Journal: Nature

Article Title: LKB1 loss links serine metabolism to DNA methylation and tumourigenesis

doi: 10.1038/nature20132

Figure Lengend Snippet: a, Heat map of RNA-seq data showing levels of the differentially regulated SAM-utilizing enzymes. Plotted data are expressed as mean centered values. b, Expression of DNMT1 and DNMT3A in K, KL, and KL cells transduced with LKB1 cDNA (rescue) was measured by qRT-PCR. Levels were normalized to 18S rRNA. Data are expressed as relative to K cells (n=4, representative of two experiments). c, Immunoblots of lysates from K, KL or ‘rescue’ cells were probed for DNMT1 or DNMT3A. Actin is used as loading control. For gel source see Supplementary Data Figure 1. d, Measurement of SAM in K and KL cells treated with 5-Aza-2-deoxycytidine (Decitabine) or RG108 for 3 days. In each case, data are expressed as relative to the amount of SAM in K-vehicle treated cells that is arbitrarily set to 1 (n=6 independent replicates). e, Immunofluorescence staining and quantitation of 5-methyl-cytosine (5mC) in K or KL cells (77–130 cells). Scale bar, 25 μm. f, Dot blot of DNA isolated from K or KL cells probed with anti-5-methyl-cytosine antibody (5mC). Quantitated signal was normalized to total DNA as measured by methylene blue staining (n=4, independent replicates). g, Dot blot of DNA isolated from KL cells transduced with empty vector or LKB1 cDNA probed with anti-5mC antibody. Quantitated signal was normalized to total DNA as measured by methylene blue staining (n=3, independent replicates). h, Immunoblot analysis of histone 3 (H3) methyl marks from K or KL cells. Data are normalized to total H3 (K4me3, n=2, K27me3, n=5, K36me3, n=5, independent replicates). For gel source see Supplementary Data Figure 1. i, Dot blot of DNA isolated from K or KL cells probed with anti-5-hydroxymethyl-cytosine antibody (5hmC). Quantitated signal is normalized to total DNA as measured by methylene blue staining (n=4, independent replicates). For all panels, error bars are standard deviation unless otherwise stated and statistical significance is indicated as follows: *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Laminin (23017-95), 2NBDG (N13195), DCFDA (C6827), CellROX Green ( C10444 ), CyQuant NF ( C35006 ), bovine pituitary extract (13038-14), HBSS, RPMI, DMEM, DMEM:F12, MEM penicillin-streptomycin, pyruvate solution, fetal bovine serum (FBS) Alexa 488 and 568-conjugated secondary antibodies from Life Technologies; nicotinamide (N3376), trypsin inhibitor (T6522), dexamethasone (D4902), S-adenosylmethionine (A2408), 3,3,5-tri-iodo-L-thyronine (91990), oligomycin (75351), antimycin A (A8674), 2-deoxyglucose (D6134), dichloroacetate (347795), collagenase (C7657), glucose (G7528), aminooxyacetate ( C13408 ), N-acetylcysteine (A9165), adenosine (A4036), guanosine (G6264), thymidine (T1895), uridine (U3003), cytidine (C4654), cycloleucine (A48105), cholera toxin (C8052), L-serine (S4500), betaine (61962), dimethylglycine (D1156), tamoxifen (T5148) and methylene blue (M4159) from Sigma; FCCP (0453), decitabine (2624), RG108 (3295), EGCG (4524), Compound C (3093), Torin 1 (4247) and galloflavin (4795) from Tocris; mouse EGF (354001), NuSerum IV (355104), ITS+ Premix (354352) and matrigel (354234) from Corning; SGI1027 (S7276) from Selleckchem; caspase-Glo 3/7 kit (G811A) and Celltiter-Glo kit (G755B) from Promega; 13 C-U-Glucose (CLM-1396-1) and 15 N-Glutamine (NLM-1016-1) from Cambridge Isotope Laboratories Inc; dispase (165-859) from Roche; lactate assay kit (K607-100) from Biovision Inc; Vectashield with DAPI (H1500) from Vector Laboratories; Bridge-IT SAM assay kit (1-1-1003B) from Medionics; meDIP kit (55009) from Active Motif; 3DZA (9000785) from Cayman Chemicals; Adeno-FlpO (1760) from Vector Biolabs; Adeno-Cre-eGFP from the Viral Vector Core Facility, University of Iowa.

