The table displays the number of mice developing tumors. D–G) CD133+ cells from glioblastoma specimen T21286 (D,F) or T12752 (E,G) expressing Control shRNA, CD133 shRNA1, CD133 shRNA1+shRNA‐resistant wild type CD133, or CD133 shRNA1+shRNA‐resistant CD133(1–862) are implanted into immunocompromised mice brain (5000 cells per mouse). Mice are sacrificed when they are moribund or 120 days after implantation. D,E) Survival of mice ( n = 6) is evaluated by Kaplan‐Meier analysis (** p < 0.01, log rank test). F,G). H&E staining of mouse brain shows tumors formation by CD133+ cells expressing Control shRNA, CD133 shRNA1, CD133 shRNA1+shRNA‐resistant wild type CD133, or CD133 shRNA1+shRNA‐resistant CD133(1–862). Scale bar, 1 cm. shR, shRNA‐resistant. Scale bar, 1 cM. H) T12752 CD133+ cells treated with the indicated peptides are i implanted into immunocompromised mice brain (5000 cells per mouse). Mice are sacrificed when they are moribund or 120 days after implantation. Survival of mice ( n = 6) is evaluated by Kaplan‐Meier analysis (** p < 0.01, log rank test). I,J) T12752 CD133+ cells treated with the indicated peptides cells are subcutaneously injected into immunodeficient mice. I) The images of the xenograft of GSC treated with control or peptides. Scale bar, 1 cm. J) Tumor volumes are measured after tumor cell inoculation every three days. Results are expressed as mean ± SD ( n = 6 mice; ** p < 0.01). Student's t ‐test.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Tube Formation Assay, Expressing, Control, shRNA, Mutagenesis, Knockdown, Infection, Staining, Injection

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The CD133–DNMT1 interaction maintains the quiescence of GSC. A,B) Immunofluorescence analysis of EdU‐labeled CD133+ cells expressing control shRNA, CD133 shRNA1, or CD133 shRNA2. A) Representative images of immunofluorescence are shown. B) The percentage of EdU‐positive cells is measured. Results are expressed as mean ± SD from six independent experiments; *** p < 0.001, Student's t ‐test. Scale bar represents 10 µM. C) Analysis of the percentage of EdU‐positive cells in CD133+ cells and CD133‐ cells. Results are expressed as mean ± SD from six independent experiments; *** p < 0.001, Student's t ‐test. D,E) In vivo CIdU and IdU incorporation assay is used to examine the proliferation of GSCs. D. CD133+ cells are intracranially implanted into immunocompromised mice brain. 7 days later, mice are intraperitoneally injected with CIdU. After 4 weeks, mice are intraperitoneally injected with IdU. 24 h later, mice are sacrificed and perfused. E) Glioblastoma orthotropic xenograft is assessed by immunofluorescence staining of CD133 (red), IdU (green), and CIdU (purple). White arrow indicates the CD133+ cells. Scale bar represents 10 µM. F) Immunofluorescence analysis the percentage of EdU‐positive cells in CD133+ cells expressing control shRNA, CD133 shRNA1, CD133 shRNA1+shR CD133, or CD133 shRNA1+shR CD133(1–862). Results are expressed as mean ± SD from six independent experiments; *** p < 0.001, Student's t ‐test. shR, shRNA‐resistant. G) Analysis of the percentage of EdU‐positive cells in CD133+ cells expressing control shRNA or CD133 shRNA1 and control or p21 or p27. Results are expressed as mean ± SD from six independent experiments; ** p < 0.01, * p < 0.05, Student's t ‐test. H) Immunofluorescence analysis of γ H2AX foci formation in CD133+ cells expressing control shRNA or CD133 shRNA1 and control or p21 or p27. The number of γ H2AX foci‐positive cells is measured. Results are expressed as mean ± SD from three independent experiments; # p > 0.05, *** p < 0.001, Student's t‐test.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Immunofluorescence, Labeling, Expressing, Control, shRNA, In Vivo, Injection, Staining

