Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log 2 -scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in . (B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM. (C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in . See also and and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Expressing, Control, One-tailed Test, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Mutagenesis, Next-Generation Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Rank plot of target-level GI scores in HeLa cells. Table insert, top synthetic lethal paralogs based on GI score. (B) Volcano plot of target-level GI scores in HeLa cells. FDR indicates the multiple hypothesis-adjusted p values from a two-tailed t test . Blue, synthetic lethal paralog GIs with GI < −0.5 and FDR < 0.1; red, buffering paralog GIs with GI > 0.25 and FDR < 0.1. (C) Scatterplot of target-level GI scores for paralog pairs in PC9 versus HeLa cells. Blue, synthetic lethal paralog pairs with GI < −0.5 and FDR < 0.1 in either PC9 or HeLa cells; gray, all paralog pairs with GI ≥ −0.5 or FDR ≥ 0.1. (D) CRISPR scores for representative synthetic lethal paralog pairs identified in the PC9 and HeLa cell screens. Top row: data shown are the mean CRISPR score for each single KO or DKO target across three biological replicates with replicate data shown in overlaid points. Shared synthetic lethal paralogs (e.g., CCNL1/CCNL2 and MEK1/MEK2 ) have FDR < 0.1 in both cell lines; PC9-specific paralogs (e.g., CDK4/CDK6 and OXSR1/STK39 ) have FDR < 0.1 in PC9 only; and HeLa-specific paralogs (e.g., GFTP1/GFPT2 and SOS1/SOS2 ) have FDR < 0.1 in HeLa only. Dashed lines indicate CRISPR score < −0.5. Bottom row: paralog gene expression in PC9 and HeLa cells from RNA-seq analysis. Dashed lines indicate log 2 (TPM) = 1, the threshold for gene expression. (E) Boxplots comparing the effect of CRISPR-mediated KO of the indicated gene in DepMap cell lines with high (top quartile) compared to low (bottom quartile) copy number of its paralogous gene. For boxplots, the middle line, hinges, notches, and whiskers indicate the median, 25th/75th percentiles, 95% confidence interval, and data points within 1.5× the interquartile range from the hinge, respectively. p values were computed using a two-tailed Wilcoxon rank-sum test. CRISPR score and copy number data were obtained from DepMap. (F) As in (E), but for gene expression. (G) Bar plot indicating the p values (computed using a two-tailed Wilcoxon rank-sum test) obtained by comparing the effect of a single paralog KO to the copy number (as in E) or gene expression (as in F) of its pair across human cancer cell lines profiled by DepMap. Bar color indicates whether each pair was synthetic lethal in PC9 only, HeLa only, or both cell lines in the pgPEN screens. Dashed line indicates p = 0.05. See also and , , and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Two Tailed Test, CRISPR, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Software

Journal: BMC cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3.

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: Figure 1 Immunohistochemistry (IHC) staining determined MAP2K3 expression in human HCC tumor and matched adjacent tissues. A-D: Representative images of MAP2K3 protein expression determined by IHC staining. A: An image represented a negative (-) expression of MAP2K3 expression; B: An image represented a low level (+) expression of MAP2K3, which showed a weak immunoreactive staining in cytoplasm; C: An image represented a negative (++) expression of MAP2K3 expression; D: An image represented a high level (+++) expression of MAP2K3, which exhibited a strong IHC staining in cytoplasm and perinuclear localization (arrowhead). E: Semi-quantitative analysis of MAP2K3 protein expression using integrated absorbance (IA) in human HCC tissues. Value was expressed as the average values from each individual sample of HCC tumor tissues or its matched adjacent tissue. The total average value of IA in the HCC tumor tissues was significantly greater as compared with the matched adjacent tissues (p<0.05, n = 14). Data was expressed as mean ± SD for 14 sets of samples.

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: BMC cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3.