Techniques: Expressing, DNA Methylation Assay, RNA Sequencing, Transduction, Quantitative RT-PCR, Western Blot, Control, Immunofluorescence, Staining, Quantitation Assay, Dot Blot, Isolation, Plasmid Preparation, Standard Deviation

Journal: Nature

Article Title: LKB1 loss links serine metabolism to DNA methylation and tumourigenesis

doi: 10.1038/nature20132

Figure Lengend Snippet: a–c, Proliferation of K (a), KL (b) and KPC (c) cells transduced with shControl or two hairpins against each of DNMT1 (D1) or DNMT3A (D3A). Data are expressed as relative to day 0 (K and KL n=6, KPC n=4). d–f, Volume of subcutaneous tumours derived from KPC cells transduced with Doxcycline (Dox)-inducible hairpins against DNMT1 or DNMT3A (n=4). Dox was introduced to the drinking water when tumours reached 125 mm3. Error bars are s.e.m. For source data on tumour volume, see Supplementary Data Table 4. g, Apoptosis measured by caspase 3/7 activity in K or KL cells treated with decitabine for 48 hrs. Values are normalized to cell number (n=3). h and i, Proliferation of KL cells transduced with empty vector (h) or LKB1 cDNA (i) treated with decitabine. Data are expressed as relative to day 0 (n=3). j, Proliferation of KL cells transduced with empty vector or LKB1 cDNA treated with RG108. Data are expressed as percentage of growth of untreated cells (n=6). k–m, Proliferation of K or KL cells treated with RG108 (n=6) (k), EGCG (n=12) (l) or SGI1027 (n=12) (m). Data are expressed as percentage of growth of untreated cells. n–q, Mice bearing subcutaneous KPC tumours were treated with decitabine (n=12) or vehicle (n=12) when tumours reached 125 mm3. n and o, Tumour volume (n) and final tumour weight (o). Error bars are s.e.m. For source data on tumour volume, see Supplementary Data Table 4. p, H&E stained slides from representative tumours (upper panels). Lower panels: Anti-CK19 (green) was used to visualize the neoplastic epithelium and anti-PCNA (red) was used to mark proliferating cells. DAPI was used to stain nuclei (blue). q, Quantitation of the CK19+ neoplastic epithelial compartment (%CK19+ cells/total cells) (Upper). Quantification of CK19+ cells with nuclear PCNA staining (Lower) (n=6). Scale bars, 100 μm. Insets are three-fold magnification. Data pooled from 2 (a–c, j) or four (k–m) experiments or representative of two (h) or three (g) experiments. For all panels, error bars are standard deviation unless otherwise stated and statistical significance is indicated as follows: *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Laminin (23017-95), 2NBDG (N13195), DCFDA (C6827), CellROX Green ( C10444 ), CyQuant NF ( C35006 ), bovine pituitary extract (13038-14), HBSS, RPMI, DMEM, DMEM:F12, MEM penicillin-streptomycin, pyruvate solution, fetal bovine serum (FBS) Alexa 488 and 568-conjugated secondary antibodies from Life Technologies; nicotinamide (N3376), trypsin inhibitor (T6522), dexamethasone (D4902), S-adenosylmethionine (A2408), 3,3,5-tri-iodo-L-thyronine (91990), oligomycin (75351), antimycin A (A8674), 2-deoxyglucose (D6134), dichloroacetate (347795), collagenase (C7657), glucose (G7528), aminooxyacetate ( C13408 ), N-acetylcysteine (A9165), adenosine (A4036), guanosine (G6264), thymidine (T1895), uridine (U3003), cytidine (C4654), cycloleucine (A48105), cholera toxin (C8052), L-serine (S4500), betaine (61962), dimethylglycine (D1156), tamoxifen (T5148) and methylene blue (M4159) from Sigma; FCCP (0453), decitabine (2624), RG108 (3295), EGCG (4524), Compound C (3093), Torin 1 (4247) and galloflavin (4795) from Tocris; mouse EGF (354001), NuSerum IV (355104), ITS+ Premix (354352) and matrigel (354234) from Corning; SGI1027 (S7276) from Selleckchem; caspase-Glo 3/7 kit (G811A) and Celltiter-Glo kit (G755B) from Promega; 13 C-U-Glucose (CLM-1396-1) and 15 N-Glutamine (NLM-1016-1) from Cambridge Isotope Laboratories Inc; dispase (165-859) from Roche; lactate assay kit (K607-100) from Biovision Inc; Vectashield with DAPI (H1500) from Vector Laboratories; Bridge-IT SAM assay kit (1-1-1003B) from Medionics; meDIP kit (55009) from Active Motif; 3DZA (9000785) from Cayman Chemicals; Adeno-FlpO (1760) from Vector Biolabs; Adeno-Cre-eGFP from the Viral Vector Core Facility, University of Iowa.

