Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: Discovery of CEL as an IGF2BP3‐targeting small molecule. (A and B) IGF2BP3 exhibited potent binding ability with CEL, as determined by SPR (KD = 1.65 µM). The 3D (C) and 2D (D) binding conformations between CEL and IGF2BP3 (6FQR). (E and F) The colocalization of Bio‐CEL with IGF2BP3 was detected by fluorescence microscopy. (G and H) Bio‐CEL interacted with IGF2BP3 in RA‐FLS and RAW264.7 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Binding Assay, Fluorescence, Microscopy

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: CEL inhibits RA‐FLS proliferation and M1 macrophage polarization. (A) The protein expression levels of IGF2BP3 in RA‐FLS after CEL treatment for 24 h. The quantification of scratch healing assays (B) and Transwell assays (C) of RA‐FLS treated with CEL for 24 h. (D) The effect on TNF‐α, IL‐17, and MMP3 expression in RA‐FLS treated with CEL for 24 h. (E) The content of IL‐6 secreted by RA‐FLS. (F) The protein expression levels of IGF2BP3 in RAW264.7 cells after treatment with CEL. (G) The mRNA expression level of NOS2 and NLRP3 in RAW264.7 cells. (H) The content of TNF‐α and IL‐6 secreted by RAW264.7 cells treated with CEL for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Expressing

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: CEL alleviates bone damage and inflammatory infiltration, and inhibits the expression of IGF2BP3 in CIA rats. (A) Representative histology images showing H&E and safranin O/fast green staining are obtained from CIA rats after the indicated treatments. (B) Inflammation scores are measured. (C and D) Representative immunohistochemical assays and scores of IGF2BP3 in CIA rats’ synovial tissue. (E and F) Representative immunohistochemical assays and scores of RASGRF1 in CIA rats’ synovial tissue. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: IGF2BP3 is a direct target of CEL to inhibit cell proliferation, inflammatory cytokine release, and promote autophagy in RA‐FLS. (A) Western blot analysis of the levels of ULK1, p‐ULK1, S6K, p‐S6K, RASGRF1, IGF2BP3, p62, and LC3 in RA‐FLS treated with TNF‐α or CEL. (B) Representative images of the RA‐FLS expressing mCherry‐GFP‐LC3. (C) Western blot analysis of the expression level of ULK1, p‐ULK1, S6K, p‐S6K, RASGRF1, IGF2BP3, p62, and LC3 in RA‐FLS after treatment with TNF‐α, CEL, or IGF2BP3 overexpression. (D) Representative images of RA‐FLS expressing mCherry‐GFP‐LC3 are shown after treatment with TNF‐α, CEL, or IGF2BP3 overexpression. (E) The expression of F‐actin in RA‐FLS. (F) The mRNA expression levels of TNF‐α, IL‐17, and MMP3 in RA‐FLS treated with TNF‐α, CEL, or OV‐IGF2BP3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Western Blot, Expressing, Over Expression

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: CEL inhibits IGF2BP3 to attenuate inflammatory activation in macrophages. (A) Western blot analysis of ULK1, p‐ULK1, S6K, p‐S6K, NLRP3, iNOS, RASGRF1, IGF2BP3, p62, and LC3 in RAW264.7 cells after treatment of LPS or CEL. (B) Western blot analysis of ULK1, p‐ULK1, S6K, p‐S6K, NLRP3, iNOS, RASGRF1, p62, and LC3 in RAW264.7 cells treated with LPS, CEL, or IGF2BP3 overexpression. (C) Representative images of RAW264.7 cells expressing mCherry‐GFP‐LC3 after treatment of LPS, CEL, or IGF2BP3 overexpression. The proportions of CD86 + cells (D) and ROS generation (E) in RAW264.7 cells treated with LPS, CEL, or IGF2BP3 overexpression. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Activation Assay, Western Blot, Over Expression, Expressing

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: IGF2BP3‐mediated mTORC1 activation plays a significant role in CEL‐mediated inhibition of cell proliferation and inflammatory activation. (A) Western blot analysis of ULK1, p‐ULK1, S6K, p‐S6K, p62, and LC3 in RA‐FLS after treatment with TNF‐α, CEL, or MHY1485. (B) The mRNA expression levels of TNF‐α, IL‐17, and MMP3 in RA‐FLS after treatment with TNF‐α, CEL, or MHY1485. (C) Western blot analysis of ULK1, p‐ULK1, S6K, p‐S6K, NLRP3, iNOS, p62, and LC3 in RAW264.7 cells treated with LPS, CEL, or MHY1485. The proportions of CD86 + cells (D) and ROS generation (E) in RAW264.7 cells after treatment with LPS, CEL, or MHY1485. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Activation Assay, Inhibition, Western Blot, Expressing

Journal: MedComm

Article Title: A New N6‐Methyladenosine Inhibitor, Celastrol, Alleviates Rheumatoid Arthritis via Targeting IGF2BP3

doi: 10.1002/mco2.70431

Figure Lengend Snippet: The knockout of IGF2BP3 abolishes the effects of CEL in alleviating the progression of RA. (A) The groups of animal experiments. (B) An illustration of the induction and treatment of mice with arthritis. (C) Hind paw thickness is detected from the second immunization. Compared with WT‐RA, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with Igf2bp3 −/− ‐RA, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Arthritis scores are monitored from the second immunization. (E) The proportion of F4/80 + CD11b + CD86 + M1 macrophages in the spleens of mice. The content of TNF‐α (F) and IL‐6 (G) in the serum of mice. (H–J) Representative immunohistochemical assays and scores of RASGRF1 and NLRP3 in the synovial tissue. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: IGF2BP3 −/− mice were purchased from Cyagen Biosciences.

