Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Up-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Down-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Reporter assays detected stimulation of p53-mediatd transactivation by MEG3 deletion mutants M1, M1+M2, M3, M2+M3 in HepG2 cells. The value are means of three independent experiments ±S.D, * P<0.05. (B) Cropped blots show the increased level of p53 protein 48h after transfection of the pcDNA-MEG3 in HepG2 cells. (C) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in HepG2 cells (0, 0.5,1, 2h) overexpressing MEG3. (D) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in doxo-induced HepG2 cells (0, 2,4, 6h)overexpressing MEG3.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Transfection, Over Expression

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 incubated with HEK293 cell extract transfected with Flag-p53 vector. (B) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 and deletion mutations RNA incubated with doxo-induced HepG2 cell extract (MEG3 deletion mutants M1, M1+M2, M3, M2+M3 are shown in ). (C) In vitro transcribed biotinylated MEG3 retrieved purified GST-p53 but not GST. (D) RIP experiments were performed using an antibody against the p53 on extracts from doxo-induced HepG2 cells. The purified RNA was used for qRT-PCR, and the enrichment of the lncRNA MEG3 was normalized to GAPDH (upper, western blot of p53 protein after immunoprecipitation). Data was relative to mock-IP (IgG). (E) A series of p53 deletion mutants which were flag-tagged was treated as in (A), and association was detected by anti-Flag. Up represents successful expression of p53 deletion mutants. Down represents in vitro transcribed biotinylated MEG3 RNA was incubated with HEK293 cell extract which was transfected with p53 deletion mutants and associated proteins were detected by anti-Flag.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Western Blot, In Vitro, Incubation, Transfection, Plasmid Preparation, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) SK-Hep–1 hepatocellular carcinoma cell lines with stably expression of MEG3 were determined by qRT-PCR. The value are means of three independent experiments ±SD, *** P<0.001. (B) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH. The bars represent the relative fold change of these genes in SK-Hep–1 after transfection with pcDNA3.0-MEG3 compared with blank vector pcDNA3.0. (C) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH after knockdown p53 level in HepG2 cells. The bars represent the relative fold change of these genes in HepG2 cells after co-transfection of pcDNA3.0 and NC, pcDNA3.0-MEG3 and NC, pcDNA3.0-MEG3 and sip53, pcDNA3.0 and sip53, respectively. (D) A schematic diagram illustrating how MEG3 can function as tumor suppressor through interactions with p53.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Cotransfection

Journal: Cell reports

Article Title: A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration

doi: 10.1016/j.celrep.2022.111511

Figure Lengend Snippet:

Article Snippet: Anti-human CD31 antibody, REA730 , Miltenyi Biotec , Cat#: 130-110-670; RRID:AB_2657283.

Techniques: Plasmid Preparation, Virus, Clone Assay, Recombinant, CCK-8 Assay, Viability Assay, Microarray, Knock-Out, Software, Functional Assay

Journal: Cancers

Article Title: Function of p21 (Cip1/Waf1/ CDKN1A ) in Migration and Invasion of Cancer and Trophoblastic Cells

doi: 10.3390/cancers11070989

Figure Lengend Snippet: Knockdown of p21 barely impacts proliferation and cell cycle distribution of choriocarcinoma or trophoblastic cells. ( A ) Real-time PCR of CDKN1A (p21) and TP53 (p53). The results are presented as RQ with minimum and maximum range. RQ: relative quantification of gene expression by setting p21 of HTR cells as 1 or p53 of Jar cells, respectively. ( B ) Western blot analysis of HTR, BeWo, Jar, and JEG-3 cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. ( C ) HTR cells were treated with control small interferingRNA (siRNA) (sicon) or siRNA against p21 (sip21 #1) for 0, 24, 48, and 72 h. Cell viability was measured via CellTiter-Blue ® assay (Promega, Mannheim, Germany). The results are presented as mean ± standard error of the mean (SEM) ( n = 2, each experiment in triplicates) and statistically analyzed compared to sicon. All differences were not significant. ( D ) Cell viability assay of BeWo cells treated as in (C). ( E ) Fluorescence-activated cell scanning (FACS) measurements of HTR cells for cell cycle distribution. The results are presented as mean ± SEM from three independent experiments. ( F ) Cellular extracts from HTR cells were prepared for Western blot analyses with indicated antibodies. GAPDH served as loading control. ( G ) FACS measurements of BeWo cells as in ( E ). ( H ) Cellular extracts from BeWo cells were prepared for Western blot analyses with indicated antibodies. GAPDH served as loading control.

