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: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A and B ) Western blotting and quantitative determination of IGF2BP3 protein in the kidney at 7 days after UUO. Kidney homogenates after sham and UUO treatment were subjected to Western blotting using anti-IGF2BP3 antibody. ** P < 0.01 versus sham ( n = 4, t test). ( C and D ) Western blot analyses of renal expression of IGF2BP3 in IRI. IGF2BP3 expression was assessed in the kidneys at 11 days after IRI. *** P < 0.001 versus sham ( n = 4, t test). ( E and F ) Western blot analyses of renal expression of IGF2BP3 in ADR. IGF2BP3 expression was assessed in the kidneys at 2 weeks after ADR. ** P < 0.01 versus sham ( n = 4, t test). ( G and H ) Western blot analyses of renal expression of IGF2BP3 after Ang II infusion. IGF2BP3 expression was assessed in the kidneys at 4 weeks after Ang II infusion. * P < 0.05 versus sham ( n = 4, t test). ( I ) Representative micrographs show renal expression and localization of IGF2BP3 protein in various animal models of CKD. IGF2BP3 was detected by immunohistochemical staining. Arrows indicates positive staining. Scale bar, 50 μm. ( J ) Western blots showed IGF2BP3 and active β-catenin distribution in cytosolic and nuclear fractions at 11 days after IRI. α-Tubulin and TATA-binding protein (TBP) were used to normalize cytosolic and nuclear fractions. ( K – M ) qRT-PCR showed renal mRNA levels of IGF2BP3 after UUO ( K ), IRI ( L ), and ADR ( M ), respectively. * P < 0.05, *** P < 0.001 versus sham ( n = 4, t test). Numbers to left of blots indicate kDa.

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining, Binding Assay, Quantitative RT-PCR

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A ) Representative micrographs show the expression and localization of IGF2BP3 protein in human CKD biopsy specimens as indicated. Arrows indicate positive staining. Scale bar, 50 μm. Ctrl, nontumor kidney section from patients with renal cell carcinoma; DN, diabetic nephropathy; CTIN, chronic tubulointerstitial nephritis; LN, lupus nephritis; IgAN, immunoglobulin A nephropathy; FSGS, focal segmental glomerulosclerosis; MN, membranous nephropathy. ( B ) Semiquantitative determination of IGF2BP3-positive area in different groups was shown. *** P < 0.001 versus controls ( n = 5–25, t test). ( C and D ) Linear regression shows significant correlation between IGF2BP3-positive area and fibrotic area ( C ) and serum creatinine (Scr) ( D ). ( E ) Linear regression shows a negative correlation between IGF2BP3 area and kidney function (eGFR).

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Expressing, Staining

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A ) Bioinformatics analyses revealed the presence of putative TBSs in the promoter region of human, mouse, and rat IGF2BP3 gene. The sequences and positions of the putative TBSs were highlighted (red), whereas the TBS consensus sequence was given at the bottom of this panel. TBS, TCF/lymphoid enhancer factor binding site. ( B – D ) Wnt3a (100 ng/mL) induced active β-catenin and IGF2BP3 expression in HKC-8 cells in a time-dependent manner. Representative Western blot ( B ) and quantitative data ( C and D ). ** P < 0.01 versus controls ( n = 3, ANOVA with Student-Newman-Keuls test). ( E – G ) Wnt3a induced active β-catenin and IGF2BP3 expression in a dose-dependent fashion. Representative Western blot ( E ) and quantitative data ( F and G ). ** P < 0.01 versus controls ( n = 3, ANOVA with Student-Newman-Keuls test). ( H ) Immunofluorescence staining for IGF2BP3 expression after Wnt3a stimulation. Cells were stained for IGF2BP3 at 2 days after incubation with Wnt3a. Boxed areas were enlarged. Arrows indicate positive staining. Scale bar, 50 μm. ( I ) Western blots show IGF2BP3 distribution in cytosolic and nuclear fractions. α-Tubulin and TBP were probed to normalize cytosolic and nuclear fractions. ( J and K ) Representative Western blot ( J ) and quantitative data ( K ) show the expression of IGF2BP3 after various treatments. * P < 0.05 versus controls; † P < 0.05 versus pDel-β-cat group alone ( n = 3, ANOVA with Student-Newman-Keuls test). ( L – O ) ICG-001 reduced renal expression of β-catenin, active β-catenin, and IGF2BP3 protein at 7 days after UUO. Representative Western blots ( L ) and quantitative data ( M – O ). ** P < 0.01 versus sham; † P < 0.05, †† P < 0.01 versus the UUO injected with vehicle group ( n = 5, ANOVA with Student-Newman-Keuls test). ( P ) Representative micrographs show IGF2BP3 protein in different groups as indicated. Arrows indicate positive staining. Scale bar, 50 μm. ( Q ) Quantitative determination of IGF2BP3 staining in different groups. ** P < 0.01 versus sham; †† P < 0.01 versus the UUO injected with vehicle group ( n = 5, ANOVA with Student-Newman-Keuls test).