Techniques: Knock-Out, Immunohistochemical staining

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Growth curves of AML cell lines grown in complete medium (CM) or equivalent medium lacking serine and glycine (-SG). B Mouse HSPCs were transduced with MLL-AF9 ( MA9 ) retroviruses, followed by cultured in CM or -SG progenitor medium for 2 days. The cells were then plated into mouse methylcellulose medium for CFA. Colony numbers and representative photos of colonies are shown. Bar = 200 μm. C A schematic diagram, created from GDP ( https://BioGDP.com , Agreement number: GDP2025UWV74M), depicting the CRISPR/Cas9-based screening strategy. Lentivirus-transduced Molm13 Cas9 cells were selected with puromycin for 5 days and collected for an initial gDNA sample (denoted as T0). Cells were further grown in CM or -SG medium for 8, 12, or 17 population doublings, and were denoted as P8, P12, or P17, respectively. For each gene, we calculated its gene score as the mean log 2 fold change (LFC) in the abundance of all the sgRNAs targeting the corresponding gene in different population doublings compared to T0. The SG-dependent score refers to the difference in gene scores in the absence versus presence of SG at the same population doubling. D The SG-dependent scores of the top 30 genes from the screening in ( C ), with the m 6 A regulatory genes IGF2BP3 and METTL14 being highlighted in red and purple, respectively. E , F The normalized abundance of the 12 sgRNAs targeting IGF2BP3 ( E ) or METTL14 ( F ) from cells cultured in CM or -SG medium and collected at P8, P12, and P17. The definition of data points is same as in ( C ). n = 12 sgRNAs. The boxes extend from the 25th to the 75th percentiles, with a distinct line marking the median. Whiskers extend to the maximum and minimum data points. G Growth curves of Molm13 Cas9 transduced with control sgRNA (sgNS) or IGF2BP3 sgRNA (sgBP3) and grown in CM or -SG medium. H Growth curves of control or IGF2BP3 KD cells in CM or -SG medium. I Growth curves of control or METTL14 KD Molm13 cells in CM or -SG medium. J Schematic illustration showing the AID2 system in inducing rapid degradation of IGF2BP3 protein. K Relative growth of IGF2BP3-mAID (clone BP3-mAID cells) cells in CM or -SG medium with or without treatment with 5’Ph-IAA (IAA) for 24 h. Mean ± SD values are shown. n = 2 biologically independent repeats in ( A , B , G , H , I , and K ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Transduction, Cell Culture, CRISPR, Control