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The effect of the CD133–DNMT1 interaction on the apoptosis of GSCs induced by TMZ. A–C) CD133+ cells are intracranially implanted into immunocompromised mice brain. 4 weeks later, temozolomide (TMZ) (2.5 mg kg −1 ) is administered orally every day for 5 days (A). B) Treatment with temozolomide increased the DNA damage in glioma cells. Tissue sections are probed with anti‐ γ ‐H2AX Ab (green), and nuclei are counterstained with Hoechst 33258 (blue). Scale bars, 10 µM. C) The percentage of CD133+ cells in glioblastoma orthotropic xenograft treated with control or temozolomide are measured by flow cytometry. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, Student's t ‐test. D) CD133+ cells expressing control shRNA or CD133 shRNA1 and p21 or p27 are treated for 48 h with temozolomide (200 µM). The ratio of apoptotic cells is measured by flow cytometry. Values are normalized to that of cells expressing control shRNA. Results are expressed as mean ± SD from three independent experiments; ** p < 0.01, * p < 0.05, # p > 0.05, Student's t ‐test. E) CD133+ cells expressing Control shRNA, CD133 shRNA1, CD133 shRNA1+shRNA‐resistant wild type CD133, or CD133 shRNA1+shRNA‐resistant CD133(1–862) are treated for 48 h with temozolomide (TMZ). The ratio of apoptotic cells is measured by FACS. Values are normalized to that of cells expressing Control shRNA+FLAG. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01. Student's t ‐test. F) The tumor‐initiating capacity of 5000 CD133+ cells expressing control shRNA,CD133 shRNA1, CD133 shRNA1+shRNA‐resistant wild type CD133, or CD133 shRNA1+shRNA‐resistant CD133(1–862) treated with or without TMZ. Mice are sacrificed when they are moribund or 120 days after implantation. The table displays the number of mice developing tumors and the median survival time of mice. G,H) IHC analysis of DNMT1 in 16 paired primary and recurrent glioma sections. G) Representative microphotographs of IHC staining of DNMT1 in primary and recurrent glioma sections. Scale bar represents 10 µM. H) The scores for quantitative staining of cytoplasmic DNMT1 in the tissue sections are determined according to a total score (range, 0–8). Values are mean ± SD ( n = 16). *** p < 0.001. Student's t ‐test.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Control, Flow Cytometry, Expressing, shRNA, Immunohistochemistry, Staining

Journal: Advanced Science

Article Title: The Interaction between DNMT1 and High‐Mannose CD133 Maintains the Slow‐Cycling State and Tumorigenic Potential of Glioma Stem Cell

doi: 10.1002/advs.202202216

Figure Lengend Snippet: The high‐mannose N‐ glycan of CD133 promotes the interaction between CD133 and DNMT1. A,B) The interaction between CD133 and DNMT1 during GSC differentiation is examined by Co‐IP assay. The lysates of T21286 (A) and T12752 (B) CD133+ cells treated with 2% FBS for 7 days are subjected to IP using anti‐CD133 (Clone W6B3C1), followed by IB with anti‐CD133 (Clone W6B3C1) or anti‐DNMT1 antibodies. Whole cell lysates are analyzed by IB with anti‐CD133 (Clone W6B3C1) or anti‐DNMT1 antibodies as input. Dotted line indicates the shift of CD133 molecular weight. A molecular‐weight size marker is shown. C) qRT‐PCR quantification of the mRNA levels of CD133 in CD133+ cells from glioblastoma specimens (T21286 and T12752) after treatment with 2% FBS at different time points. Values are mean ± SD from three independent experiments. D) Overview of the N‐ glycan biosynthesis pathway. Relationships between N‐ glycans, GlcNAc‐transferases, and plant lectins. The boxed shaded structures are recognized by the plant lectins ConA and PHA‐L. ConA lectin, recognizing high mannose glycans; PHA‐L lectin, recognizing β 1,6 branched GlcNAc. E–G) Lectin blot is performed to analyze the structure of CD133 N‐ glycan during GSC differentiation. The lysates of T21286 (E,F) and T12752 (G) CD133+ cells treated with 2% FBS for 7 days are subjected to IP using anti‐CD133 (Clone W6B3C1) Ab (E,G) or anti‐CD133 Ab (Clone AC133) Ab (F), followed by IB with anti‐CD133 antibody or biotinylated lectins. H,I) qRT‐PCR analysis of the mRNA levels of MAN1A1, MAN1A2, MAN1C1, MGAT1, MGAT2, MGAT4A, MGAT4B, or MGAT5 in T21286 (H) and T12752 (I) CD133+ cells treated with or without 2% FBS for 7 days. Values are mean ± SD from three independent experiments; *** p < 0.001, ** p < 0.01, #, ns. Student's t ‐test. J) MAN1A1 regulated the N‐ glycosylation of CD133. The lysates of CD133+ cells treated with 2% FBS for 7 days transfected with control shRNA or MAN1A1 shRNA are subjected to IP using anti‐CD133 Ab, followed by IB with anti‐CD133 Ab (Clone W6B3C1), anti‐DNMT1 Ab or biotinylated lectin. Whole cell lysates are analyzed by IB with anti‐CD133 (Clone W6B3C1) Ab, anti‐DNMT1 Ab, or anti‐MAN1A1 Ab as input. GAPDH is used as a loading control. Dotted line indicates the shift of CD133 molecular weight. A molecular‐weight size marker is shown. K) The level of total 5‐methylcytosine in CD133+ cells expressing control shRNA, or CD133 shRNA1 and FLAG or MAN1A1‐FLAG is examined by ELISA kit. Values are normalized to that of CD133+ cells. Results are expressed as mean ± SD from three independent experiments; ** p < 0.01, #, ns. Student's t ‐test. L) The level of total 5‐methylcytosine in CD133+ cells expressing FLAG, or MAN1A1‐FLAG and control or DNMT1(Del(155–163)) is examined by ELISA kit. Values are normalized to that of CD133+ cells. Results are expressed as mean ± SD from three independent experiments; *** p < 0.001, #, ns. Student's t ‐test.