Article Snippet: The following antibodies were used for Western blot analysis: Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GTX627408) from GeneTex (BIOZOL, Eiching, Germany), p53 (sc-126) from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit polyclonal antibodies against ERK3/MAPK6 (#4067) and PARP (#9542) from Cell Signaling (Frankfurt, Germany), rabbit polyclonal antibody against ERK1/2 (#06-182) from Merck Millipore (Darmstadt, Germany), and rabbit monoclonal antibody against p21 (#2947) from Cell Signaling.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Gene Expression, Western Blot, Control, CtB Assay, Viability Assay, Fluorescence

Journal: Cancers

Article Title: Function of p21 (Cip1/Waf1/ CDKN1A ) in Migration and Invasion of Cancer and Trophoblastic Cells

doi: 10.3390/cancers11070989

Figure Lengend Snippet: Suppression of p21 affects chromosome segregation in BeWo cells. ( A ) Western blot control for small interfering RNA (siRNA) transfection efficiency. BeWo cells were treated with scrambled siRNA (sicon) or siRNA against the untranslated region (UTR) of p21 (sip21 #1) or mixed siRNAs against the coding region of p21 (sip21 #2) for 48 h. ( B ) Quantification of defects in chromosome congression in metaphase cells treated as in (A) ( n = 3, 100 metaphase cells per experiment and condition). The results are presented as mean ± standard error of the mean (SEM) and statistically analyzed. ( C ) Quantification of defects in chromosome segregation in anaphase cells treated as in (A) ( n = 3, 100 anaphase cells per experiment and condition). The results are presented as mean ± SEM and statistically analyzed. ** p < 0.01; *** p < 0.001. ( D ) Representative images of confocal laser scanning microscopy are shown. Cells were stained for tubulin, pericentrin, anti-centromere antibody (ACA), and DNA. Scale bar: 7.5 µm. Arrow: Indicating either chromosome congression/segregation defect (4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining) or failure of centrosome integrity (pericentrin staining).

Article Snippet: The following antibodies were used for Western blot analysis: Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GTX627408) from GeneTex (BIOZOL, Eiching, Germany), p53 (sc-126) from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit polyclonal antibodies against ERK3/MAPK6 (#4067) and PARP (#9542) from Cell Signaling (Frankfurt, Germany), rabbit polyclonal antibody against ERK1/2 (#06-182) from Merck Millipore (Darmstadt, Germany), and rabbit monoclonal antibody against p21 (#2947) from Cell Signaling.

Techniques: Western Blot, Control, Small Interfering RNA, Transfection, Confocal Laser Scanning Microscopy, Staining

Journal: Cancers

Article Title: Function of p21 (Cip1/Waf1/ CDKN1A ) in Migration and Invasion of Cancer and Trophoblastic Cells

doi: 10.3390/cancers11070989

Figure Lengend Snippet: p21 is required for cell motility. Single-cell tracking using time-lapse microscopy was performed with treated HTR cells. ( A – C ) Representative trajectories of individual cells ( n = 30) treated with sicon (A), sip21 #1 ( B ), or sip21 #2 (C) are shown. ( D ) Control Western blot analysis shows the efficient knockdown of endogenous p21 in HTR cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. ( E – G ) Accumulated distance (E), velocity (F), and directionality ( G ) of these cells were analyzed and statistically evaluated from three independent experiments. The results are shown as scatter plots. *** p < 0.001. ( H – J ) Representative trajectories are shown for individual HeLa cells ( n = 30) with sicon ( H ), sip21 #1 ( I ), and sip21 #2 (J). ( K ) Control Western blot analysis of HeLa cells. GAPDH served as loading control. ( L – N ) Accumulated distance (L), velocity (M), and directionality (N) were evaluated. The data are derived from three independent experiments and shown as scatter plots. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were used for Western blot analysis: Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GTX627408) from GeneTex (BIOZOL, Eiching, Germany), p53 (sc-126) from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit polyclonal antibodies against ERK3/MAPK6 (#4067) and PARP (#9542) from Cell Signaling (Frankfurt, Germany), rabbit polyclonal antibody against ERK1/2 (#06-182) from Merck Millipore (Darmstadt, Germany), and rabbit monoclonal antibody against p21 (#2947) from Cell Signaling.

Techniques: Single Cell Tracking, Time-lapse Microscopy, Control, Western Blot, Knockdown, Derivative Assay

Journal: Cancers

Article Title: Function of p21 (Cip1/Waf1/ CDKN1A ) in Migration and Invasion of Cancer and Trophoblastic Cells