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Bubble diagram showing the enrichment of metabolic pathways by the C 13 -labeled metabolites with reduced levels after IGF2BP3 KD in Molm13 cells. B Heatmaps showing levels of representative C 13 -labeled metabolites after IGF2BP3 KD in Molm13 cells. C The total levels and isotopolog distribution ( M + n , n refers to numbers of 13 C) of purines measured by LC-MS in Molm13 cells transduced with IGF2BP3 shRNAs or shNS and grown in medium containing U-[ 13 C]-glucose. D Schematic of the serine synthesis pathway and its downstream pathways. E The levels of C 13 -labeled serine ( M + 3) were measured by LC-MS in Molm13 cells transduced with IGF2BP3 shRNAs or shNS and grown in medium containing U-[ 13 C]-glucose. F – H Intracellular serine level measured by fluorescence-based serine detection assays in Molm13 cells transduced with shRNAs ( F ) or sgRNAs ( G ) targeting IGF2BP3 or shRNAs targeting METTL14 ( H ), compared to those with negative control (shNS). I The Venn diagram showing the overlap of significantly downregulated genes (adjusted P < 0.05, fold change <0.67) in Molm13 cells with IGF2BP3 KD, as well as in U937 BP3-mAID cells treated with 5’Ph-IAA for 6 or 24 h to induce degradation of the IGF2BP3 protein. J Pie chart showing numbers of transcripts with or without m 6 A modifications among the 61 overlapping genes in ( H ), and heatmap showing expression changes of the 48 m 6 A-containing transcripts in IGF2BP3 KD Molm13 cells or IGF2BP3-depleted U937 cells. K GO enrichment analysis of the 48 candidate targets of IGF2BP3 in ( I ). The degrees of interactions were generated by STRING. Mean ± SD values are shown. n = 2 biologically independent repeats in ( C ) and ( E ), while n = 3 biologically independent repeats in ( F – H ). P -values were calculated with one-sided Hypergeometric test ( A , K ); two-tailed Wald test adjusted with Benjamini-Hochberg Procedure ( I ); two-tailed student’s t -test ( F – H ); Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy, Transduction, Fluorescence, Negative Control, Expressing, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Western blot after IGF2BP3 KD in various AML cell lines. GAPDH was used as a loading control. B Western blot of U937 BP3-mAID cells after treatment with 5’Ph-IAA for the indicated time periods. ACTB was used as a loading control. C Western blot of Molm13 cells after overexpression of wild-type (BP3-WT) or KH3-4 mutated (BP3-KH34) IGF2BP3. D Western blot of Molm13 cells after METTL14 KD. E IGV tracks showing the distribution of m 6 A (GEO: GSE97408 ) or IGF2BP3 binding sites (GEO: GSE90639 ) in target mRNAs. Gray shadow depicts high-confidence m 6 A regions for qPCR validation in ( G ) and ( H ). F RIP assays using an IGF2BP3 antibody were performed in Molm13 cells, followed by qPCR to detect direct binding of IGF2BP3 to target mRNAs at regions indicated in ( E ). G MeRIP-qPCR was performed in control and METTL14 KD Molm13 cells to detect the change of m 6 A abundance at indicated regions of ATF4 , PHGDH , and PSAT1 transcripts. H Bst DNA polymerase-mediated cDNA extension and qPCR assays to evaluate relative m 6 A abundance changes at specific sites within ATF4 , PHGDH , and PSAT1 transcripts in control and METTL14 KD Molm13 cells. I Control and IGF2BP3 KD Molm13 cells were treated with actinomycin D for indicated time periods and the RNA level of target genes was examined by qPCR. 18S rRNA was used as a loading control. The mRNA half-life ( t 1/2 ) was calculated and shown. ( J , K ) Live cell counting ( J ) and cytometric analysis of Annexin V + apoptotic cells ( K ) in Molm13 cells upon IGF2BP3 KD and ATF4, PHGDH, or PSAT1 overexpression. EV, empty vector. ( L , M ) Molm13 cells were co-transduced with control or IGF2BP3 shRNAs and ATF4/PHGDH/PSAT1 overexpression vectors and subjected to colony-forming assays ( L ) and serine level detection ( M ). Representative results from one of the two independent experiments were shown ( A – D ). Mean ± SD values are shown. n = 3 biologically independent repeats in ( F , G , H , I , K , and M ), while n = 2 biologically independent repeats in ( J ) and ( L ). Two-tailed student’s t -test ( F – H ), two-way ANOVA ( K , M ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Western Blot, Control, Over Expression, Binding Assay, Biomarker Discovery, Cell Counting, Plasmid Preparation, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Schematic illustration of the in vivo primary BMT assay and in vitro CFA with HSPCs co-transduced with MA9 retroviruses and Igf2bp3 shRNA (or shNS) lentiviruses. B Kaplan–Meier curves showing the effect of Igf2bp3 KD on MA9 -induced leukemogenesis. C Wright-Giemsa staining of BM cells and PB blood smear, and hematoxylin and eosin (H&E) staining of livers and spleens of the representative primary BMT mice from ( B ) at the endpoint. Scale bars from left to right: 20 μm, 50 μm, 200 μm, 300 μm. D , E CFA using mouse HSPCs transduced with MA9 ( D ) or PML-RARa ( E ) plus shNS, Bp3-sh1, or Bp3-sh2 viruses. F HSPCs from FLT3-ITD/NPM1-mut mice were transduced with shNS, Bp3-sh1, or Bp3-sh2 viruses and seeded for CFA. Bar = 200 μm. G HSPCs from Mettl14 fl/fl -CRE ERT mice were transduced with MA9 retroviruses and seeded for CFA. Mettl14 KO was induced by addition of 4-Hydroxytamoxifen (4-OHT, 1 mM) during plating. H Schematic illustration of the in vivo primary BMT assay and in vitro CFA with HSPCs from Igf2bp3 WT and KO mice. I Colony numbers and representative photos of colonies in the CFA assays illustrated in ( H ). Bar = 200 μm. J Flow cytometric analysis of CD45.2 + donor cell percentages in PB of recipient mice 8 weeks after BMT. n = 7 mice. K Kaplan–Meier curves showing the effect of Igf2bp3 KO on MA9 -induced leukemogenesis. L , M Representative images of liver ( L ) and spleen ( M ) tissues from the primary BMT mice in ( K ) at the endpoint. N Wright-Giemsa staining of BM cells and PB blood smear, and H&E staining of livers and spleens of representative primary BMT recipient mice from ( K ) at the endpoint. Scale bars are same as in ( C ). O HSPCs from WT or Igf2bp3 KO mice were co-transduced with MA9 and ATF4 -, PHGDH -, or PSAT1 -overexpressing viruses and seeded for CFA. P Schematic illustration of the secondary BMT assay and CFA with MA9 -induced leukemic cells transduced with Igf2bp3 shRNA (or shNS) lentiviruses. Q CFA assays showing the effect of Igf2bp3 KD on BM cells from MA9 leukemia mice. Bar = 200 μm. R Kaplan–Meier curves showing the result of secondary BMT using cells collected from the first plating of ( O ). Representative results from one of at least two mice were shown ( C , N ). Mean ± SD values are shown. n = 2 biologically independent repeats in ( D , E , F , G , I , O , and Q ). Statistical analysis: Two-tailed student’s t -test ( J ); log-rank test ( B , K , and R ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: In Vivo, In Vitro, Transduction, shRNA, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Expression of IGF2BP3 mRNA in AML patient samples and healthy donors from the TNMplot database. Box plot, center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. n = 407 healthy samples; n = 151 AML samples. B Expression of IGF2BP3 in CD34 + and CD34 - cells from BM of healthy donors (normal) or AML patients as detected by microarray ( GSE30029 ). n = 31 CD34 + normal samples; n = 46 CD34 + AML samples; n = 44 CD34 - AML samples. C Expression of IGF2BP3 in blast (LSC − , non-engrafting) or LSC (LSC + , engrafting) cells from AML patients ( GSE199452 ). n = 40 LSC − samples; n = 70 LSC + samples. D Experimental scheme for ( E – G ). Patient-derived CD34 + cells were transduced with shNS or shRNAs targeting IGF2BP3 and subjected to different assays. E – G Growth curves ( E ), percentages of Annexin V + apoptotic cells ( F ), and colony numbers ( G ) of patient-derived CD34 + leukemia cells with or without IGF2BP3 KD. Bar = 200 μm. H The percentage of L-GMP population (CD45.2 + Lin - c-kit + Sca1 - CD34 + CD16/32 + ) in BM of the primary BMT mice. n = 6 mice per group. I , J Statistics of the percentage of Annexin V + ( I ) or Ki67 + ( J ) L-GMPs of mice in ( H ). n = 6 mice. K In vitro LDA assays using MA9 -transduced HSPCs from Igf2bp3 WT and KO mice. Logarithmic plots show the percentage of nonresponding wells (those without any colonies) at different doses of cells seeded. The estimated LSC/LIC frequency is calculated by ELDA and shown. L , M In vitro LDA assays of mouse HSPCs co-transduced with MA9 plus shRNA targeting Igf2bp3 ( L ) or its target genes ( M ). N HSPCs collected from Igf2bp3 WT and HO mice were co-transduced with MA9 and target gene-overexpressing viruses and selected in methylcellulose medium with G418 and puromycin for 7 days before seeded for LDA assays. Mean ± SD values are shown. n = 2 biologically independent repeats in E , G while n = 3 biologically independent repeats in ( F ). Statistical analysis: Two-tailed student’s t -test ( A , C , F , H – J ); two-way ANOVA ( B ); One-sided Chi-squared test ( K – N ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Expressing, Microarray, Derivative Assay, Transduction, In Vitro, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Schematic outline of experimental strategy testing the effect of Igf2bp3 KO on mouse static normal hematopoiesis. n = 7 mice per group. B – D PB analysis of Igf2bp3 WT and KO mice. The density of white blood cells (WBC), lymphoma cells (LYM), red blood cells (RBC) ( C ), palates (PLT) ( D ), neutrophils (NEUT), monocytes (MONO), and eosinophils (EO) ( E ) are shown. E Flow cytometric gating strategies for HSC and progenitors. F , G Frequencies of various hematopoietic progenitors in the BM of Igf2bp3 WT and KO mice as examined by flow cytometry. LSK, Lin - Sca1 + cKit + cells; MPP, multipotent progenitor (Lin - Sca1 + cKit + CD48 + CD150 - ); LT-HSC, long-term HSC (Lin - Sca1 + cKit + CD48 - CD150 + ); ST-HSC, short-term HSC (Lin - Sca1 + cKit + CD48 - CD150 - ); LMPP, lymphoid-primed multipotent progenitor (Lin - Sca1 high cKit high CD127 + CD135 + ); CLP, common lymphoid progenitor (Lin - Sca1 low cKit low CD127 + CD135 + ); CMP, common myeloid progenitor (Lin - Sca1 - cKit + CD34 int CD16/32 low ); GMP, granulocyte-monocyte progenitor (Lin - Sca1 - cKit + CD34 + CD16/32 high ); MEP, megakaryocyte–erythroid progenitor (Lin - Sca1 - cKit + CD34 - CD16/32 - ). H Flow cytometric gating strategies for mature cells. I Frequencies of various mature cells in the BM of Igf2bp3 WT and KO mice as examined by flow cytometry. J Schematic outline of competitive repopulation assay. n = 7 mice per group. K Flow cytometric gating strategy for BM cells derived from Igf2bp3 WT or KO donor mice in the competitive repopulation assay. L Flow cytometry analysis for different donor-derived cells (CD45 + ) in PB of recipient mice 4, 8, 12, and 16 weeks after BMT. M , N Percentage of donor-derived stem cell ( M ) and progenitor ( N ) compartments in the bone marrow of recipients 16 weeks after BMT. Mean ± SD values are shown. Statistical analysis: Two-tailed student’s t -test ( B – D , F , G , I , L – N ); n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: m 6 A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability

doi: 10.1038/s41467-025-58966-1

Figure Lengend Snippet: A Experimental scheme for ( B ) and ( C ). M-NSG immunodeficient recipient mice were transplanted with AML PDX cells (FLT3-TKD mutation) transduced with shNS or shRNA targeting Igf2bp3, and fed with a regular (control) or SG-free (-SG) diet. B Colony numbers of AML PDX cells (FLT3-TKD) are shown to reflect the effect of SG deprivation (-SG) on control (NS) or Igf2bp3 KD (shBp3) cells compared to complete medium (CM). C Kaplan–Meier curves showing survival of recipient mice fed with a regular (control) diet or an equivalent diet lacking SG (-SG). n = 7 mice per group. D Experimental scheme for ( E ) and ( F ). BM cells from MA9 leukemic mice were transduced with shNS or shRNA targeting Igf2bp3 and selected with puromycin before subjected to CFA assays ( E ) or BMT assays ( F ). E Colony numbers in CFA assays are shown to reflect the effect of SG deprivation (-SG) on control (NS) or Igf2bp3 KD (shBp3) cells compared to complete medium (CM). F Kaplan–Meier curves showing survival of recipient mice fed with a regular (control) diet or an equivalent diet lacking SG (-SG). n = 8 mice per group. G Experimental scheme of the xenotransplantation assays in ( H ). M-NSG immunodeficient recipient mice were transplanted with U937 BP3-mAID cells and fed with a regular (control) or SG-free (-SG) diet. 5’Ph-IAA (3 mg/kg) or vehicle control (DMSO) were given via intraperitoneal injection (i.p.) every day for 7 consecutive days starting from the 8th day after transplantation. H Kaplan–Meier curves showing survival of the M-NSG recipient mice in ( G ). n = 7 mice per group. I Schematic illustration of the working model and therapeutic strategy proposed in the study, created with the assistance of GDP (Agreement number: GDP2025KABQ7W). Mean ± SD values are shown. n = 2 biologically independent repeats in ( B ) and ( E ). Statistical analysis: log-rank test ( C , F , and H ). Source data are provided as a Source Data file.

Article Snippet: Human ATF4 , PHGDH and PSAT1 coding sequences were reverse-transcribed and PCR amplified from total RNA of Molm13, and subcloned into the cl20c-N-2×FLAG-GFP lentiviral vector. shRNA vectors targeting human or mouse IGF2BP3 , ATF4 , PHGDH , and PSAT1 were constructed by synthesizing shRNA-encoded DNA oligos and cloning them into the pLKO.1 vector (Addgene).