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: Figure 2 Validation of MAP2K3 mRNA as a target of miR-21. (A): Sequence of potential binding site of miR-21 in the 3’UTR of MAP2K3 mRNA (top panel), mutations were introduced into the binding site for generation of mutated MAP2K3 3’TUR (bottom panel). (B and C): Validation of miR-21 target using MAP2K3 3’UTR luciferase reporter. Cells co-transfected with pMIR-Report/MAP2K3 3’UTR (WT) or pMIR-Report/ Mut-MAP2K3 3’UTR (Mut) and pAd/pri-miR-21 (B), pAd/miR-21/inhibitor (C), and pAd/con plasmids showed a decreased luciferase activity in pAd/pri-miR-21 cells (B). Luciferase activity after site directed mutagenesis of the 3’UTR of MAP2K3 mRNA in the miR-21 seed sequence (pMIR-Report/Mut-MAP2K3) was significantly higher with respect to the pMIR-Report/MAP2K3 vector transfected cells (B and C). Results represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Luciferase, Transfection, Activity Assay, Mutagenesis, Plasmid Preparation

Journal: BMC cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3.

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: Figure 4 miR-21 targets MAP2K3 mRNA. The HepG2 cells were infected with Ad/pri-miR-21, Ad/miR-21/inhibitor or Ad/con adenoviral vector. The expression of MAP2K3 was detected by immunoblotting analysis against anti-MAP2K3 antibody. Compared with Ad/con group, *: p<0.05. Data in A represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Infection, Plasmid Preparation, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes

doi: 10.1093/nar/gkaa249

Figure Lengend Snippet: Exosc10cKO oocytes exhibit dysregulated transcriptomes during oocyte maturation. (A) Schematic illustrating the pipeline of single oocyte RNA-seq. After individual oocyte lysis, oligo-dT beads captured poly(A) RNAs for library construction and sequencing. Genotypes were determined from genomic DNA. (B) Total RNA levels were normalized for each library by an ERCC RNA spike-in mix. (C) Further normalization of total RNA level in B by the mean value of the GV stage within each genotype. (D) Heatmap of all libraries, each row represents one gene and each column represents one library. Genes are ranked from highest to lowest expression level, and every 100th gene from the top half were selected to represent the transcriptome. The transcription level was color-coded from high to low. (E) PCA of the 64 libraries. Each dot represents one library, color-coded by genotype and stage. (F) Log2 fold change of Exosc10 and Gapdh in cKO versus control oocytes. The bars and lines are log2 fold change and standard error of the mean from DESeq2 analyses. **** P-adjust <0.0001, n.s. no significance, which are the P-adjust values in DESeq2 analysis. (G andH) MA-plots of transcript changes from GV to GV3h, and from GV3h to MII stage in control oocytes. The more and less abundant transcripts in each comparison are labeled by red and blue, respectively (both have P-adjust <0.01). (I) MA plot of transcript change from GV3h to MII stage in control oocytes when analyzed by median ratio normalization. (J–L) MA-plots of transcript changes in cKO versus control oocytes at GV, GV3h and MII stages. The increased and decreased abundant transcripts in each comparison are labeled by red and blue, respectively (P-adjust <0.01). (M andN) Gene ontology of the more abundant transcripts in G, J and K.

Article Snippet: The Exosc10 coding sequence was inserted into plasmid #44118 (Addgene) to form an in-frame fusion with mVenus.

Techniques: RNA Sequencing, Lysis, Sequencing, Expressing, Control, Comparison, Labeling