doi: 10.3390/cancers11070989

Figure Lengend Snippet: The invasion capacity is impaired in cells with reduced p21 levels. ( A ) Schedule of invasion assay. Different cell lines were treated with control small interfering RNA (siRNA) (sicon) or two distinct siRNA against p21 (sip21 #1 or #2). ( B ) Quantification of invaded HTR cells. The total number of invaded cells in the control group was assigned as 100%. ( C ) Representatives of invaded HTR cells are shown. Scale bar: 20 μm. ( D ) Control Western blot analysis showing the efficient knockdown of endogenous p21 in HTR cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. ( E ) Quantification of invaded Jar cells. ( F ) Representatives of invaded Jar cells are shown. Scale bar: 20 μm. ( G ) Control Western blot analysis of Jar cells. GAPDH served as loading control. ( H ) Quantification of invaded SGHPL-4 cells. ( I ) Representatives of invaded SGHPL-4 cells are shown. Scale bar: 20 μm. ( J ) Control Western blot analysis of SGHPL-4 cells. GAPDH served as loading control. ( K ) Quantification of invaded HIPEC-65 cells. ( L ) Representatives of invaded HIPEC-65 cells. Scale bar: 20 μm. ( M ) Control Western blot analysis of HIPEC-65 cells. GAPDH served as loading control. The results from each cell line are presented as mean ± standard error of the mean (SEM) from three independent experiments. * p < 0.05.

Article Snippet: The following antibodies were used for Western blot analysis: Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GTX627408) from GeneTex (BIOZOL, Eiching, Germany), p53 (sc-126) from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit polyclonal antibodies against ERK3/MAPK6 (#4067) and PARP (#9542) from Cell Signaling (Frankfurt, Germany), rabbit polyclonal antibody against ERK1/2 (#06-182) from Merck Millipore (Darmstadt, Germany), and rabbit monoclonal antibody against p21 (#2947) from Cell Signaling.

Techniques: Invasion Assay, Control, Small Interfering RNA, Western Blot, Knockdown

Journal: Cancers

Article Title: Function of p21 (Cip1/Waf1/ CDKN1A ) in Migration and Invasion of Cancer and Trophoblastic Cells

doi: 10.3390/cancers11070989

Figure Lengend Snippet: Extracellular signal-regulated kinase 3 (ERK3) is reduced in p21-depleted cell lines. ( A – C ) Microarray analysis. HTR cells were treated with sicon or sip21 #1 for 48 h and the RNA from three independent experiments was extracted. ( A ) Western blot control of HTR cells with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. ( B ) The effects of sip21 #1 on different pathways were determined by gene ontology enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) . ( C ) Heatmap of the most differently expressed genes. Gene expression was analyzed using HumanHT-12 v4 beadchip array (Illumina, San Diego, CA, USA). Genes with a p -value < 0.01, and a fold change greater than 2 (red color code) and below 0.5 (blue color code), respectively, are included. ( D ) Western Blot of HTR cells treated with control siRNA (sicon) or two distinct siRNA against p21 (sip21 #1 or #2) for 48 h. Ratio of ERK3/GAPDH is shown. GAPDH served as loading control. ( E , F ) The gene levels of p21 (E) and ERK3 (F) were measured. ( G ) Western blot analyses of Jar cells treated as in (D). ( H , I ) The gene levels of p21 (H) and ERK3 (I). ( J ) Western blot analyses of HeLa cells treated as in (D). ( K , L ) The gene levels of p21 (K) and ERK3 (L). The mRNA data are based on three experiments and presented as RQ with minimum and maximum range. RQ: relative quantification of gene expression. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were used for Western blot analysis: Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GTX627408) from GeneTex (BIOZOL, Eiching, Germany), p53 (sc-126) from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit polyclonal antibodies against ERK3/MAPK6 (#4067) and PARP (#9542) from Cell Signaling (Frankfurt, Germany), rabbit polyclonal antibody against ERK1/2 (#06-182) from Merck Millipore (Darmstadt, Germany), and rabbit monoclonal antibody against p21 (#2947) from Cell Signaling.

Techniques: Microarray, Western Blot, Control, Gene Expression, Quantitative Proteomics

Journal: Cancer Gene Therapy

Article Title: m 6 A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer

doi: 10.1038/s41417-022-00451-8

Figure Lengend Snippet: PC9 cells were transfected with siNC, siALKBH5#1, or siALKBH5#3 for 96 h ( n = 3 for each group). A , B Differentially expressed genes (DEGs) for siALKBH5#1 ( A ) or siALKBH5#3 ( B ) were detected using expression microarray and shown using volcano plots. Dashed lines indicate the threshold for the differential expression [fold change > 1.5 (log2 fold change = 0.5849) or < 0.67 (log2 fold change = −0.5849), P < 0.01 via Student’s t -test] for upregulated (pink dots) or downregulated (light blue dots) genes. C Venn diagram indicating the number of common DEGs in ALKBH5-knockdown cells with different siRNA sequences. D mRNA expression levels of genes related to cell proliferation in ALKBH5-knockdown PC9 cells were analyzed using qPCR. Gene expression was normalized to the GAPDH expression and was shown relative to the expression with siNC. E m 6 A level in the 3′ UTRs of target mRNA in PC9 cells transfected with siALKBH5#1 or siALKBH5#3 was quantified via MeRIP qPCR using anti-m6A antibody and was compared with that in cell transfected with siNC. The m 6 A level was normalized to that of the input fraction ( n = 3). IgG was used to evaluate the nonspecific binding of the target mRNA. ( F, G ; Upper panel) Prediction scores of m 6 A modification in the CDKN1A and TIMP3 genes were calculated using the SRAMP algorithm. The combined scores were distributed through the full-length mRNA as different levels of very high, high, moderate, and low confidence. Arrows show the location of qPCR primers. Adenosines in consensus sequences for m 6 A modification are presented in red. (Lower panel) PolyA-enriched RNA extracted from PC9 cells transfected with siALKBH5#1, siALKBH5#3, or siNC ( n = 3) was immunoprecipitated using anti-m 6 A antibody or normal IgG. The m 6 A level was calculated from transcript abundance in input or MeRIP fraction quantified using qPCR. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates a significant difference between the indicated groups.