Techniques: Mutagenesis, Transduction, shRNA, Control, Injection, Transplantation Assay

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 1 The expression of IGF2BP3 was related to progression and COG risk in NB patients. A Differential expression of m6A methylation regulators between subgroups stratified by progression (N: non-progression; P: progression) in dataset GSE49711. B Differential expression of m6A methylation regulators between subgroups stratified by COG risk (Children’s Oncology Group risk stratification) in dataset GSE49711. C Univariate Cox regression analysis evaluating independently predictive ability of m6A methylation regulators for overall survival of NB patients in TARGET databases. D IGF2BP3 expression level in progression and non-progression NB patients of dataset GSE49711. E IGF2BP3 expression level in high COG risk and nonhigh COG risk NB patients of dataset GSE49711. F The expression levels of IGF2BP3 and MYCN were positively correlated.

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Expressing, Quantitative Proteomics, Methylation

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 2 IGF2BP3 is upregulated in MYCN-amplified NB tissues and cell lines. A Immunohistochemistry images of IGF2BP3 in MYCN-amplified and non-MYCN-amplified NB tissues (n = 35). B The expression level of IGF2BP3 in MYCN-amplified tissues was higher than that in non-MYCN- amplified tissues (n = 35). C qRT-PCR analysis of the expression level of IGF2BP3 in NB cell lines. D Western blot analysis of the expression level of IGF2BP3 in NB cell lines (n = 3).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 3 Knockdown of IGF2BP3 inhibited N-myc expression in MYCN-amplified NB cells. A qRT-PCR analysis of the expression level of IGF2BP3 mRNA in SK-N-BE(2) (n = 3); B qRT-PCR analysis of the expression level of MYCN mRNA in SK-N-BE(2) (n = 3); C qRT-PCR analysis of the expression level of IGF2BP3 mRNA in BE(2)-C (n = 3); D qRT-PCR analysis of the expression level of MYCN mRNA in BE(2)-C (n = 3); E RNA-FISH and immunofluorescence analysis of the expression level of MYCN mRNA and IGF2BP3 in SK-N-BE(2) (Green: MYCN mRNA; Red: IGF2BP3 protein; Blue: DAPI); F RNA-FISH and immunofluorescence analysis of the expression level of MYCN mRNA and IGF2BP3 in BE(2)-C (Green: MYCN mRNA; Red: IGF2BP3 protein; Blue: DAPI).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Knockdown, Expressing, Quantitative RT-PCR

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 4 Knockdown of IGF2BP3 repressed proliferation of MYCN-amplified NB cells. A Monolayer colonies were detected in SK-N-BE(2) and BE(2)-C (n = 3); B, C Xenograft formation of SK-N-BE(2) cells (n = 6); D, E Xenograft formation of BE(2)-C cells (n = 6).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Knockdown

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 6 IGF2BP3 regulated NB cell proliferation via reading m6A modification of MYCN. A IGF2BP3 could bind to MYCN RNA predicted by RBPsuite website tools; B The enrichment of IGF2BP3 in the mRNA of MYCN performed by RIP-qPCR assay in SK-N-BE(2) (n = 3); C The enrichment of IGF2BP3 in the mRNA of MYCN performed by RIP-qPCR assay in BE(2)-C (n = 3); D, E The m6A modification site of MYCN predicted by SRAMP website tools based on sequence-derived features, and primers designed for MeRIP-qPCR assay. F Obvious m6A modification of MYCN confirmed by MeRIP-qPCR, and knockdown of m6A writer METTL3 repressed MYCN m6A modification in SK-N-BE(2) (n = 3); G Obvious m6A modification of MYCN confirmed by MeRIP-qPCR, and knockdown of m6A writer METTL3 repressed MYCN m6A modification in BE(2)-C (n = 3); H The mRNA stability and degradation halftime of MYCN in SK-N-BE(2) treated by Actinomycin D (n = 3); I The mRNA stability and degradation halftime of MYCN in BE(2)-C treated by Actinomycin D (n = 3).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Sequencing, Derivative Assay, Knockdown

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 7 N-myc directly promotes the IGF2BP3 expression in NB cells. A qRT-PCR analysis of the expression level of IGF2BP3 mRNA in SK-N- BE(2) (n = 3); B qRT-PCR analysis of the expression level of MYCN mRNA in SK-N-BE(2) (n = 3); C qRT-PCR analysis of the expression level of IGF2BP3 mRNA in BE(2)-C (n = 3); D qRT-PCR analysis of the expression level of MYCN mRNA in BE(2)-C (n = 3); E Schematic illustration of E-boxes (CANNTG) element in IGF2BP3 promoter; F, G Analysis of the effect of N-myc on IGF2BP3 promoter by ChIP-qPCR (n = 3); H Analysis of the targeting effect of N-myc on IGF2BP3 promoter by luciferase reporter assay (n = 3); I Schematic diagram of a positive feedback loop between RNA N6-methyladenosine reader IGF2BP3 and MYCN facilitates NB proliferation.

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Expressing, Quantitative RT-PCR, ChIP-qPCR, Luciferase, Reporter Assay

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2 RNAi induces minor defects in ribosome biogenesis and disrupts TORC2 activation. A) Analytical sucrose density gradient fractionation of GBM301 cells treated with either a lentiviral control lacZ shRNA or a validated RIOK2 shRNA. Cells were infected with shRNAs, treated with 20 μM zVAD, and harvested 72 hrs following infection. Immunoblots of total cell lysates (right) confirmed RIOK2 knockdown and equivalent expression levels of ribosomal proteins between control and RIOK2 knockdown samples. 4 ODs from each lysate were ultracentrifuged in a sucrose density gradient of 10%-45%, and eluted into several fractions while a UV lamp was used to detect ribosome subunits. Annotated UV traces show that RIOK2 knockdown slightly reduced levels of 40S and 60S subunits relative to levels of mature 80S monosomes. B) RIOK2 co-fractionates with mTOR and RICTOR in association with the 40S ribosome subunit in GBM cells by analytical sucrose density gradient fractionation. RIOK2 knockdown efficiency confirmed by immunoblot on total cell lysate, right two lanes. Annotated UV traces for each sample (9 ODs used for each) is shown above immunoblot results for concentrated matched fractions, which show that RIOK2 knockdown induced a slight reduction in levels of 40S and 60S subunits relative to mature 80S monosomes. RIOK2, mTOR, and RICTOR proteins are enriched in the concentrated fractions collected for the 40S peak, as confirmed by the presence of the small subunit protein RPS3. RIOK2 knockdown inhibited mTOR-RICTOR co-fractionation with the 40S ribosome subunit. IMP3 also co-fractionated with mature and immature ribosomes. These results may indicate either direct association or independent co-fractionation of RIOK2-IMP3-mTOR-RICTOR complexes with ribosome subunits.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Activation Assay, Fractionation, Control, shRNA, Infection, Western Blot, Knockdown, Expressing