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Sequencing, Binding Assay, Expressing, Western Blot, Immunofluorescence, Staining, Incubation, Injection

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A – F ) Overexpression of IGF2BP3 triggers the upregulation of fibronectin, vimentin, collagen I, and α-SMA expression in tubular cells. HKC-8 cells were infected with either control lentivirus or IGF2BP3 lentivirus overnight, then incubated for 2 days as indicated. Representative Western blots ( A ) and quantitative data of IGF2BP3 ( B ), fibronectin ( C ), vimentin ( D ), collagen I ( E ), and α-SMA ( F ) are shown. * P < 0.05, ** P < 0.01 versus controls ( n = 3, t test). ( G ) Immunofluorescence staining shows fibronectin induction by IGF2BP3 in HKC-8 cells. Cells were stained for fibronectin at 2 days after infection with control lentivirus or IGF2BP3 lentivirus. Boxed areas were enlarged. Arrows indicated positive staining. Scale bar, 50 μm. ( H – M ) IGF2BP3 activates β-catenin and active β-catenin and induces its downstream target genes. HKC-8 cells were infected with either control lentivirus or IGF2BP3 lentivirus overnight, then incubated for 2 days as indicated. Representative Western blots ( H ) and quantitative determination of β-catenin ( I ), active β-catenin ( J ), PAI-1 ( K ), MMP-7 ( L ), and Snail1 ( M ) in HKC-8 cells. * P < 0.05, ** P < 0.01 versus controls ( n = 3, t test). ( N ) IGF2BP3 activates β-catenin–mediated gene transcription. The HEK293T cells were infected with either control lentivirus or IGF2BP3 lentivirus, followed by transfection with the TOPFlash reporter plasmid. TOPFlash reporter luciferase activities were measured. The relative luciferase activity (fold-change) was reported. * P < 0.05 versus controls ( n = 5, t test). ( O – R ) Overexpression of IGF2BP3 triggers the upregulation of KIM-1, p-H3, and p16 INK4A expression in tubular cells. HKC-8 cells were infected with either control lentivirus or IGF2BP3 lentivirus overnight, then incubated for 2 days as indicated. Representative Western blots ( O ) and quantitative data of KIM-1 ( P ), p-H3 ( Q ), and p16 INK4A ( R ) are shown. * P < 0.05, *** P < 0.001 versus controls ( n = 3, t test).

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing, Infection, Incubation, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A – J ) Knockdown of IGF2BP3 inhibited Wnt/β-catenin signaling. HKC-8 cells were incubated with Wnt3a (100 ng/mL) in the presence of control siRNA or IGF2BP3-specific siRNA for 2 days, as indicated. Representative Western blot ( A ) and quantitative data for IGF2BP3 ( B ), β-catenin ( C ), active β-catenin ( D ), Snail1 ( E ), fibronectin ( F ), α-SMA ( G ), KIM-1 ( H ), p53 ( I ), and p16 INK4A ( J ) are presented. * P < 0.05, ** P < 0.01 versus controls; † P < 0.05, †† P < 0.01 versus the Wnt3a plus control siRNA group ( n = 3, ANOVA with Student-Newman-Keuls test). ( K ) Immunofluorescence staining shows fibronectin expression after various treatments. HKC-8 cells were incubated with Wnt3a (100 ng/mL) in the presence of control siRNA or BP3 siRNA for 2 days as indicated. Arrows indicate positive staining. Scale bar, 50 μm. ( L – R ) Representative Western blots ( L ) and quantitative data show the expression of IGF2BP3 ( M ), active β-catenin ( N ), MMP-7 ( O ), fibronectin ( P ), p-H3 ( Q ), and p16 INK4A ( R ). HKC-8 cells were pretreated with ICG-001 (10 μM) for 1 hour, then incubated with Wnt3a (100 ng/mL) for 2 days. * P < 0.05 versus controls; † P < 0.05, †† P < 0.01 versus the Wnt3a group ( n = 3, ANOVA with Student-Newman-Keuls test). ( S ) Schematic presentation of reciprocal feed-forward activation loop between IGF2BP3 and β-catenin.