Article Snippet: Primary antibodies for ALKBH5 (1:1000 dilution, HPA007196; Atlas Antibodies), FTO (1:1000 dilution, Ab124892; Abcam), IGF2BP1 (1:1000 dilution, 22803-1-AP; Proteintech), IGF2BP2 (1:1000 dilution, 11601-1-AP; Proteintech), IGF2BP3 (1:2000 dilution, 14642-1-AP; Proteintech), YTHDF2 (1:400, Ab170118, Abcam), TIMP3 (1:3000 dilution, Ab39184; Abcam), p21 (1:1000 dilution, A1483; ABclonal, Woburn, MA, USA), E2F1 (1:500 dilution, A2067; ABclonal), CCNG2 (0.2 μg/mL, Ab251826; Abcam), p53 (1:200 dilution, Sc-126; SANTA CRUZ BIO TECHNOLOGY, Dallas, TX, USA), and GAPDH (1:1000 dilution, Ab8245; Abcam) were incubated for overnight at 4°C.

Techniques: Transfection, Expressing, Microarray, Binding Assay, Modification, Immunoprecipitation

Journal: Cancer Gene Therapy

Article Title: m 6 A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer

doi: 10.1038/s41417-022-00451-8

Figure Lengend Snippet: A A schematic outline showing the workflow for the analysis of downstream targets of ALKBH5. B Target protein levels in PC9 or A549 cells transfected with siALKBH5#1 or siALKBH5#3 were compared with those transfected with siNC via western blot analysis. C Western blot analysis demonstrated CDKN1A (p21) or TIMP3 protein levels in PC9 cells transfected with siALKBH5, siCDKN1A, both siALKBH5 and siCDKN1A, or siNC. D Western blot analysis demonstrated TIMP3 protein levels in A549 cells transfected with siALKBH5, siTIMP3, both siALKBH5 and siTIMP3, or siNC. E , F Cell proliferation relative to baseline in PC9 ( E ) and A549 ( F ) cells transfected with siALKBH5 was assessed via the CCK-8 assay and compared with that in cells cotransfected with siALKBH5 and siCDKN1A or siTIMP3 ( n = 3). *** P < 0.001, **** P < 0.0001 indicates a significant difference between the indicated groups.

Article Snippet: Primary antibodies for ALKBH5 (1:1000 dilution, HPA007196; Atlas Antibodies), FTO (1:1000 dilution, Ab124892; Abcam), IGF2BP1 (1:1000 dilution, 22803-1-AP; Proteintech), IGF2BP2 (1:1000 dilution, 11601-1-AP; Proteintech), IGF2BP3 (1:2000 dilution, 14642-1-AP; Proteintech), YTHDF2 (1:400, Ab170118, Abcam), TIMP3 (1:3000 dilution, Ab39184; Abcam), p21 (1:1000 dilution, A1483; ABclonal, Woburn, MA, USA), E2F1 (1:500 dilution, A2067; ABclonal), CCNG2 (0.2 μg/mL, Ab251826; Abcam), p53 (1:200 dilution, Sc-126; SANTA CRUZ BIO TECHNOLOGY, Dallas, TX, USA), and GAPDH (1:1000 dilution, Ab8245; Abcam) were incubated for overnight at 4°C.