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: IMP3 is a binding partner and downstream effector of RIOK2. (A) Proteomic Analysis of RIOK2 immunoprecipitations (IPs) from U87MG-EGFR vIII GBM cells performed with antibodies against either endogenous RIOK2 or with a non- specific IgG control. Values are reported in spectral counts and number of unique peptides. Spectral counts are the total number of spectra identified for a protein and is a relative measurement of the quantity of a given protein, while the number of unique peptides refers to the number of peptides that matches the protein in question. Names of RNA-binding proteins are colored red, and names of ribosome assembly factors are colored blue. (B) RIOK2 WT -FLAG or RIOK2 123A,246A -FLAG overexpression was induced in U87MG cells with 8 μg/ml doxycycline for 72 hrs, and proteins were IPed with Mouse M2 Anti-FLAG magnetic beads. immunoblots on total cell lysates set aside prior to IP show that endogenous IMP3 was expressed across all samples, and that FLAG-tagged RIOK2 WT - FLAG and RIOK2 123A,246A -FLAG were sufficiently induced by doxycycline treatment. immunoblots of show that a significantly reduced amount of IMP3 co-IPed with RIOK2 123A,246A -FLAG compared to RIOK2 WT -FLAG. (C) IPs from GBM39 cells using antibodies against either RIOK2 or IMP3 demonstrated reciprocal co-IP of endogenous IMP3 and RIOK2, respectively. IMP3 IPs showed no detectable co-IP of endogenous NOB1, a ribosome assembly factor and established RIOK2 binding partner. Both endogenous RIOK2 and IMP3 were detected in total cell lysates. (D) co-IPs of endogenous RIOK2 showed enrichment of endogenous IMP3 from GBM39 cells compared to human neural progenitor cells (hNPCs). Both endogenous RIOK2 and IMP3 were detected in total cell lysates of hNPCs and GBM39s. (E) Purified recombinant RIOK2 and IMP3 protein was mixed together in vitro and co-IPed with anti-RIOK2 antibodies, which demonstrate direct binding of IMP3 and RIOK2. RIOK2 protein and antibodies were pretreated with 0.1 mg/ml of Bovine Serum Albumin (BSA 0.1mg/ml) binding competitor prior to protein mixing and co-IP in order to reduce non-specific binding to recombinant IMP3.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Binding Assay, Control, RNA Binding Assay, Over Expression, Magnetic Beads, Western Blot, Co-Immunoprecipitation Assay, Purification, Recombinant, In Vitro

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2 and IMP3 are co-overexpressed in human GBMs. (A) A panel of lysates from human neural progenitor cells (hNPCs) or short-term patient-derived GBM neurosphere cultures derived from tumors that showed loss of PTEN and amplification of EGFR (GBM301, GBM725, GBM914, GBM1219) or other RTKs (NTRK2: GBM131, MET: GBM730, GBM56) show elevated RIOK2 expression compared to hNPCs; all cultures express endogenous IMP3. (B) Analysis of TCGA GBM tumor profiling data show significantly increased expression of both RIOK2 and IMP3 mRNA in grade IV gliomas compared to lower grade gliomas. Increased expression of RIOK2 and IMP3 mRNA are significantly correlated with each other across all TCGA glioma grades. IMP3 mRNA expression is significantly increased in high grade gliomas with EGFR amplification compared to tumors without EGFR amplification. (C) U87MG-EGFR vIII cells were fixed and stained to label nuclear DNA (DRAQ7, blue), RIOK2 (magenta), and IMP3 (green). RIOK2 and IMP3 were co-localized predominantly in the cytosol, with both RIOK2 and IMP3 also exhibiting a perinuclear localization pattern (denoted by white arrows).

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Derivative Assay, Amplification, Expressing, Staining

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2 autophosphorylates itself but does not directly phosphorylate IMP3. (A) Phospho-image of in vitro kinase assay with purified bacterially produced recombinant RIOK2 protein using 32 P radiolabeled ATP show that RIOK2 is autophosphorylated. (B) Representative image of potential RIOK2 autophosphorylation-sites from an in vitro kinase assay using purified bacterially produced labelled with cold ATP, identified using phospho-proteomics. (C) Phospho-image of in vitro kinase assay using purified bacterially produced recombinant RIOK2 and/or IMP3 protein mixed together and radiolabeled with 32 P ATP show that RIOK2 is autophosphorylated, but that RIOK2 does not directly phosphorylate IMP3.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: In Vitro, Kinase Assay, Purification, Produced, Recombinant, Phospho-proteomics