Article Snippet: [ ] ELISA plate wells were coated with a CD133 antibody (Miltenyi Biotec, Cat # 130‐092‐395) by incubating 1 μg/100 μL of the antibody per well at 4 °C for 12–14 h. After wells were washed with PBST (PBS with 0.05% Tween 20), wells were blocked by incubation with PBS containing 1% bovine serum albumin (BSA) for 2 h at RT.

Techniques: Glycoproteomics, Co-Immunoprecipitation Assay, Molecular Weight, Marker, Quantitative RT-PCR, Transfection, Control, shRNA, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: G9a methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.1101/2020.04.20.050328

Figure Lengend Snippet: (A) Schematic representation of serial orthotopic transplantation of primary KP cells can be viewed in three basic parts: i) Primary tumors are initated via intranasal infection of adenovirus expressing Cre recombinase in Kras LSL.G12D ; p53 flox/flox (KP) mice. Tumors develop with a latency of approximately 16 weeks. Primary tumors are isolated from KP mice and immediately transduced with lentiviral constructs harboring doxycycline-inducible hairpins and a tRFP label to facilitate identification of hairpin-expressing cells. Ii) Transduced primary KP cells are then orthotopically seeded into the lungs of wild-type recipient mice via intratracheal administration. Animals are thereafter monitored for tumor formation using microCT. Once tumor formation is confirmed in 1 st recipient mice, animals are stratified and dosed with doxycycline for 13 consecutive days to induce expression of latent hairpins targeting either EHMT2 or control transcripts. After 13 days of expression, tumors from 1 st recipients are harvested and tRFP+ cells are sorted to identify hairpin expressing cells. Iii) tRFP+ cells are then orthotopically transplanted into a 2 nd recipient. The animals are maintained on Doxycycline and tumor growth is monitored. (B) Flow cytometry showing gating strategy of primary tRFP-sorted cells prior to secondary transplantation (left plot, tRFP gate control; middle and right plot, sorted tRFP-positive cells from shcontrol and sh EHMT2 , respectively). (C) Relative expression of sh Ehmt2.2 in tRFP-sorted tumor cells prior to secondary transplantation (n=6).

Article Snippet: The following antibodies were used for flow cytometry: CD74-BUV395 (BD, 740274, In-1, 1:25), Pro-SPC (Abcam, ab170699: 1:200), G9a (Abcam, ab185050, EPR18894).