Journal: Nucleic Acids Research

Article Title: EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes

doi: 10.1093/nar/gkaa249

Figure Lengend Snippet: Oocyte-specific knockout of Exosc10 causes female subfertility by impairing GVBD during oocyte maturation. (A) Schematic of strategy to generate an Exosc10 floxed allele using CRISPR/Cas9. Two loxP sites were inserted to bracket exons 4–10. (B) Mating strategy to obtain oocyte-specific conditional knockouts of Exosc10 (cKO). Siblings with other genotypes were used as controls. The paternal allele is labeled in blue and the maternal allele is labeled in magenta in the offspring. Note that the floxed maternal Exosc10 allele will become a deletion allele (−) during oocyte growth. (C) qRT-PCR of Exosc10 in single oocytes obtained from controls and cKO mice. Error bars: standard deviation of three technique replicates of each sample. (D) Dot plot of individual litter sizes over 6 months of harem breeding of controls and cKO females with wild-type males. The sizes of the dots are normalized by the average litter number per female. The number in parenthesis is the number of females having the indicated genotypes. The number of pups born is indicated below each group. The horizontal lines represent the mean and standard deviation. (E) Bright-field images of cKO and control oocytes cultured ex vivo for 0 (GV) or 3 h (GV3h). (F) Percentage of GVBD oocytes in E. Numbers of oocytes are indicated below each group. (G and H) Confocal fluorescence and DAPI images of oocytes after lamin B immunostaining at GV (G) and GV3h (H) stages. Lamin B and DAPI are maximum intensity projections. Quantification of lamin B fluorescence is on the right. The horizontal lines inside the violins represent the median and the quartiles. The number of oocytes from at least three females are indicated below each group. **** P < 0.0001 in D, G, H, two-tailed Student's t-test. Scale bars: 100 μm in E; 20 μm in G and H.

Article Snippet: The Exosc10 coding sequence was inserted into plasmid #44118 (Addgene) to form an in-frame fusion with mVenus.

Techniques: Knock-Out, CRISPR, Labeling, Quantitative RT-PCR, Standard Deviation, Control, Cell Culture, Ex Vivo, Fluorescence, Immunostaining, Two Tailed Test

Journal: ASN NEURO

Article Title: Subacute Transplantation of Native and Genetically Engineered Neural Progenitors Seeded on Microsphere Scaffolds Promote Repair and Functional Recovery After Traumatic Brain Injury

doi: 10.1177/1759091419830186

Figure Lengend Snippet: Primary Antibodies Used in Immunofluorescent Staining.

Article Snippet: Anti-MAP2 , Recombinant human MAP2 , Abcam , Chicken polyclonal IgY , 1:5000 , AB_2138153.

Techniques: Staining, Derivative Assay, Isolation, Recombinant, Purification, Sequencing, Plasmid Preparation, Virus, Phospho-proteomics

Journal: ASN NEURO

Article Title: Subacute Transplantation of Native and Genetically Engineered Neural Progenitors Seeded on Microsphere Scaffolds Promote Repair and Functional Recovery After Traumatic Brain Injury

doi: 10.1177/1759091419830186

Figure Lengend Snippet: RT-PCR Primer Sequences Used.

Article Snippet: Anti-MAP2 , Recombinant human MAP2 , Abcam , Chicken polyclonal IgY , 1:5000 , AB_2138153.

Techniques: Sequencing

Journal: ASN NEURO

Article Title: Subacute Transplantation of Native and Genetically Engineered Neural Progenitors Seeded on Microsphere Scaffolds Promote Repair and Functional Recovery After Traumatic Brain Injury

doi: 10.1177/1759091419830186

Figure Lengend Snippet: Expression of insulin-like growth factor 1 (IGF-1)-HA increases neuronal differentiation from RG3.6 cells. Representative images of cell expressing IGF-1-HA and Nestin (a), MAP2 and βIII Tubulin (b), O4 (c), and GFAP. Cells shown in panels (a–d) were treated with DOX-containing medium to stimulate expression and secretion of IGF-1-HA. (e) Quantification of neurons (Tuj1), astrocytes (GFAP), and oligodendrocytes (O4) after differentiation of pSLIK-IGF-1-HA infected RG3.6 cells in control or DOX-containing differentiation medium (scale bar = 20 μm). (f) IGF-1-HA effects on lineage marker expression after 20 days of differentiation. Doxycycline-mediated expression of IGF-1-HA significantly increased cDNA levels of interneuron, hippocampal, and neocortical neuronal markers. Scale bars represent 20 µm. Values represent means ± SEM . * p < .05. ** p < .01. NP = neural progenitors; GFAP = glial fibrillary acidic protein; GFP = green fluorescent protein.

Article Snippet: Anti-MAP2 , Recombinant human MAP2 , Abcam , Chicken polyclonal IgY , 1:5000 , AB_2138153.