Techniques: Transfection, Western Blot, CCK-8 Assay

Journal: Cancer Gene Therapy

Article Title: m 6 A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer

doi: 10.1038/s41417-022-00451-8

Figure Lengend Snippet: A Expression levels of IGF2BP1, IGF2BP2, and IGF2BP3 (IGF2BPs) protein determined using western blot analysis were compared between cell lines. B IGF2BP protein levels in cells transfected with siALKBH5 (left end), those in cells transfected with siIGF2BP1, siIGF2BP2, or siIGF2BP3 with or without siALKBH5 (middle two lanes), or those with siNC (right end indicating both siALKBH5 and siIGF2BPs were negative) were confirmed via western blot analysis. C Relative mRNA expression levels of CDKN1A in PC9 cells or those of TIMP3 in A549 cells transfected with siALKBH5 were analyzed via qPCR and compared with those in cells cotransfected with siALKBH5 and one of the siIGF2BPs. Gene expression was normalized to the GAPDH expression and was shown relative to the expression in siNC ( n = 3). D Expression levels of YTHDF2 protein determined using western blot analysis were compared between cell lines. E Relative mRNA expression levels of CDKN1A in PC9 cells or those of TIMP3 in A549 cells transfected with siALKBH5 were analyzed via qPCR and compared with those in cells cotransfected with siALKBH5 and siYTHDF2. Gene expression was normalized to the GAPDH expression and was shown relative to the expression in siNC ( n = 3). F The remaining RNA level of CDKN1A in PC9 cells or of TIMP3 in A549 cells after actinomycin D treatment for 0, 2, 4, and 6 h was determined using qPCR and normalized to the expression at 0 h. RNA decay rate in cells transfected with siALKBH5 and/or one of the siIGF2BPs and siNC were compared with the stability of CDKN1A and TIMPs ( n = 3). G Cell proliferation relative to baseline in PC9 and A549cells transfected with siALKBH5 was assessed via the CCK-8 assay and compared with that in cells cotransfected with siALKBH5 and one of the siIGF2BPs ( n = 3). H Schematic illustration for the proposed mechanism of tumorigenicity via ALKBH5 in non-small-cell lung cancer. Upregulation of ALKBH5 in NSCLC reduces m 6 A modifications on the 3′ UTR of specific genes. The loss of m 6 A decreases the opportunity for recognition by IGF2BPs and destabilizes the target transcripts such as CDKN1A (p21) and TIMP3. Downregulation of CDKN1A (p21) and TIMP3 induces cell cycle alteration and inhibits apoptosis. This ALKBH5–IGF2BPs axis promotes cell proliferation and tumorigenicity, which, in turn, causes the unfavorable prognosis of NSCLC. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates a significant difference between the indicated groups.

Article Snippet: Primary antibodies for ALKBH5 (1:1000 dilution, HPA007196; Atlas Antibodies), FTO (1:1000 dilution, Ab124892; Abcam), IGF2BP1 (1:1000 dilution, 22803-1-AP; Proteintech), IGF2BP2 (1:1000 dilution, 11601-1-AP; Proteintech), IGF2BP3 (1:2000 dilution, 14642-1-AP; Proteintech), YTHDF2 (1:400, Ab170118, Abcam), TIMP3 (1:3000 dilution, Ab39184; Abcam), p21 (1:1000 dilution, A1483; ABclonal, Woburn, MA, USA), E2F1 (1:500 dilution, A2067; ABclonal), CCNG2 (0.2 μg/mL, Ab251826; Abcam), p53 (1:200 dilution, Sc-126; SANTA CRUZ BIO TECHNOLOGY, Dallas, TX, USA), and GAPDH (1:1000 dilution, Ab8245; Abcam) were incubated for overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, CCK-8 Assay

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) The interaction between LOXL2 and PEAR1 was detected by pulldown with Dynabeads in the supernatant of MDA-MB-231 cells, to which 15 μg exogenous PEAR1-ECD-his was added. ( B ) LOXL2 levels in the supernatants of various TNBC cell lines were quantified by ELISA (MDA-MB-231, red; MDA-MB-468, orange; SUM159, yellow). The complete culture medium served as a negative control (blue) ( n = 2; mean ± SD). ( C and D ) Quantification of invasive cells in Transwell assay; and relative migration area in wound healing assay of MDA-MB-231 cells treated with 0, 5, or 10 ng/mL LOXL2 ( n = 3; mean ± SEM). ( E ) Phosphorylated PEAR1, full-length CD44, and CD44-ICD levels were detected in MDA-MB-231 cells and SUM159 cells treated with 0, 5, 10, 20, or 50 ng/mL LOXL2. ( F ) Schematic showing the structures of full-length LOXL2 protein, the LOXL2-SRCR1-3 truncation, and the LOXL2-SRCR1-2 truncation. ( G – I ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay ( n = 3; mean ± SEM) and detection of PEAR1 phosphorylation levels with Western blotting in MDA-MB-231 cells treated with 0.2 nM full-length LOXL2 or its truncation construct. ( J ) Interaction of PEAR1-ECD–His with LOXL2 in the supernatant of MDA-MB-231 cells was inhibited by 20 μg/mL simtuzumab. ( K and L ) Quantification of invasive cells in Transwell assay and relative migration area in wound healing assay in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab ( n = 3; mean ± SEM). ( M ) Levels of phosphorylated PEAR1, CD44, and CD44-ICD were determined in MDA-MB-231 cells treated with 0, 5, 10, or 20 μg/mL simtuzumab. One-way ANOVA followed by Dunnett’s test was used for C , D , G , H , K , and L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Article Snippet: Human breast tissue microarrays containing 126 breast tumor samples (stock HBre-Duc090Sur-01), 86 normal adjacent breast samples (HBreD145Su01), and 80 corresponding paracancer tissue samples (TNBC-1602) were acquired from Shanghai Outdo Biotech Co. IHC staining of the tissues for PEAR1 (HPA035217, Sigma-Aldrich), phospho-PEAR1 Ser891 (developed as described above), LOXL2 (67139-1-Ig, Proteintech), and CD44 (3570, Cell Signaling Technology) was performed.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Transwell Assay, Migration, Wound Healing Assay, Phospho-proteomics, Western Blot, Construct