Techniques: DNA Methylation Assay, Transduction, Derivative Assay, Activity Assay, Plasmid Preparation, Staining, Quantitation Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: The integrative genomic and functional immunological analyses of colorectal cancer initiating cells to modulate stemness properties and the susceptibility to immune responses

doi: 10.1186/s12967-025-06176-0

Figure Lengend Snippet: Expression of HLA and APM molecules in CRC-CICs and FBS tumor cells following or not the treatment of the cells with epigenetic or immunomodulatory agents. The assessment of the expression of HLA (HLA-ABC, HLA-Class II, ICAM1; A ) and APM (β2-microglobulin, Calnexin, Calreticulin LMP2, LMP7, LMP10, TAP1, TAP2 and Tapasin; B ) markers was performed through flow cytometry in primary CRC-CSCs (N = 6) and FBS tumor cell lines (N = 3). Cell lines were treated or not (UT, A , B ) with either 1000 IU/mL IFN-γ for 48 h ( C , D ) or 5 µM of 5-Azacytidine (5-Aza) or 5 µM of RG108 ( E , F ) or for HDACi (1.25 µM Vorinostat + 0.3 µM Mithramycin A) for 24 h or overnight with 1 mM Buthyrate ( G , H ). The primary CRC-CSCs (N = 3, I , J ) and differentiated pair cell lines (N = 3; K , L ) were transfected for 48 h with miR-15a mimics and then the expression of HLA-ABC, HLA-A, HLA-Class, Calnexin, Calreticulin and ErP57 molecules was assessed by flow cytometry. Data in the Figure are represented as relative mean of intensity of fluorescence (rMFI) calculated for each marker as the ratio of the mean of intensity of fluorescence between the stained and unstained samples. Means ± SD values from three experiments are shown. * p < 0.05, ** p < 0.01

Article Snippet: Cells were cultured at a concentration of 0.2 × 10 6 cells/ml in 6-well plates with 5 ml/well of specific CIC or FBS medium and incubated with the following agents: IFN-γ (1000 IU/ml; Peprotech, Cat # 300-02-500) for 48 h; Vorinostat (1.25 μM, Selleckchem, Cat #S104713), Mithramycin A (0.3 μM, Sigma, Cat # 1489/1), RG108 (5 μM, Selleckchem, Cat #S2821) and 5-azacytidine (5-Aza, 5 μM, Selleckchem, Cat #S1782) for 24 h; Butyrate (1 mM, Stemcell, Cat #72242) for 12 h. The agents were dissolved according to the manufacturers’ guidelines.

Techniques: Expressing, Flow Cytometry, Transfection, Fluorescence, Marker, Staining

Journal: Biomedicines

Article Title: Senescent Markers Expressed by Periodontal Ligament-Derived Stem Cells (PDLSCs) Harvested from Patients with Periodontitis Can Be Rejuvenated by RG108

doi: 10.3390/biomedicines11092535

Figure Lengend Snippet: Effect of RG108 of PDLSCs. RG108 did not significantly affect growth rate ( A ) and PDT ( B ). The anti-apoptotic gene BCL2 was significantly less expressed in pPDLSCs than in hPDLSCs, p < 0.05 ( C ). Treatment with RG108 at 100 μM increased BCL2 expression in pPDLSCs p < 0.05 ( C ). A trend for a higher level of Annexin V expression was detected in pPDLSCs compared to hPDLSCs ( D – F ).

Article Snippet: RG108 was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany), reconstituted in DMSO (Sigma Aldrich, Burlington, MA, USA) according to the instructions, and stored at −20 °C.

Techniques: Expressing

Journal: Biomedicines

Article Title: Senescent Markers Expressed by Periodontal Ligament-Derived Stem Cells (PDLSCs) Harvested from Patients with Periodontitis Can Be Rejuvenated by RG108

doi: 10.3390/biomedicines11092535

Figure Lengend Snippet: RG108 affects the senescent phenotype of pPDLSCs. RG108 did not show any significant variation of p16 and p21 expression in hPDLSCs for any of the conditions ( A ). In pPDLSCs, RG108 at 100 μM induced a significant reduction of both p16 and p21, p < 0.05 ( B ). The expression of the stemness genes, SOX2, OCT4, and NANOG was not significantly modulated in hPDLSCs ( C ), whereas an increase of SOX2 ( p < 0.05) and of OCT4 ( p < 0.01) was induced by treatment with RG108 at 100 μM ( D ). RG108 decreased the subset of pPDLSCs co-expressing OCT4 and p21 ( p < 0.05), while no significant modulation was detected on hPDLSCs ( E ).

Article Snippet: RG108 was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany), reconstituted in DMSO (Sigma Aldrich, Burlington, MA, USA) according to the instructions, and stored at −20 °C.

Techniques: Expressing

Journal: Biomedicines

Article Title: Senescent Markers Expressed by Periodontal Ligament-Derived Stem Cells (PDLSCs) Harvested from Patients with Periodontitis Can Be Rejuvenated by RG108

doi: 10.3390/biomedicines11092535

Figure Lengend Snippet: RG108 stimulates the adipogenic potential in pPDLSCs. RG108 did not affect chondrogenic and osteogenic capabilities in both hPDLSCs and pPDLSCs ( A – D ), while a significant up-regulation of PPARγ was detected in pPDLSCs, p < 0.01 ( E , F ). pPDLSCs treated with RG108 increased their adipogenic potential as evidenced by the major formation of cells containing lipid droplets in pPDLSCs ( G , H ).

Article Snippet: RG108 was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany), reconstituted in DMSO (Sigma Aldrich, Burlington, MA, USA) according to the instructions, and stored at −20 °C.

Techniques: