Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: Identification of Nono as a phospho-HLXB9 interacting protein. A, overexpression of HLXB9 shows both its phosphorylated and unphosphorylated isoform. WCE were prepared from MIN6-4N cells transfected with myc-His-tag empty vector (mh-Vector) or plasmids expressing myc-his-tagged HLXB9 (mh-HB9-WT) or the phospho-dead mutant of HLXB9 with alanine substitution at Ser-78 and Ser-80 (mh-HB9-AA). WCE were run on the same gel to generate two Western blots to probe with anti-myc-tag or anti-HB9-PO4 (phospho-HLXB9 antibody). To analyze the bands, the blots were placed side-by-side (indicated by the dotted line). The top band of the doublet in the lane marked mh-HB9-WT corresponds to phospho-HLXB9 because it is not detected with the myc-tag antibody in mh-HB9-AA, and it is detected specifically with anti-HB9-PO4. The bottom band of the doublet corresponds to the unphosphorylated isoform of HLXB9 (HB9-unPO4) because it is not detected with anti-HB9-PO4. β-Actin was used as the loading control. B, large scale co-IP shows the two isoforms of HLXB9 and co-immunoprecipitating proteins. Silver-stained gel of proteins separated on SDS-PAGE after large scale co-IP with a myc-tag antibody using WCE prepared in A. As also seen by Western blot analysis in A, the bands marked SDMS1 and SDMS3 show the doublet in mh-HB9-WT Co-IP (PO4-HB9 and unPO4-HB9) and a single band in mh-HB9-AA corresponding to unPO4-HB9. Bands marked SDMS1, SDMS2, SDMS3, and SDMS4 (that were absent in the mh-Vector lane) were excised from the gel and subjected to mass spectrometry analysis. C, Nono uniquely co-immunoprecipitates with phospho-HLXB9. The number of peptides (total and unique) in the bands excised from the gel shown in B and their corresponding proteins is shown. Several proteins were present in both the mh-HB9-WT and mh-HB9-AA immunoprecipitates. The protein Nono emerged as a phospho-HLXB9-specific partner found only in the co-IP of mh-HB9-WT and not in the co-IP of the phospho-dead mutant of HLXB9 (mh-HB9-AA). D, overexpressed HLXB9 can Co-IP with endogenous Nono. Western blot (WB) probed with anti-myc-tag showing specific co-IP of HLXB9 with Nono using WCE of MIN6-4N cells transfected with mh-HB9-WT. Anti-HA-tag was used as a negative control. Higher exposure (top panel) and lower exposure (bottom panel) of the blot are shown to clearly visualize the input HLXB9 bands. IP, immunoprecipitation. E, endogenous phospho-HLXB9 can co-immunoprecipitate with endogenous Nono. Western blots probed with anti-Nono and anti-HB9 show specific co-IP of endogenous Nono with endogenous phospho-HLXB9 using WCE of MIN6-4N cells. An anti-HA-tag was used as a negative control.

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, Staining, SDS Page, Mass Spectrometry, Negative Control, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: HLXB9 co-localizes with Nono in the nucleus, and co-overexpression of HLXB9 and Nono decreases the overexpression of Nono with translocation of HLXB9 into the cytoplasm. A, endogenous phospho-HLXB9 co-localizes with endogenous Nono in the nucleus. IF images of MIN6-4N cells show endogenous Nono (red) and phospho-HLXB9 (green). DAPI was used to detect the nuclei (blue). A merged image of the red and green IF shows co-localization of Nono and phospho-HLXB9 in subnuclear spots and some regions with phospho-HLXB9 (green) that did not co-localize with Nono. B, overexpression of HLXB9 decreases the level of overexpressed Nono protein. Western blots are shown of WCE and the pellet leftover after WCE preparation from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA (Vec) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag and with anti-Nono to detect both endogenous and transfected FLAG-tagged Nono. β-Actin was used as the loading control. Endogenous Nono levels were not affected by HLXB9 overexpression. However, the level of transfected FLAG-Nono was reduced upon HLXB9 overexpression. A similar pattern of bands was seen in the pellet leftover after WCE preparation, indicating that the reduced level of Nono upon HLXB9 overexpression was not due to differential cell lysis in the WCE preparation. C, HLXB9 did not reduce the expression of endogenous Nono protein. Western blot analysis to detect endogenous HLXB9 and Nono using WCE prepared from MIN6-4N cells transfected with control siRNA (siC) or HLXB9 siRNA (siHB9) is shown. p84 was used as the loading control. HLXB9 was significantly knocked down, but that did not affect the level of endogenous Nono. D, co-overexpression of HLXB9 and Nono reduces the level of Nono protein in the nucleus with translocation of HLXB9 to the cytoplasm. Shown is Western blot analysis of subcellular fractionation of CE, NE, and CB/PE from MIN6-4N cells expressing mh-HB9-WT, FLAG-Nono, or both together. Empty vector DNA (Vec) was used to maintain the same amount of DNA in the transfections. The expression of transfected HLXB9 was detected with the anti-myc-tag; Nono was detected with the anti-FLAG-tag; detection of marker proteins (Hsp90 for CE, p84 for NE, and histone H3 for CB/PE) showed minimal cross-contamination of the fractions and also served as loading controls for each fraction. Overexpressed Nono was found in the NE and in the CB/PE, but its level in NE was reduced by HLXB9 overexpression. Overexpressed HLXB9 was mostly located in the CB/PE and with a significant amount in the nucleus, but it was also detected in the CE by Nono overexpression.

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Over Expression, Translocation Assay, Western Blot, Expressing, Plasmid Preparation, Transfection, FLAG-tag, Lysis, Fractionation, Marker

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: Identification of Cblb as a phospho-HLXB9 target gene. A, the anti-HB9-PO4 antibody specifically recognizes the phosphorylated isoform of HLXB9. WCE and chromatin were prepared from MIN6-4N cells transfected with a plasmid expressing myc-his-tagged HLXB9 (mh-HB9-WT). WCE was used IP with rabbit antibodies anti-myc-tag or anti-HB9-PO4 and detected by Western blot (WB) with mouse anti-myc-tag. Rabbit anti-HA-tag was used as the negative control. The input WCE and anti-myc-tag IP display a doublet corresponding to phospho-HLXB9 and unphosphorylated HLXB9. Anti-HB9-PO4 could specifically immunoprecipitate phospho-HLXB9 corresponding to the top band of the doublet. B, significant enrichment of promoter regions among the anti-HB9-PO4 ChIP-Seq tags. Chromatin prepared from MIN6-4N cells transfected in A was used for ChIP with anti-HB9-PO4. DNA obtained before and after ChIP was used for preparing libraries followed by deep sequencing (ChIP-Seq) and mapping of the anti-HB9-PO4-specific ChIP-Seq tags to the mouse genome. The pie chart shows the percent distribution of tags in the mouse genome (a typical input library, Genomatix) and in the anti-HB9-PO4 ChIP-Seq at the indicated regions; 20% of the anti-HB9-PO4 ChIP-Seq tags were located near promoter regions and selected for further analysis. C, phospho-HLXB9 occupancy is highest at the Arid1b and Cblb gene in cells overexpressing HLXB9. ChIP-quantitative PCR assay for validating the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells expressing mh-HB9-WT was used for anti-HB9-PO4 ChIP. Also shown is a Western blot confirming overexpression of HLXB9 (myc-tag antibody) and β-actin as the loading control. D, endogenous phospho-HLXB9 occupancy is highest at the Cblb gene. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells was used for endogenous anti-HB9-PO4 ChIP. Endogenous phospho-HLXB9 occupancy was only detected at Cblb. E and F, H3K4me3 at Cblb unaffected but reduced H3K27me3 upon HLXB9 knockdown. ChIP-quantitative PCR assay of the 10 phospho-HLXB9 targets is shown as the percent of input chromatin DNA PCR for each primer pair. Chromatin prepared from MIN6-4N cells transfected with control siRNA (siC) or HLXB9 siRNA (siHB9) was used for endogenous anti-H3K4me3 ChIP (E) or H3K27me3 ChIP (F). Also shown is a Western blot confirming knockdown of HLXB9 (HLXB9 antibody) and β-actin as the loading control. In siC versus siHB9, reciprocal H3K4me3 or H3K27me3 at only Cblb was HLXB9 binding-dependent because endogenous phospho-HLXB9 was only found to occupy Cblb (D).

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, ChIP-sequencing, Sequencing, Real-time Polymerase Chain Reaction, Over Expression, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: HLXB9 suppresses the expression of Cblb mRNA by suppressing Cblb promoter activity. A and B, among targets identified by anti-HB9-PO4 ChIP-Seq, the expression of only Arid1b and Cblb was affected by HLXB9. Quantitative RT-PCR is shown of the indicated genes using RNA prepared from MIN6-4N cells transfected with control siRNA (siC) or HLXB9 siRNA (siHB9), empty vector, or mh-HB9-WT. A, Western blot confirming knockdown or overexpression of HLXB9 with anti-HLXB9 or anti-myc-tag, respectively, and β-actin as the loading control. B, HLXB9 knockdown or overexpression regulated the relative mRNA level of only two genes (Arid1b and Cblb). Both were reduced upon HLXB9 overexpression, but only Cblb was increased upon HLXB9 knockdown. Error bar = mean and S.D. from three experiments. * = p < 0.05. C and D, Nono did not regulate the expression of HLXB9 target genes. Shown is quantitative RT-PCR of the indicated genes using RNA prepared from MIN6-4N cells transfected with control shRNA (shC) or Nono shRNA (shNono), FLAG-vector, or FLAG-Nono. A, Western blot confirming knockdown or overexpression of Nono with anti-Nono or anti-FLAG-tag, respectively, and β-actin as the loading control. B, Nono knockdown or overexpression did not regulate the relative mRNA level of any gene. Error bar = mean and S.D. from three experiments. E and F, Cblb promoter activity is suppressed by HLXB9. The promoter region of Cblb (−1078 to +219) located near the sequence identified by anti-HB9-PO4 ChIP-Seq was cloned in the promoter-less luciferase reporter vector PG02 (GeneCopoeia) and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb plasmid showed significantly high RLU, and co-expression of increasing amounts of HLXB9 suppressed the Cblb promoter activity. Error bar = mean and S.D. from three experiments. * = p < 0.05. A representative Western blot shows the expression of HLXB9 (with anti-myc-tag) in the MIN6-4N cells analyzed for luciferase activity. p84 was used as the loading control. G and H, Cblb expression is down-regulated by the phosphorylated isoform of HLXB9. WCE and RNA were prepared from MIN6-4N cells transfected with control-shRNA (C−) or Men1-shRNA (M−) together with empty vector or mh-HB9-WT. G, Western blot shows the extent of menin knockdown (anti-menin blot) and transfected HLXB9 and increased phospho-HLXB9 in M− (anti-myc-tag blot, top band of the doublet band). H, quantitative RT-PCR shows significantly reduced Cblb mRNA upon menin knockdown in mh-HB9-WT-transfected cells. Error bar = mean and S.D. of a representative experiment performed in triplicate. * = p < 0.05.

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Expressing, Activity Assay, ChIP-sequencing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Over Expression, shRNA, FLAG-tag, Sequencing, Clone Assay, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: HLXB9 binding motif in the Cblb promoter. A, consensus motif in anti-HB9-PO4 ChIP-Seq tags located at promoter regions. De novo motif analysis from anti-HB9-PO4 ChIP-Seq tag sequences located at promoter regions was performed using Genomatix software. A core sequence ATTTTA was identified that resembles homeodomain-binding consensus (35). B, sequence of the HLXB9 binding motifs in the Cblb promoter. The top line shows the putative HLXB9 binding motifs (red) in the DNA sequence of the Cblb promoter (−741 to −710 region from the transcriptional start site is shown). The bottom line shows nucleotide substitutions (blue) to mutate the motifs by site-directed mutagenesis in the Cblb-promoter construct used in C. C and D, HLXB9 did not suppress the activity of the Cblb promoter containing mutations at the HLXB9 binding motifs. The putative HLXB9 binding motifs shown in B were mutated by site-directed mutagenesis of the PG02-Cblb promoter construct and analyzed for promoter activity in MIN6-4N cells. RLU for each of the transfections are shown. Compared with the empty vector PG02, the PG02-Cblb-SDM2 plasmid showed significantly high RLU and co-expression of increasing amounts of HLXB9 did not suppress the Cblb promoter activity. Error bar = Mean and S.D. from 3 experiments, * = p < 0.05. A representative Western blot shows expression of HLXB9 (with anti-myc-tag) in the MIN6-4N cells analyzed for luciferase activity. p84 was used as the loading control.

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Binding Assay, ChIP-sequencing, Software, Sequencing, Mutagenesis, Construct, Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: Cblb overexpression or HLXB9 knockdown inactivates the oncogenic c-Met pathway. A, overexpression of Cblb or knockdown of HLXB9 decreases c-Met levels. Western blot analysis of the indicated proteins using WCE prepared from MIN6-4N cells transfected (by nucleofection) with empty vector or Cblb expression plasmid, control siRNA (siC), or HLXB9 siRNA (siHB9) and control shRNA (shC) or Nono shRNA (shNono). Cblb overexpression reduced the level of endogenous c-Met. HLXB9 knockdown increased the level of endogenous Cblb and reduced the level of endogenous c-Met. p84 was used as the loading control. The upper band marked pro-c-Met is the glycosylated c-Met precursor form that is cleaved and processed into mature c-Met (lower band) (44). B, reduced cell proliferation from Cblb overexpression but not from HLXB9 or Nono knockdown. An MTT assay assessed cell proliferation of MIN6-4N cells transfected in A. Overexpression of Cblb caused a slight but significant reduction in cell proliferation, but cell proliferation was unaffected upon HLXB9 or Nono knockdown. Note that the Western blots in A were performed using WCE prepared at 96 h post-transfection. Error bar = mean and S.D. from 3 experiments, * = p < 0.05. vec, vector. C, reduced cell migration from Cblb overexpression or HLXB9 knockdown but not from Nono knockdown. Cell migration assay of MIN6-4N cells transfected in A assessed by staining for cells that migrate across a polycarbonate membrane in a Boyden chamber. Stain from the cells was extracted and measured at 560 nm as an index of cell migration. Cblb overexpression or HLXB9 knockdown significantly reduced cell migration. Error bar = mean and S.D. from three experiments. * = p < 0.05. D, soft agar colony formation is unaffected from Cblb overexpression, HLXB9 knockdown, or Nono knockdown. Bright-field microscopy images of colonies (white dots) formed in soft agar by MIN6-4N cells transfected in A. MIN6-4N cells make very small colonies after 4–6 weeks. The number of days to form colonies or the number and size of the colonies was unaffected by Cblb overexpression, HLXB9 knockdown, or Nono knockdown.

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, shRNA, MTT Assay, Migration, Cell Migration Assay, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conventional mouse model of menin loss (Men1+/−). Shown are images of immunofluorescence for insulin and IHC for the indicated proteins in the pancreas section of an 18-month-old Men1+/− mouse. Insulin staining shows the location of the normal-looking islet (panels on the left) and the large islet tumor that covers the entire viewing field (panels on the right). Compared with a normal-looking insulin-positive islet in the same section, the insulin-positive islet tumor shows increased nuclear staining for HB9 and HB9-PO4 and increased nuclear and cytoplasmic staining for c-Met but almost no staining for Cblb (cytoplasm).