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: The RIOK2-IMP3 complex recruits TORC2 and promotes phosphorylation of IMP3. (A) Diagram of experimental workflow for DSP crosslinking and IPs from GBM39 cells. (B) GBM39 cells were treated with either DMSO or DSP crosslinker (1mM) for 2 hrs. immunoblot analysis show that GBM39 total cell lysates set aside prior to IPs endogenously expressed mTOR, RICTOR, IMP3, and RIOK2. Immunoblots of RIOK2 IPs from GBM39 cells indicate enrichment of endogenous mTOR and RICTOR protein in the RIOK2-IMP3 complex as compared to control non-specific IgG IPs. (C) GBM39 treated with either DMSO or DSP crosslinker (1 mM) for 2 hrs prior to IPs with either IMP3 antibody or a non-specific IgG control. Immunoblots show enrichment of endogenous mTOR protein in the IMP3-RIOK2 co-IPed complex, as well as phosphorylated IMP3 (detected with MPM2 antibody). (D) Phospho-peptide profiling of changes in phosphorylation of endogenous IMP3 upon RIOK2 knockdown. GBM39 cells were treated with verified lentiviral RIOK2 shRNAs. Control cells were infected with a non-targeting GFP control lentivirus. GBM cells were treated with 20 μM zVAD to prevent apoptosis. Cells were harvested 72 hrs after infection, and IMP3 was IPed. RIOK2 knockdown in pre-IP total cell lysates and anti-IMP3 IPs were verified by immunoblots. IMP3 was isolated on acrylamide gel slice and profiled for total protein and phosphorylation. Total peptide coverage for each IP in shown, and total percentage of phosphorylated peptides were calculated using according to signal intensity for phospho-peptides and total peptides for indicated fragments shown. GBM39 cells with RIOK2 knockdown showed no detectable IMP3 phosphorylation on Ser183 and Ser528 (magenta) compared to IMP3 from control cells.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Phospho-proteomics, Western Blot, Control, Knockdown, Infection, Isolation, Acrylamide Gel Assay

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2 phospho-proteomic analysis. Phospho-peptide profiling of endogenous IMP3 upon RIOK2 knockdown. GBM39 cells were treated with verified lentiviral RIOK2 shRNAs. Control cells were infected with a non-targeting GFP control lentivirus. GBM cells were treated with 20 μM zVAD to prevent apoptosis. Cells were harvested 72 hrs after infection, and IMP3 was IPed. RIOK2 knockdown in pre-IP total cell lysates and anti-IMP3 IPs were verified byimmunoblots (see ). IMP3 was isolated on acrylamide gel slice and profiled for total protein and phosphorylation. The alignment shows total peptide coverage for each IP (residues covered by peptides are underlined in the specific reference alignments shown) and for non-phosphorylated and phosphorylated peptides. No serine/threonine phosphorylated peptides were detected in lysates from cells treated with RIOK2 lentiviral shRNAs.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Knockdown, Control, Infection, Isolation, Acrylamide Gel Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: dRIOK2/RIOK2 and Imp/IMP3 modulate dMyc/MYC expression in a conserved pathway. (A) 3 μm optical projections of brain hemispheres, aged-matched 3 rd instar larvae. CD8-GFP (green) labels glial cell bodies and magenta labels dMyc protein (alone in top panels, merged in bottom panels). Glial-specific dMyc knockdown was used as a control to determine background in dMyc antibody stains. dRIOK2 knockdown ( repo>dEGFR λ ;dp110 CAAX ; dRIOK2 dsRNA ) (Third panel from the left) reduced dMyc levels in glia compared to repo>dEGFR λ ;dp110 CAAX brains (rightmost panel) and showed similar levels as dMyc knockdown( repo>dEGFR λ ;dp110 CAAX ; dMyc dsRNA ) (second panel of the left). Glial-specific Imp knockdown ( repo>dEGFR λ ;dp110 CAAX ; Imp dsRNA ) (rightmost panel) reduced dMyc levels in glia compared to repo>dEGFR λ ;dp110 CAAX brains (leftmost panel) and showed similar levels as dMyc knockdown ( repo>dEGFR λ ;dp110 CAAX ; dMyc dsRNA ) (second panel of the left). (B) IMP3 was IPed with two different antibodies (anti-IMP3 #1 and anti-IMP3 #2) from GBM39 cells. IP with non-specific IgG was used as a negative control. Co-IPed IMP3- bound mRNAs were extracted and cDNAs were generated. cDNA was PCRed with primers against MYC , and DNA gel electrophoresis showed specific MYC PCR products, indicating that MYC mRNA was bound to IMP3 in GBM cells expressing RIOK2. (C) GBM39 cells were infected with using verified lentiviral shRNAs against RIOK2 or IMP3. Control cells were infected with a non-targeting GFP control lentivirus. GBM cells were treated with 20 μM zVAD to prevent apoptosis. Cells were harvested 96 hrs after infection. GBM39 cells with RIOK2 and IMP3 knockdown showed reduced expression of endogenous MYC mRNA, as measured by qPCR, and MYC protein, as measured by immunoblot, compared to control cells infected with GFP shRNA. Two replicates were used per conditions. Statistics generated using unpaired two-tailed Student TTESTS, **p<0.01.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Expressing, Knockdown, Control, Negative Control, Generated, DNA Gel Electrophoresis, Infection, Western Blot, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2 and IMP3 modulate MYC expression in GBM cells in a conserved pathway. RIOK2 or IMP3 knockdown was performed in GBM 301cells using verified lentiviral shRNAs. GBM301 cells were treated with 20 μM zVAD to prevent apoptosis. GBM301 cells with RIOK2 and IMP3 knockdown showed reduced expression of endogenous MYC mRNA, as measured by qPCR, and MYC protein, as measured by immunoblot, compared to control cells infected with GFP shRNA. The samples shown in this figure were harvested 72 hrs after infection, although by 96 hrs after infection we observed a stronger effect on MYC protein levels as shown in , indicating that MYC mRNA and protein levels drop following a prolonged reduction of IMP3 protein. Two replicates were used per condition. Statistics generated using unpaired two-tailed Student TTESTS, **p<0.01. (C, D) Analysis of TCGA glioma tumor profiling data show significant correlation between increased expression of both RIOK2 and IMP3 mRNA and MYC mRNA expression across all TCGA glioma grades (C). Increased expression of IMP3 and MYC mRNA are significantly correlated with each other and significantly increased in grade IV gliomas with EGFR gain and amplification compared to tumors diploid for EGFR (D).