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Incubation, Western Blot, Immunofluorescence, Staining, Expressing, Activation Assay

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A ) Experimental design. The green arrow indicates the timing of injecting AAV-Ctrl or AAV-BP3. The black arrow indicates the timing of UUO surgery. ( B and C ) Representative Western blot ( B ) and quantitative data ( C ) show the expression of Flag and IGF2BP3 in different groups as indicated. *** P < 0.001 versus the sham injected with AAV-Ctrl group; ††† P < 0.001 versus the UUO injected with AAV-Ctrl group ( n = 5, ANOVA with Student-Newman-Keuls test). ( D ) Micrographs show Flag and IGF2BP3 expression in UUO kidney. Arrows indicate positive staining. Scale bar, 50 μm. ( E – H ) Representative Western blot ( E ) and quantitative data show the expression of vimentin ( F ), fibronectin ( G ), and α-SMA ( H ) proteins in different groups as indicated. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the sham-injected with AAV-Ctrl group; † P < 0.05, †† P < 0.01 versus the UUO injected with AAV-Ctrl group ( n = 5, ANOVA with Student-Newman-Keuls test). ( I ) Micrographs show the expression of vimentin, fibronectin, α-SMA, and collagens at 7 days after UUO in different groups. Arrows indicate positive staining. Scale bar, 50 μm. ( J ) Graphical presentation of renal fibrotic lesions in different groups. *** P < 0.001 versus the sham injected with AAV-Ctrl group; ††† P < 0.001 versus the UUO injected with AAV-Ctrl group ( n = 5, ANOVA with Student-Newman-Keuls test). ( K and L ) Representative Western blots ( K ) and quantitative data ( L ) show the expression of β-catenin, active β-catenin, MMP-7, Snail1, p-H3, p53, and p16 INK4A in different groups as indicated. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the sham injected with AAV-Ctrl group; † P < 0.05, †† P < 0.01, ††† P < 0.001 versus the UUO injected with AAV-Ctrl group ( n = 5, ANOVA with Student-Newman-Keuls test).

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Injection, Staining

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A ) Experimental design. The green arrow indicates the timing of injecting Ctrl-shR or IGF2BP3-shR. The black arrow indicates the timing of UUO. ( B and C ) Representative Western blot ( B ) and quantitative data ( C ) show the expression of IGF2BP3 protein in different groups as indicated. *** P < 0.001 versus the sham group; ††† P < 0.001 versus the UUO injected with Ctrl-shR group ( n = 5, ANOVA with Student-Newman-Keuls test). ( D ) Micrographs show collagen deposition at 7 days after UUO in different groups. Arrows indicate positive staining. Scale bar, 50 μm. ( E ) Graphical presentation of renal fibrotic lesions in different groups. *** P < 0.001 versus the sham group; † P < 0.05 versus the UUO injected with Ctrl-shR group ( n = 5, ANOVA with Student-Newman-Keuls test). ( F ) Representative Western blots show the expression of E-cadherin, vimentin, fibronectin, α-SMA, and collagen I protein in different groups as indicated. ( G ) Micrographs show E-cadherin, vimentin, fibronectin, α-SMA, and β-catenin at 7 days after UUO in different groups. Arrows indicate positive staining. Scale bar, 50 μm. ( H ) Representative Western blots show the expression of β-catenin, active β-catenin, PAI-1, MMP-7, Snail1, p-H3, and p16 INK4A proteins in different groups as indicated.

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Injection, Staining

Journal: JCI Insight

Article Title: Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β -catenin signaling

doi: 10.1172/jci.insight.162060

Figure Lengend Snippet: ( A ) Flowchart shows experimental design and procedures. qRT-PCR was performed with the primers of CTNNB1 and 18S rRNA. ( B ) Representative Western blots show the protein levels of IGF2BP3 from input, RIP with IgG, or αIGF2BP3. ( C ) Assessment of CTNNB1 mRNA in RIP by qRT-PCR. 18S rRNA was used for normalization. ** P < 0.01 versus IgG control group ( n = 3, t test). ( D ) Quantitative data show that knockdown of IGF2BP3 decreases β-catenin mRNA in HKC-8 cells. β-Actin was used for normalization. * P < 0.05 versus Ctrl siRNA group ( n = 3, t test). ( E and F ) Representative Western blots ( E ) and quantitative data ( F ) show the expression of β-catenin protein. ** P < 0.01 versus Ctrl siRNA group ( n = 3, t test). ( G ) Knockdown of IGF2BP3 decreases β-catenin mRNA stability. * P < 0.05, ** P < 0.01 versus Ctrl siRNA group ( n = 3, t test). ( H ) Colocalization of IGF2BP3 protein and β-catenin mRNA in renal tubules after IRI. Kidney serial sections were stained for IGF2BP3 using immunohistochemical staining and β-catenin mRNA using ISH. Arrows indicate IGF2BP3 and β-catenin mRNA colocalization. Scale bar, 50 μm. ( I ) Colocalization of IGF2BP3 (red) and β-catenin protein (green) in renal tubules after IRI. Arrows indicate IGF2BP3 and β-catenin colocalization. Scale bar, 50 μm. ( J ) Working model of the reciprocal feed-forward activation loop between IGF2BP3 and β-catenin. In tubular epithelial cells, Wnts’ engagement with their receptors leads to β-catenin activation, resulting in induction of IGF2BP3. In turn, upregulated IGF2BP3 protein activates β-catenin signaling by binding to and stabilizing its mRNA. -P, dephosphorylation; CBP, cAMP response element binding protein binding protein; TBM, tubular basement membrane.

Article Snippet: Briefly, RIP was carried out by using the RIP-Assay Kit (RN1001; MBL International) according to the manufacturer’s recommendations with normal rabbit IgG/RIP-certified IGF2BP3 antibody and protein A/G plus-agarose beads (Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, Immunohistochemical staining, Activation Assay, Binding Assay, De-Phosphorylation Assay, Protein Binding

Journal: Frontiers in Genetics

Article Title: Molecular Etiology Disclosed by Array CGH in Patients With Silver–Russell Syndrome or Similar Phenotypes

doi: 10.3389/fgene.2019.00955

Figure Lengend Snippet: Genomic imbalances detected using array CGH and categorized as pathogenic (P) or potentially pathogenic (PP) variants.

Article Snippet: All assays were provided by Thermo Fisher Scientific (TaqMan Gene Expression Assays: ID# Hs00959293_m1, GRB10; Hs00946580_m1, IGF2BP3; Hs99999905_m1, GAPDH; Hs00939627_m1, GUSB; Hs00609297_m1, HMBS; Hs99999902_m1, RPLP0; and Hs99999910_m1, TBP).

Techniques:

Journal: Frontiers in Genetics

Article Title: Molecular Etiology Disclosed by Array CGH in Patients With Silver–Russell Syndrome or Similar Phenotypes

doi: 10.3389/fgene.2019.00955

Figure Lengend Snippet: Physical map of the genomic regions and molecular characterization of the potentially pathogenic CNVs identified by array CGH. The 19q13.42 (A-1) and 7p12.1 (B-1) regions showing in blue the duplication identified in patients 8 (A-1) and 18 (B-1) . The RefSeq genes are depicted in dark blue, and the OMIM disease genes in green; the candidate genes are illustrated by red boxes, and the differentially methylated regions (DMRs) in black. The positions of pyrosequencing assays and qPCR probes used in the CNVs’ molecular characterization are reported in black and blue, respectively. The images are a modification of a version obtained from the UCSC Genome Browser (human genome assembly GRCh37/hg19). (A-2) Copy number analysis by using qPCR probes specific for ZNF331 gene promoter confirmed the entire duplication of ZNF331 gene in both patient 8 and her mother (M). (A-3) Pyrosequencing analysis performed on blood DNA of the ZNF331:alt-TSS-DMR2 showed an hypermethylation level in both patient 8 and her mother (M) compared with healthy controls. (B-2) Patient 18’s family tree showing duplication transmission. (B-3) Relative expression of GRB10 blood mRNA in patient 18 and her mother (M), compared with healthy controls. Data were normalized against GAPDH as housekeeping gene; similar results were obtained using TBP , HMBS , and RPLP0 as normalizer (data not shown). (B - 4 , B - 5) Pyrosequencing analysis of the GRB10:alt-TSS-DMR performed on blood DNA showed a methylation level in patient 18 and her maternal grandfather (MGF) compared with healthy controls with both assays, while in patient 18’s mother (M), a hypomethylation level was detected with both assays. CNVs, copy number variants; CGH, comparative genomic hybridization; OMIM, Online Mendelian Inheritance in Man; qPCR, quantitative polymerase chain reaction; UCSC, University of California Santa Cruz.