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b) *

doi: 10.1074/jbc.M115.661413

Figure Lengend Snippet: Increased phospho-HLXB9, decreased Cblb, and increased c-Met in insulinoma from the conditional mouse model of menin loss (RIP-Cre-Men1f/f). Images of immunofluorescence for insulin and IHC for the indicated proteins in pancreas sections from a 12-month-old Men1f/f mouse and RIP-Cre-Men1f/f mouse. Compared with the insulin-positive normal islet (panels on the left), the large insulin-positive islet tumor that covers the entire viewing field (panels on the right) shows increased nuclear staining for HB9 and HB9-PO4, increased nuclear and cytoplasmic staining for c-Met, and decreased staining for Cblb (cytoplasm).

Article Snippet: The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) ( 11 ), pcDNA3.1-mh-menin ( 27 ), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA ( 28 ), and control and Nono shRNA ( 29 ).

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes

doi:

Figure Lengend Snippet: A. Representative immunoblots showing the re-expression of RD3 in MSDACs and silencing of RD3 in SH-SY5Y cells. Expression of GFP-tagged RD3 (Origene) was carried out by using TurboFectin 8.0 and RD3 silencing with shRNA (MISSION ® shRNA, Sigma-Aldrich) following standard protocols. B. Representative microphotographs acquired from Operetta high-content confocal immunofluorescence imaging validate RD3 silencing in SH-SY5Y cells and RD3 re-expression in MSDACs. C. Scratch-wound-assay showing the cell-migration patterns of MSDACs and RD3-re-expressed MSDACs under proliferation controlled conditions at 0, 24 and 48 h after wound initiation. MSDACs exhibits robust cell migrations with significant wound closure after 48 h, while re-expression of RD3 in MSDACs significantly inhibited their migration. D. Histograms of scratch wound gap measurements (mean and SD) showing the cell migration patterns of MSDACs with and without RD3 re-expression and parental SH-SY5Y cells with and without RD3 silencing examined at 0, 24 and 48 h after wound initiation. Group-wise comparisons were examined by two-way ANOVA with Bonferroni's post-hoc test made using GraphPad PRISM software and a P value of < 0.05 is considered significant. E. Scratch-wound-assay showing the cell-migration patterns of SH-SY5Y cells and RD3-silenced SH-SY5Y cells under proliferation controlled conditions at 0, 24 and 48 h after wound initiation. SH-SY5Y cells exhibited only base-line migrations after 48 h, while silencing RD3 in SH-SY5Y cells consistently increased their migration with significant wound closure. F. Representative microphotographs of matrigel invasion assay showing robust invasion of MSDACs, completely alleviated invasion in RD3-re-expressed MSDACs and profound increase in invasive potential of RD3-silenced SH-SY5Y cells. Invasion assays are performed using BD Matrigel invasion assay following standard protocols. G. Histograms of matrigel invaded cells (mean and SD) showing complete inhibition of MSDACs' invasion potential with RD3 re-expression and significant increase in the invasiveness of RD3-silenced SH-SY5Y cells. Quantification of invaded cells was performed using Image Quant colony count analysis software and the group-wise comparisons were examined by ANOVA with Bonferroni's post-hoc corrections using GraphPad PRISM software. A P value of < 0.05 is considered significant.

Article Snippet: Expression of RD3 (GFP-tagged - Human retinal degeneration 3, transcript variant 1, Origene) was carried out by using TurboFectin 8.0 reagent (Origene).

Techniques: Western Blot, Expressing, shRNA, Immunofluorescence, Imaging, Scratch Wound Assay Assay, Migration, Software, Invasion Assay, Inhibition

Journal: Oncotarget

Article Title: RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes

doi:

Figure Lengend Snippet: A. Representative immunoblots showing RD3 knocked down in human SH-SY5Y cells stably transfected with RD3 shRNA (MISSION ® shRNA, Sigma-Aldrich) with puromycin mammalian selection and re-expression of RD3 in MSDACs that were stably transfected with RD3 (GFP-tagged - Human retinal degeneration 3, transcript variant 1, Origene Technologies) with neomycin mammalian selection. The stable transfection was carried out using either TurboFectin 8.0 reagent (Origene) or Neon electroporation transfection system (Life Technologies). Representative photomicrograph showing cells stably transfected with GFP tagged RD3 construct showing the expression of GFP. B. Representative phase contrast photomicrographs showing tumorosphere forming capabilities of SH-SY5Y (vc), RD3 stably silenced SH-SY5Y cells, MSDACs (vc) and RD3 stably re-expressed MSDACs maintained in serum free stem cell culture conditions. C. Schematic representation and representative mouse images showing relative tumorigenic capacity and aggressive disease formation of RD3 stably expressing MSDACs. RD3 stably expressing MSDACs resulted in the development of relatively small xenograft (~250 mm 3 ) without metastatic tumors compared with the large xenografts with multiple metastasis in mice that received MSDACs (vc). Vertical Box and Whiskers plot showing mean number of metastatic tumors observed in animals that received MSDACs and RD3 re-expressed MSDACs.

Article Snippet: Expression of RD3 (GFP-tagged - Human retinal degeneration 3, transcript variant 1, Origene) was carried out by using TurboFectin 8.0 reagent (Origene).

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Selection, Expressing, Variant Assay, Electroporation, Construct, Stem Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Visualization of Juno and CD9 localization in oolemma captured by 3D STED. Imaging of Juno (green) and CD9 (red) in oolemma (A–C) in whole oocyte surface visualized by maximal intensity projection, (D–F) in one plane and (G–I) in selected area of one plane. The asterisk (*) indicates microvilli -free region. Scale bar represents 10 μm (A–F) , 5 μm ( G–I) . For more details see .

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Imaging

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Visualization of the mutual position of Juno and CD9 in super-resolution images captured by 3D STED. (A) Imaging of Juno (green) and CD9 (red) in oolemma in whole oocyte surface visualized by maximal intensity projection and (B) in one plane. (C) A top and bottom segment of oocyte was captured for analysis of Juno and CD9 mutual localization within oolemma. (D–F′) The representative image analyzed by Imaris software shows the colocalization area (white) of the studied proteins in a top and bottom segment (D) , in selected area of oolemma (E) and in an individual plane (F,F′) . The asterisk (*) indicates polar body. Scale bar represents 10 μm (A–D) , 5 μm (E) . For details see .

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Imaging, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Study of close proximity of Juno and CD9 in mouse oocyte oolemma by PLA. (A) The presence of positive signal (red dots) on the sample stained by Juno and CD9 and visualized by maximal intensity projection, confirmed the existence of their close proximity, (B) α and β tubulin-stained sample was used as a positive control of the method. (C) Combination of α tubulin and CD9 staining was used as a negative control. Chromosomes are visualized with Dapi (blue). Scale bar represents 10 μm.