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Expressing, Knockdown, Western Blot, Control, Infection, shRNA, Generated, Two Tailed Test, Amplification

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: A model for the role of the RIOK2-IMP3-TORC2 complex in regulating MYC expression to promote GBM tumorigenesis. RIOK2 catalytic activity promotes autophosphorylation to allow for binding to IMP3 and TORC2. IMP3 is phosphorylated by TORC2 and this complex modulates MYC mRNA to promote increased levels of MYC protein to promote GBM tumorigenesis.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: Expressing, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: RIOK2 drives glioblastoma cell proliferation by modulating MYC through the RNA-binding protein IMP3

doi: 10.1101/2020.12.07.413385

Figure Lengend Snippet: RIOK2-IMP3 complexes include other RNA-binding proteins and are stabilized by RNA. A). GBM39 treated with either DMSO or DSP crosslinker (1 mM) for 2 hrs prior to IPs with either a RIOK2 antibody or a non-specific IgG control. Immunoblots show enrichment of endogenous ATXN2 protein in the RIOK2 IP samples treated with DSP. B). GBM39 total cell lysates pre-treated with RNAse A (100µg/ml) prior to IPs with either a RIOK2 antibody or a non-specific IgG control. Immunoblots show decreased levels of IMP3 protein co-IPed with RIOK2 from the lysate treated with RNase A.

Article Snippet: In vitro immunoprecipitations were prepared using 0.012 μg of bacterially produced and purified recombinant RIOK2 protein (Origene, cat#TP602270) and 0.012 μg of bacterially produced and purified recombinant IMP3 protein (Origene, cat#TP760798) in hypotonic lysis buffer (5 mM Tris, 2.5 mM MgCl 2 , 1.5 mM KCl) with 0.1 mg/ml of Bovine Serum Albumin (BSA) as a competitor.

Techniques: RNA Binding Assay, Control, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Mitochondrial dysfunction induced by HIF‐1α under hypoxia contributes to the development of gastric mucosal lesions

doi: 10.1002/ctm2.1653

Figure Lengend Snippet: METTL3 regulates dynamin‐related protein 1 (Drp1) N6‐methyladenosine (m6A) modification and expression in an IGF2BP3‐dependent manner. (A) Schematic representation of the action of different ‘Readers’ (left panel). The heatmap showing the different ‘Readers’ expression levels in the uninvolved and portal hypertensive gastropathy (PHG) groups. (B) The expression levels of different Readers in the indicated groups were analysed via microarray analysis. n = 3 per group. (C) IGF2BP3 IHC staining (brown) in the related groups was presented. The area affected by IGF2BP3 was also presented. n = 6 per group. * p < .05. (D) The expression of IGF2BP3 was measured by western blotting. (E) The expression of IGF2BP3 in the normal and gastric cancer (GC) groups was detected by IHC staining (brown) and western blotting. The ratio of densitometry units of normalised IGF2BP3/β‐actin and the IGF2BP3 area were listed. n = 6 per group. * p < .05. (F) Double immunofluorescence (IF) staining of Drp1 (red) and IGF2BP3 (green) in the normal and GC groups was shown. Nuclei (blue) were counterstained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). (G) Drp1 expression in the indicated groups after different treatments was measured by western blotting. The ratio of densitometry units of normalised Drp1/β‐actin was also analysed. n = 6 per group. * p < .05 versus sham operation (SO) mice, # p < .05 versus portal hypertension (PHT) mice or SGC7901 cells without shIGF2BP3 transfection. (H) Drp1 expression in vector‐ and HIF‐1α ‐transfected GES‐1 cells with or without shIGF2BP3 transfection was measured by western blotting. The ratio of densitometry units of normalised Drp1/β‐actin was listed. n = 6 per group. * p < .05 versus SO mice, # p < .05 versus HIF‐1α‐transfected GES‐1 cells without shIGF2BP3 transfection. (I) Agarose electrophoresis and RNA immunoprecipitation (RIP)‐qPCR using an anti‐IGF2BP3 antibody showing direct binding between the IGF2BP3 protein and Drp1 mRNA in primary epithelial cells isolated from PHG patients than that from healthy volunteers (uninvolved). * p < .05. (J) Agarose electrophoresis and RIP‐qPCR using an anti‐IGF2BP3 antibody revealing direct binding between the IGF2BP3 protein and Drp1 mRNA in primary epithelial cells isolated from the indicated mouse models. * p < .05. (K) Agarose electrophoresis and RIP‐qPCR using an anti‐IGF2BP3 antibody revealed that IGF2BP3 significantly bound to Drp1 mRNA in SGC7901 cells without METTL3 knockdown ( shMETTL3 transfection). * p < .05. (L) Agarose electrophoresis and qPCR analysis of RIP assays using an anti‐IGF2BP3 antibody revealed the affinity of IGF2BP3 for Drp1 mRNA in vector‐ and HIF‐1α ‐transfected GES‐1 cells. * p < .05. HIF‐1α, hypoxia‐inducible factor‐1α.

Article Snippet: To observe the phenotype of the transient transfectants, Lipofectamine 2000 was used to knockdown IGF2BP3 ( shIGF2BP3 , sc‐60846‐SH, Santa Cruz) and METTL3 ( shMETTL3 , sc‐92172‐SH, Santa Cruz).

Techniques: Modification, Expressing, Microarray, Immunohistochemistry, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Electrophoresis, RNA Immunoprecipitation, Binding Assay, Isolation, Knockdown