Techniques: Expressing, Infection, Control, Marker

Journal: Neuron

Article Title: Selective neuronal vulnerability in Alzheimer’s disease: a network-based analysis

doi: 10.1016/j.neuron.2020.06.010

Figure Lengend Snippet: Prediction, validation, and functional analysis of genes associated with AD pathology. A, B. Top NetWAS 2.0 gene predictions show significantly higher enrichment of NFT- (A) and Aβ-associated genes (B) (curated by an independent expert, Table S4) than the original GWAS used as input to NetWAS 2.0. C. Human hippocampal expression levels of top AD-associated gene predictions are highly correlated with amyloid plaque amounts in the ACT cohort (Miller et al., 2017). The x-axis represents the proportion of top NetWAS 2.0 genes obtained. The average absolute values of correlations between gene expression level and amyloid plaques across the subset of genes are plotted (NetWAS 2.0 predictions in red with 95% confidence interval; Braak GWAS in black; background genes in grey). D. Clustering of the top 10% NetWAS genes using a shared-nearest-neighbor-based community-finding algorithm identifies functional modules corresponding to distinct AD-associated processes. We indicate pathways enriched in each module, as well as the association of each module with aging and AD pathology in both our data (independent from our functional network analysis) and external datasets. Each dot represents a gene (where size inversely correlates with the NetWAS 2.0 ranking, i.e. larger dots represent top ranked genes). Network layout (ForceAtlas) by gephi (Bastian et al., n.d.) of ECII-specific network posterior probabilities above prior are shown (comembership score ≥ 0.75 based on 1000 subsamples for visual clarity). E. Representation of pathways enriched in each module (d) in ECII neurons. Microtubules (MT) are represented in blue. Enrichment for genes modulated by Aβ and aging is indicated for each module. Module A is enriched in neuronal cell body processes, while module C includes many axonal processes. Modules A, B, and D may be generally associated with tau pathology in many types of projection neurons, while module C may capture the surplus of vulnerability from ECII neurons. The module includes both structural and functional axonal remodeling pathways, suggesting that axonal plasticity is key to the degeneration process in AD. Concomitant actions of Aβ and aging on module C genes might perturb crosstalk between axon remodeling processes and eventually impinge on SNCA and MAPT function. Inset: magnified view of an axon terminal. β-synuclein, a regulator of neurotransmitter release, binds to synaptic vesicles (grey circles), to the membrane of the presynaptic active zone, and to MTs. Both forms of tau (3R in red, and 4R in green) are present along MT in the axons, with 4R (as well as non-phosphorylated tau) having higher affinity to MT than 3R (as well as hyperphosphorylated tau). Tau-bound MTs are less stable and more prone to severing, a requirement for axon sprouting and axonal plasticity. PTBP1 regulates both tau isoform usage and α-synuclein levels. See also Table S3–6 and Figure S2B.

Article Snippet: human MAPT, FAM/MGB Taqman probe spanning exons 9 and 10 (4R-tau) , Applied Biosystems , Assay ID Hs00902312_m1, RRID : NA.

Techniques: Biomarker Discovery, Functional Assay, Expressing, Gene Expression, Membrane

Journal: Neuron

Article Title: Selective neuronal vulnerability in Alzheimer’s disease: a network-based analysis

doi: 10.1016/j.neuron.2020.06.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: human MAPT, FAM/MGB Taqman probe spanning exons 9 and 10 (4R-tau) , Applied Biosystems , Assay ID Hs00902312_m1, RRID : NA.

Techniques: Immunofluorescence, Virus, Plasmid Preparation, shRNA, Sequencing, Control, Gene Expression, Quantitative Proteomics, Functional Assay, Over Expression, Purification, Recombinant, Modification, BAC Assay, Software

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: Sequence primers designed for real-time qPCR.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Sequencing