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Schematic showing the structural features of PEAR1. ( B ) The interaction between LOXL2 and various PEAR1-ECD domain peptides was detected using ELISA ( n = 2; mean ± SD). ( C – E ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with or without 10 ng/mL LOXL2 and 1 or 5 μg/mL PEAR1-EMI domain protein. EMI del, EMI deletion. ( F – H ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of CD44 and CD44-ICD by Western blotting of MDA-MB-231 cells with oe-PEAR1-EMI domain deficiency. EMI del, EMI deletion. ( I – K ) Quantification of invasive cells in Transwell assay; quantification of relative migration area in wound healing assay ( n = 3; mean ± SEM); and detection of phosphorylated PEAR1, CD44, and CD44-ICD using Western blotting of MDA-MB-231 cells treated with 10 ng/mL LOXL2 and 5 or 10 μg/mL PEAR1 Fab-HSA. Quantitative analysis of the gray values of phospho–PEAR1 Ser/Thr to PEAR1, CD44 to GAPDH, and CD44-ICD to histone H3 ( n = 3; mean ± SD). ( L ) The number of metastatic foci in the lung and liver was significantly reduced by i.v. injection of PEAR1 Fab-HSA at a dose of 3.35 mg/kg for 40 days in a mouse model of metastasis generated by i.v. injection of MDA-MB-231 cells ( n = 5 mice per group; mean ± SEM). One-way ANOVA followed by Dunnett’s test was used for C , D , F , G , and I – L . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The Western blotting results are representative of 3 independent experiments.

Article Snippet: Human breast tissue microarrays containing 126 breast tumor samples (stock HBre-Duc090Sur-01), 86 normal adjacent breast samples (HBreD145Su01), and 80 corresponding paracancer tissue samples (TNBC-1602) were acquired from Shanghai Outdo Biotech Co. IHC staining of the tissues for PEAR1 (HPA035217, Sigma-Aldrich), phospho-PEAR1 Ser891 (developed as described above), LOXL2 (67139-1-Ig, Proteintech), and CD44 (3570, Cell Signaling Technology) was performed.

Techniques: Enzyme-linked Immunosorbent Assay, Transwell Assay, Migration, Wound Healing Assay, Western Blot, Injection, Generated

Journal: The Journal of Clinical Investigation

Article Title: LOXL2-induced PEAR1 Ser891 phosphorylation suppresses CD44 degradation and promotes triple-negative breast cancer metastasis

doi: 10.1172/JCI177357

Figure Lengend Snippet: ( A ) Quantification of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 staining scores by IHC in TNBC and corresponding adjacent samples ( n = 80 for TAT and tumor samples; mean ± SEM). ( B ) Overall survival of patients with TNBC based on PEAR1, phospho-PEAR1 (Ser891), LOXL2, and CD44 expression levels ( n values as indicated; log-rank test). ( C ) Heatmap of the correlation between the expression levels of PEAR1, phospho-PEAR1 Ser891, LOXL2, and CD44 in TNBC samples ( n = 80; Pearson’s correlation analysis). ( D ) The prognostic effect of the risk score of phospho-PEAR1 Ser891 and its combination with CD44 for patients with TNBC (time-dependent ROC curve analysis). ( E ) Schematic diagram of the mechanisms by which the LOXL2/PEAR1/CD44 pathway regulates TNBC metastasis. Unpaired 2-tailed t tests were used for A ; log-rank test was used for B ; Pearson’s correlation analysis was used for C ; time-dependent ROC curve analysis was used for D . **** P < 0.0001.

Article Snippet: Human breast tissue microarrays containing 126 breast tumor samples (stock HBre-Duc090Sur-01), 86 normal adjacent breast samples (HBreD145Su01), and 80 corresponding paracancer tissue samples (TNBC-1602) were acquired from Shanghai Outdo Biotech Co. IHC staining of the tissues for PEAR1 (HPA035217, Sigma-Aldrich), phospho-PEAR1 Ser891 (developed as described above), LOXL2 (67139-1-Ig, Proteintech), and CD44 (3570, Cell Signaling Technology) was performed.