Techniques: Transplantation Assay, Infection, Expressing, Isolation, Transduction, Construct, Control, Flow Cytometry

Journal: bioRxiv

Article Title: G9a methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.1101/2020.04.20.050328

Figure Lengend Snippet: (A) Graphs show enrichment analyses of distinct alveolar cell-lineage gene signatures in transcriptomes generated from KP-derived primary tumorspheres following G9a inhibition (G9ai) vs. vehicle control (control) (n=4; mean Z-score±SEM, two-tailed paired t-test, p<0.05), each paired with immunofluorescence (IF) micrographs of representative canonical marker from their respective cell lineage. (See Figure (S3E) for quantitation of IF). (B) Representative flow cytometry of cells derived from primary tumorspheres treated with either vehicle control (Control) or G9a inhibitor (G9ai) for 5 days and immuno-stained fo23mmuneAT2 markers SPC and CD74. (C) Quantification of the SPC-CD74 double positive (DP) population depicted in (B). (n=4; mean±SEM, two-tailed paired t-test, p<0.05). (D) Representative transmission electron microscopy (TEM) image of cells extracted from primary tumorspheres, treated as in (B). (Upper panel, scale bar 2μm; lower panel, respective insets in the upper panel, scale bar 0.5μm). (N, nucleus; yellow arrows, lamellar bodies). (E) Quantification of TEM in (D) (n=2; mean±SEM, two-tailed paired t-test p<0.05). (F) Representative flow cytometry of tumor-propagating cells (TPCs) sorted after G9a inhibitor (G9ai)- or vehicle control-treatment of primary tumorspheres and immuno-stained fo23mmuneAT2 markers SPC and CD74. (G) Quantification of (F), showing fold-change in G9ai/control ratio of AT2 markers SPC and CD74 (n=2; mean±SEM, One-way ANOVA with Tukey’s multiple comparison test, p<0.005). (H) Relative expression of Sftpc and Cd74 transcripts in G9ai vs. control; TPC and non-TPC respectively. (n=3; mean±SEM, two-tailed paired t-test p<0.05). (I) Spearman’s rank correlation analysis between orthogonal human AT2 gene signatures and G9a EHMT2 transcript in 546 human lung adenocarcinomas. (n=546, linear regression analysis, p<0.0001, r=−0.4)

Article Snippet: The following antibodies were used for flow cytometry: CD74-BUV395 (BD, 740274, In-1, 1:25), Pro-SPC (Abcam, ab170699: 1:200), G9a (Abcam, ab185050, EPR18894).

Techniques: Generated, Derivative Assay, Inhibition, Control, Two Tailed Test, Immunofluorescence, Marker, Quantitation Assay, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy, Comparison, Expressing

Journal: bioRxiv

Article Title: G9a methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.1101/2020.04.20.050328

Figure Lengend Snippet: (A) Relative expression of surfactants and Cd74 transcripts following G9a inhibition (n=4±SEM; p<0.05) or sh Ehmt2 vs. shcontrol (control) (two-tailed paired t-test n=4±SEM; p<0.05) (B) Relative expression of surfactants and Cd74 transcripts following G9a inhibition (n=4±SEM; p<0.05) or sh Ehmt2 vs. shcontrol (control) (n=2±SEM)

Article Snippet: The following antibodies were used for flow cytometry: CD74-BUV395 (BD, 740274, In-1, 1:25), Pro-SPC (Abcam, ab170699: 1:200), G9a (Abcam, ab185050, EPR18894).

Techniques: Expressing, Inhibition, Control, Two Tailed Test

Journal: bioRxiv

Article Title: G9a methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.1101/2020.04.20.050328

Figure Lengend Snippet: Relative expression of AT2 markers in tRFP-sorted tumor cells derived from primary recipients expressing either shcontrol or sh Ehmt2 (n=3; mean±SEM;, Sftpc, Slc34a2 , Lamp3, Cd74; two-tailed paired t-test p<0.05).

Article Snippet: The following antibodies were used for flow cytometry: CD74-BUV395 (BD, 740274, In-1, 1:25), Pro-SPC (Abcam, ab170699: 1:200), G9a (Abcam, ab185050, EPR18894).

Techniques: Expressing, Derivative Assay, Two Tailed Test