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Staining, Positive Control, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Analysis of Juno-CD9 protein-protein interaction via co-immunoprecipitation and MS. (A) HEK293T/17 cells were co-transfected with Juno and CD9-GFP mouse plasmids (pJuno/pCD9-GFP). CD9 protein was visualized in the cell membrane immediately after transfection via fluorescent GFP-tag (green); BF (bright field). (B) Juno protein expression was confirmed by WB and visualized by anti-Juno antibody. (C) Juno-CD9 complex was precipitated via GFP-tag on CD9 using anti-GFP antibody. CD9-bound target protein Juno was detected by using WB analysis with anti-Juno antibody (∼28 kDa); ST–molecular standards. (D) Schematic figure depicting MS analysis of the protein complex, which was bound to immunobeads. Database-search algorithms (bioinformatics) were used to identify specific proteins based on their mass spectra; see raw data files Table 1 MS_IP; Table 2 MS_IP control; https://biobox.biocev.org/index.php/s/4NCxYE2ckAJ4eCP ). For both WB and MS the non-transfected HEK293T/17 cells were used as a negative control (p−/−), and neither CD9 nor Juno was detected and identified. (E) As a control for GFP-tag antibody specificity, the Juno and GFP antibody detection was performed in the anti-GFP immunoprecipitate (IP) and in Juno transfected cell lysate (pJuno/–), respectively; ST–molecular standards.

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Immunoprecipitation, Transfection, Membrane, Expressing, Control, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Localization of Juno and CD9 in MvM and PlnM compartments of microvilli -rich oolemma. (A) STED microscopy visualization of microvilli (B) SEM visualization of microvilli . (C) STED imaging revealed differences in Juno (green) and CD9 (red) localization within MvM and PlnM compartments of oolemma. (D) Scheme represents dividing the individual oolemma compartments for image analyzis of STED and TEM data. (E) Comparison of the localization of Juno and CD9 in MvM and PlnM separately in the segmented regions of oolemma shows significant difference between Meander’s coefficient 1 (M1) and Meander’s coefficient 2 (M2) in PlnM and significant difference in Meander’s coefficient 2 (M2) between MvM and PlnM regions. (F–H) Juno and CD9 differences captured by TEM, Juno (green arrows) was present in both MvM and PlnM in contrast to CD9 (red arrows) which was mainly detected in MvM. (I) TEM image analysis confirmed significant differences in localization Juno and CD9 both between MvM and PlnM compartments. Scale bar represents 10 μm (A) , 1 μm (C) 500 nm (B,F–H) . p -value equal or lower 0.05 was considered to be significant ( p ≤ 0.001***). n–number of golden particles, l–total length per frame (nm), n/l–number of particles on total length per frame.

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Microscopy, Imaging, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Juno and CD9 protein network organization in oolemma of mouse oocyte

doi: 10.3389/fcell.2023.1110681

Figure Lengend Snippet: Juno-CD9 interactive model within the oocyte oolemma. (A–C) Biologically relevant position of Juno and CD9, shown by interacting pose in the membrane model for (A) Juno, (B) CD9, (C) Juno-CD9 prepared in CHARMM-GUI. (D) Energetically adequate and biologically most favorable and significant docked pose of Juno-CD9. (E) Interacting amino acids of Juno and CD9, where 26 hydrogen bonds were formed between Juno and CD9, and the distances between donor hydrogen and acceptor can be seen. (F) Responsible amino acids from Juno and CD9 responsible for hydrogen bonds formation and Juno-CD9 complex formations. (G) Juno-CD9 docked pose, where blue marked residues from Juno and CD9 are interacting for making the complex, whereas the orange residues were identified (either by docking or human Izumo1-Juno crystal analysis) for interacting with Izumo1. (H) RMSD plot for Juno (green), CD9 (red), and Juno and CD9 complex (blue). Based on RMSD analysis, Juno maintain the highest level of stability around 2.2 Å, CD9 showed highest deviation around 5.5 Å, whereas Juno and CD9 complex displayed deviation, 3.6 Å, in the middle between CD9 and Juno readings. The Juno-CD9 complex was reflected by highly stable conformation during the MD simulation which indicated a stable biological relevant structural conformation of Juno and CD9 complex.

Article Snippet: When cells reached 70–80% of confluence, they were co-transfected by 3 µg of mouse CD9-GFP plasmid DNA (MG226288, Origene, MD, USA) and 3 µg of mouse Juno plasmid DNA (MC207552, Origene) and left for 16 h in DMEM cultivation medium with 10% of FBS without antibiotics.

Techniques: Membrane