Techniques: Staining, Expressing

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: shRNA mediated silencing of KLF4 expression in human osteosarcoma cell lines. a. and e. Western blot detected the expression of KLF4 in MG63 and SaOS2 cells. GAPDH was detected as loading control. b–c. and f–g. Colony formation assay was used to measure the clonogenicity of MG63 and SaOS2 cells with or without knockdown KLF4. The numbers of control cells were set as 100%.(n=3, mean ± SD, t-test, n**P<0.01 vs. shRNA CTR). d. and h. Cells with or without knockdown KLF4 were cultured for the days as indicated and cell growth was evaluated by CKK8 assay. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. i–j. and l–m. Colony formation assay was used to measure the clonogenicity of MG63 and SaOS2 cells with or without overexpression of KLF4. The numbers of control cells were set as 100% (n=3, mean ± SD, t-test, **P<0.01 vs. CTR). Western blot was used to detect the expression level of KLF4. k. and n. CKK8 assay was used to measure the cell viability of MG63 and SaOS2 with or without overexpression of KLF4.

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: shRNA, Expressing, Western Blot, Control, Colony Assay, Knockdown, Cell Culture, Over Expression

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: a. shRNA KLF4 or shRNA NC infected MG63 cells were injected subcutaneously into either side of the posterior flank of the same nude mouse (n =6 per group). The tumor size is shown in Figure 2a. The black bar represents 1 cm. b. The tumor growth curve is displayed in Figure 2b. c. The final tumor weight is shown in Figure 2c. d. Western blot decided the efficiency of KLF4 in the tumors shown in Figure 2a. e–f. KLF4 protein level in osteosarcoma and normal bone tissues was detected by immunohistochemical staining.

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: shRNA, Infection, Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: a–d. KLF4 were overexpressed or knockdown in MG63 cells using lentivirus vectors. The effects of KLF4 on cell migration were examined by wound-healing assay. Bar graph quantification of migration distance. U, units. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. e–g. Effects of KLF4 on the migration were examined by Matrigel migration assay in MG63 (e) and SaOS2 cells (g). Migrated cells were plotted as the average number of cells per field of view. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. i–f. KLF4 were overexpressed in MG63 cells and its effect on cell migration were examined by Matrigel migration assay. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr.

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: Knockdown, Migration, Wound Healing Assay

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: a–d. MG63 and SaOS2 cells with or without stable knockdown KLF4 were generated. The expression level of CRYAB and KLF4 were analyzed by western blot and q-RT-PCR. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. e–h. MG63 and SaOS2 cells with or without stable overexpressing KLF4 were generated using lentivirus vector. The expression level of CRYAB and KLF4 were analyzed by western blot and q-RT-PCR. i–j. Increasing amounts of KLF4 were transfected into U2OS cells. The expression levels of CRYAB and KLF4 were measured by western blot and q-RT-PCR.

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: Knockdown, Generated, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: a. Schematic illustration of the putative KLF4-binding region located at 585-564 and 479-463 bp upstream of the CRYAB translational start site. b. ChIP analysis showed binding of KLF4 to the putative binding sites. Input and immunoprecipitate from MG63 cells with or without overexpressing KLF4 were amplified by PCR with primer pairs complementary to the CRYAB promoter. c. ChIP analysis showed binding of KLF4 to the putative binding sites. Input and immunoprecipitate from MG63 cells with or without knockdown KLF4 were amplified by PCR with primer pairs complementary to the CRYAB promoter. d. Schematic illustration of PGL3-basic-based reporter constructs used in luciferase assays to examine the transcriptional activity of the binding site 1. The red sequences indicate the mutated nucleotide residues. e. MG63 cells with or without overexpressing KLF4 were transfected with pGL3-CRYAB WT or pGL3-CRYABMUT. 24h after transfection, transcription activity was determined with dual-luciferase assay. f. MG63 cells with or without knockdown KLF4 were transfected with pGL3-CRYAB WT. 24h after transfection, transcription activity was determined with dual-luciferase assay. g–h. U2OS cells were transfected with pGL3-Siat7A WT as well as flag-KLF4, flag- KLF4▵TA (deletion of the transcription activity domain), or flag- KLF4▵DBD (deletion of the DNA binding domain). 24 h after transfection, transcription activity was determined with dual-luciferase assay (g). The expression levels of CRYAB and KLF4 were detected by western blot (h).