Article Snippet: All assays were provided by Thermo Fisher Scientific (TaqMan Gene Expression Assays: ID# Hs00959293_m1, GRB10; Hs00946580_m1, IGF2BP3; Hs99999905_m1, GAPDH; Hs00939627_m1, GUSB; Hs00609297_m1, HMBS; Hs99999902_m1, RPLP0; and Hs99999910_m1, TBP).

Techniques: Methylation, Modification, Transmission Assay, Expressing, Hybridization, Real-time Polymerase Chain Reaction

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: bioRxiv

Article Title: CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression

doi: 10.1101/2022.04.25.489339

Figure Lengend Snippet: IGF2BP3 was targeted by gga-miR-449a and promoted the steroid synthesis and secretion. (a) The transfection efficiency of gga-miR-449a mimic; (b) The relative expression of potential target genes of gga-miR-449a in GCs after transfection with gga-miR-449a mimic; (c) The protein expression of IGF2BP3 in GCs after transfection with gga-miR-449a mimic; (d) The relative luciferase activity of the co-transfection of gga-miR-449a and IGF2BP3 in DF-1 cell; (e) The mRNA and protein expressions of IGF2BP3 in GCs after treating IGF2BP3 overexpression or interference; (f) The mRNA expression of key genes related steroid synthesis after transfecting IGF2BP3 overexpression or interference; (g) The concentrations of steroid hormones after transfecting IGF2BP3 overexpression or interference.

Article Snippet: The primary antibodies of IGF2BP3 and GAPDH were purchased from Novusbio (Littleton, CO, USA) and Affinity (Cincinnati, OH, USA), respectively, and the secondary antibody (HRP-Goat anti Rabbit) was provided by Elabscience (Wuhan, China).

Techniques: Transfection, Expressing, Luciferase, Activity Assay, Cotransfection, Over Expression

Journal: bioRxiv

Article Title: CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression

doi: 10.1101/2022.04.25.489339

Figure Lengend Snippet: The evaluation of the potential related signaling pathways of IGF2BP3 regulating steroid hormone synthesis and hormone secretion. (a) The gene expression of IGF2BP3 and the key genes related steroid synthesis in GC after transfected the inhibitors of potential signaling pathway alone or co-transfected with overexpression of IGF2BP3; (b) The concentrations of steroid hormones in GC after transfected the inhibitors of potential signaling pathway alone or co-transfected with overexpression of IGF2BP3.

Article Snippet: The primary antibodies of IGF2BP3 and GAPDH were purchased from Novusbio (Littleton, CO, USA) and Affinity (Cincinnati, OH, USA), respectively, and the secondary antibody (HRP-Goat anti Rabbit) was provided by Elabscience (Wuhan, China).

Techniques: Protein-Protein interactions, Gene Expression, Transfection, Over Expression

Journal: bioRxiv

Article Title: CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression

doi: 10.1101/2022.04.25.489339

Figure Lengend Snippet: Gga-miR-449a reversed the regulatory role of circEML1 on the expressions of gga-miR-449a and IGF2BP3 and the promoting effect of steroid synthesis. (a) The mRNA expression of gga-miR-449a after transfection with cicrEML1 overexpression and gga-miR-449a mimic/NC in GCs; (b) The relative mRNA and protein expression of IGF2BP3 in GCs; (c) The relative mRNA expressions of the key genes related steroid synthesis in GCs; (d) The concentrations of E2, P4 and androgen in GCs.

Article Snippet: The primary antibodies of IGF2BP3 and GAPDH were purchased from Novusbio (Littleton, CO, USA) and Affinity (Cincinnati, OH, USA), respectively, and the secondary antibody (HRP-Goat anti Rabbit) was provided by Elabscience (Wuhan, China).

Techniques: Expressing, Transfection, Over Expression