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: MAP3K2 may be the potential target of miR-372-3p in colon cancer tissues. (A–C) mRNA expression levels of the potential targets of miR-372-3p including p21, HDAC4, and Wee1, are displayed as 2E-deltaCT (normalized to GAPDH) in colon cancer or matched normal tissues. ** p < 0.01 vs. normal tissues (Mann Whitney test). (D–F) Correlation between miR-372-3p expression and its potential targets p21, HDAC4, and Wee1 in colon cancer. The Y-axis exhibits the log2 value of the ratio of miR-372-3p (orange triangle) and p21, HDAC4, or Wee1 (blue circle) expression levels between colon cancer and matched normal tissues, and the X-axis exhibits the number of samples. The correlation between miR-372-3p and p21, HDAC4, or Wee1 was statistically analyzed using the GraphPad Prism software. (G) MAP3K2 mRNA expression (normalized to GAPDH) in colon cancer, matched adjacent and normal tissues. *** p < 0.0001 vs. normal tissues (one-way analysis of variance). (H) Expression patterns of MAP3K2 in colon cancer and matched adjacent tissues. Each bar presents the log2 value of the ratio of MAP3K2 expression levels between colon cancer and matched normal tissues or adjacent and normal tissues from the same patients. (I) Correlation between miR-372-3p and MAP3K2 expression levels in colon cancer tissues. The Y-axis exhibits the log2 value of the ratio of miR-372-3p (orange triangle) and MAP3K2 (blue circle) expression levels, and the X-axis exhibits the number of samples. All qPCRs were performed in three independent experiments with three replicates per group.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, MANN-WHITNEY, Software

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: miR-372-3p suppressed cell proliferation in SW480 colon cancer cells, and also negatively regulated MAP3K2 expression. (A) SW480 cell proliferation was inhibited by miR-372-3p mimics. SW480 cells were transiently transfected with miR-372-3p mimics in the presence or absence of miR-372-3p inhibitors. The cell viability was then detected using a CCK-8 assay kit at 24 and 48 h. Data are presented using mean ± standard deviation values. * p < 0.05, ** p < 0.001, vs. the vector group, # p < 0.05, ## p < 0.01, vs. the miR-372-3p 3 µg group (Mann Whitney test). (B) Colony-formation assay. The colony-formation ability of SW480 cells was analyzed with a colony formation assay (upper), and the quantified numbers of colonies for each group are displayed as a bar graph (lower). Data are presented using mean ± standard deviation values. ** p < 0.01, *** p < 0.001, vs. The vector group, ## p < 0.01, ### p < 0.001, vs. the miR-372-3p 3 µg group (Mann Whitney test). (C,D) Effects of miR-372-3p on the MAP3K2 expression in SW480 cells. Cells were transfected with miR-372-3p mimics (0, 1, 3 µg). MAP3K2 mRNA was detected using RT-qPCR (at 48 h) (C) , and the protein levels were analyzed with western bloting (at 48 and 72 h) approach (D) . GAPDH was used as an internal control. (E) The MAP3K2 protein level reduced by miR-372-3p was restored by co-transfection with miR-372-3p inhibitors in SW480 cells. (F) Binding sites of miR-372-3p on the MAP3K2 3′-UTR. The 3′-UTR fragments of human MAP3K2 (+21 to +472 bp, +6394 to +6656 bp) were cloned downstream of the luciferase between the MulI and HindIII sites.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Transfection, CCK-8 Assay, Standard Deviation, Plasmid Preparation, MANN-WHITNEY, Colony Assay, Quantitative RT-PCR, Western Blot, Control, Cotransfection, Binding Assay, Clone Assay, Luciferase

Journal: Frontiers in Genetics

Article Title: MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2

doi: 10.3389/fgene.2022.836256

Figure Lengend Snippet: miR-372-3p modulated the expression of MAP3K2 by targeting its 3′-UTR in SW480 colon cancer cells. (A,B) Relative luciferase activities of pMIR-MAP3K2 3′-UTR wild type (wt) and mutants (mt) were detected in pMIR-Vector and miR-372-3p mimics (0, 1, and 2 µg) groups. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. vector group (Mann Whitney test). Three biological replicates were conducted.

Article Snippet: Anti-MAP3K2 (BA3634-2) polyclonal antibody was purchased from BOSTER Biological Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Luciferase, Plasmid Preparation, MANN-WHITNEY