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: Binding Assay, Amplification, Knockdown, Construct, Luciferase, Activity Assay, Transfection, Expressing, Western Blot

Journal: Oncotarget

Article Title: Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression

doi: 10.18632/oncotarget.8824

Figure Lengend Snippet: shRNA mediated knockdown CRYAB vector was transfected into the MG63 cells with or without overexpression of KLF4. a–c. Colony formation assay was used to measure the clonogenicity. d–f. The cells were injected subcutaneously into either side of the posterior flank of the same nude mouse (n =6 per group). The tumor size is shown in Figure 6d. The black bar represents 1 cm. The final tumor weight is shown in Figure 6e. The levels of KLF4 and CRYAB were detected by western blot (f). *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. g–h. Matrigel migration assay was used to determine the migration viability of the cells. Migrated cells were plotted as the average number of cells per field of view. Results are representative of three independent experiments. *P<0.05, **p<0.01 and ***p<0.001 vs Ctr. i. ERK, MEK and their phosphorylation were detected by western blot assay. j. The immunostaining analysis of KLF4 and CRYAB protein expression from human osteosarcoma tissue microarray. High expression of KLF4 and CRYAB were shown by being stained as brown. k. The correlation between the expression of KLF4 and CRYAB in human osteosarcoma tissues from 40 patients was shown.

Article Snippet: The following antibodies were used in this study: antibody against CRYAB(Cell Signaling, USA; 8851); GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; SC-25778); KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA; SC-126); KLF4 (Protein Tech, China, 11880-1-AP).

Techniques: shRNA, Knockdown, Plasmid Preparation, Transfection, Over Expression, Colony Assay, Injection, Western Blot, Migration, Phospho-proteomics, Immunostaining, Expressing, Microarray, Staining

Journal: bioRxiv

Article Title: Loss of the mitochondrial citrate carrier, SLC25A1/CIC disrupts embryogenesis via 2-Hydroxyglutarate

doi: 10.1101/2023.07.18.549409

Figure Lengend Snippet: ( A-B ) 2HG enrichment in the amniotic fluid (A) and brain samples (B) obtained from SLC25A1 +/+ (blue) and SLC25A1 −/− (red) embryos at E18.5 dpf (n=4). ( C ) D- and L-2HG enrichment in the amniotic fluid samples obtained from SLC25A1 +/+ (blue) and SLC25A1 −/− (red) embryos (n=4-5). ( D ) Relative increase in D- and L-2HG concentration (%) in the amniotic fluid samples obtained from SLC25A1 −/− (red) embryos compared to their wild-type littermates (n=4-5). ( E ) D-2HG enrichment in A549 cells harboring the shRNA mediated SLC25A1 knock-down relative to control. ( F ) Urine D- and L-2HG concentration in patient samples. Data were extrapolated based on enrichment detected in two different studies. Patients 13-20 were from Ref. 11; Patients 1 and 2 from Ref. 8. The second patient in 8 showed inconsistent elevations of D/L-2HG in repeated urinalyses and only one measurement was used to calculate the percentage of D/L-2HG. Top numbers represent % enrichment. ( G ) Venn diagram representing genes differentially regulated in the brains isolated from SLC25A1 +/+ and SLC25A1 −/− embryos at 18.5 dpc (n=3). ( H ) Hierarchical clustering map of differentially expressed genes as in G. ( I ) Pathway enrichment analysis of genes significantly elevated in SLC25A1 −/− mice relative to wild-type. All significantly enriched pathways (FDR<0.05), assessed from the Reactome Database, were clustered via a distance metric derived from shared significant genes common amongst enriched gene sets. Clustered gene sets were summarized by biological function. ( J ) GeoExplorer was used to perform differential expression analysis of microarray data from GSE54838 (heart, Ref. 33) and GSE85080 (CNS, Ref.32). Significantly upregulated genes with IDH1/2 KI were assessed for pathway enrichment using the Reactome 2022 database via EnrichR. Overlap of enriched pathways between SLC25A1 −/− in this study and that of the IDH1/2 data were assessed via a two-tailed fisher’s exact test. Dark green indicates a p-value below 0.05, light green indicates genes significantly upregulated and mapped to the same pathway, but not enough for enrichment; white indicates no significantly upregulated genes. Created with biorender.

Article Snippet: The membrane was blocked in blocking buffer with 10% horse serum to prevent non-specific binding, followed by incubation with different antibodies: SLC25A1 (Santa Cruz, sc-86392 or Proteintech, 15235-1-AP), ACC1 (Cell Signaling, #3676), FASN (Cell Signaling, #3180), IDH1 (Proteintech, 12332-1-AP), AMPK-α (Cell Signaling, #5832), pAMPK-α (Cell Signaling, #2535), IDH1 (Proteintech, 12332-1-AP), p21 Waf1/Cip1 (Cell Signaling, #37543), mTOR (Cell Signaling, #2983), pH2A.X (Cell Signaling, #9718), GLS1 (Proteintech, 15235-1-AP), p53 (Santa Cruz, sc-126 and Sigma Aldrich, OP29-100UG).

Techniques: Concentration Assay, shRNA, Knockdown, Control, Isolation, Derivative Assay, Quantitative Proteomics, Microarray, Two Tailed Test