Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: In silico analysis of rod-specific photoreceptor cGMP phosphodiesterase (PDE6D) expression in hepatocellular carcinoma (HCC). ( A ) Oncomine TM human cancer microarray database analysis of six patient datasets depicting PDE6D mRNA expression levels in HCCs and non-tumorous livers (* p < 0.05 vs. non-tumorous livers). ( B , C ) Oncomine TM human cancer microarray database analysis of PDE6D expression as detected in large-scale RNA profiling studies comparing diverse carcinomas of different origins (* p < 0.05 vs. average expression).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: In Silico, Expressing, Microarray

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: PDE6D expression in HCC in vivo and in vitro. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis of HCC patient samples and paired non-tumorous liver tissues (* p < 0.05). ( B ) Detection of PDE6D mRNA in human HCC cells (PLC, Hep3B, HepG2) after qRT-PCR amplification using gel electrophoresis (left panel) and relative PDE6D mRNA levels (qRT-PCR) in human HCC cell lines (PLC, Hep3B, HepG2) compared with primary human hepatocytes derived from different donors (#1–3) (right panel) (* p < 0.05 vs. hepatocytes). ( C ) Exemplary Western blot image (left panel) and summarized densitometric quantification (right panel) of PDE6D protein levels in HCC cells (PLC, Hep3B, HepG2, Huh-7) compared with hepatocytes derived from different donors (#1–2) (* p < 0.05 vs. hepatocytes).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Quantitative RT-PCR, Amplification, Nucleic Acid Electrophoresis, Derivative Assay, Western Blot

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Effects of PDE6D knockdown on HCC proliferation and clonogenicity. Prior to functional experiments, HCC cell lines (PLC, Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis (* p < 0.05 vs. control). ( B ) PDE6D protein levels as quantified by Western blot analysis. The left panel depicts an exemplary Western blot image, and the right panel depicts the summarized densitometric quantification (* p < 0.05 vs. control). ( C ) Real-time cell proliferation (xCELLigence). Exemplary proliferation curves (left panel) and quantified “slopes” (summarizing the proliferative ability) (right panel) are shown (* p < 0.05 vs. control). ( D ) Relative (to mean) PDE6D as correlated to CyclinD1 mRNA expression levels (qRT-PCR) in human HCC patient tissue samples. ( E , F ) Anchorage-dependent clonogenic assay (an exemplary image (Hep3B) is depicted in the left panel of ( E )). Quantification of colony number (right panel of ( E )) and sizes ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Functional Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Clonogenic Assay

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and function of PDE6D in sorafenib resistance. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B and HepG2 cell clones (* p < 0.05). ( B ) Exemplary image (left panel) and summarized densitometric quantification (right panel) of Western blot analysis of PDE6D levels in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B cells (rel DM: relative (PDE6D/Actin) densitometry) (* p < 0.05 vs. non-resist.). ( C , D ) Prior to functional experiments, resistant cells (Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( C ) Depicts relative proliferation (cell numbers) and ( D ) depicts exemplary clonogenic assays. ( E , F ) Forced overexpression of PDE6D protein (PDE6D-OE) in HCC cells (e.g., PLC) was performed by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (an empty control vector without the PDE6D ORF was used as control treatment). ( E ) Depicts Western blot analysis depicting the overexpressed Myc-DDK-tagged PDE6D protein after PDE6D-OE as well as endogenous (arrow) PDE6D in both PDE6D-OE and control-treated cells. ( F ) Depicts exemplary images (representing 8 replicate values of 2 independent experiments) of cells cultured in 6-wells for 72 h (100,000 cells were initially seeded per 6-well) and treated with different doses of sorafenib (0, 4, 8 µM).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Western Blot, Functional Assay, Transfection, Over Expression, Plasmid Preparation, Cell Culture

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: TGF-â-mediated regulation of PDE6D and the effect of PDE6D on HCC cell migration. ( A – C ) Quantitative RT-PCR analysis of VIMENTIN, SNAIL, S100A4 ( A ) and KRAS ( B ) mRNA levels in HCC cells (PLC) that were stimulated with different doses of recombinant human TGF-â1 protein for 72–96 hours (* p < 0.05 vs. control). ( C , D ) Quantitative RT-PCR revealing mRNA levels ( C ) as well as protein levels as quantified by Western blot analysis (including a representative Western blot image) (the densitometric values represent two independent Western blot analysis) ( D ) of PDE6D expression in HCC cells (PLC) that were treated with different doses of recombinant human TGF-â1 for 72–96 hours. ( D ) also depicts co-treatment with 15 µM of the TGF-â-receptor-1 (TGFBR1) inhibitor LY2157299 (“galunisertib”) (* p < 0.05 vs. control). ( E , F ) Prior to Boyden chamber experiments, non-resistant HCC cells (PLC, Hep3B) ( E ) and sorafenib-resistant Hep3B cells ( F ) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). Migration (migrating cells per visual field) as measured by Boyden chamber migration assay (duration of migration: 4 hours) is depicted as absolute cell counts ( E ) or as normalized migration ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Migration, Quantitative RT-PCR, Recombinant, Western Blot, Expressing, Transfection

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and cellular localization of PDE6D in HCC in vivo and in vitro. ( A ) PDE6D staining (exemplary images) of non-tumorous liver tissues (left side) and HCC tissues (right side) deposited on the Human Protein Atlas database. ( B ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues ( N = 117) as compared with corresponding non-tumorous liver tissues ( N = 127) (Fisher’s exact P < 0.001). ( C , D ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues correlated with tumor grading (Fisher’s exact P = 0.030) ( C ) and tumor stages ( D ). ( E ) Exemplary immunohistological images of PDE6D protein expression in human HCC samples and corresponding non-tumorous liver tissues applying a tissue microarray revealing nuclear staining next to cytoplasmatic staining patterns (paired samples of two different patients (#1, #2) are depicted). ( F , G ) Tissue microarray analysis comparing tumor stages ( F ) and ERK activation (p-ERK) ( G ) in human HCC tissues with (“yes”) and without (“no”) cytoplasmatic localization pattern of PDE6D. ( H ) In silico-based analysis of importin-á-dependent nuclear localization signals (NLS, red letters ) using the “cNLS Mapper” predicted bipartite NLS in both isoforms of PDE6D (score for both isoforms was 5.3). A legend depicts that higher scores indicate stronger NLS activity and defines major localizations in dependence of each score. ( I , J ) Exemplary immunofluorescence ( I ) and Western blot analysis ( J ) depicting nuclear localization of PDE6D (I) and expression of PDE6D in both nuclear and cytoplasmatic fractions ( J ) of HCC cell lysates.

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Staining, Microarray, Activation Assay, In Silico, Activity Assay, Immunofluorescence, Western Blot

Journal: Cancer Genomics & Proteomics

Article Title: SPOCD1 Enhances Cancer Cell Activities and Serves as a Prognosticator in Esophageal Squamous Cell Carcinoma

doi: 10.21873/cgp.20503

Figure Lengend Snippet: SPOCD1 mRNA expression and the effects of siRNA-mediated SPOCD1 knockdown in ESCC cells. (A) SPOCD1 mRNA levels in 20 ESCC cell lines. (B) Heatmap from CCLE data illustrating expression of genes highly correlated with SPOCD1 and their related pathways in 25 ESCC lines (C) siRNA-mediated knockdown effect of SPOCD1 in KYSE590 and KYSE70 cells on SPOCD1 expression. (D) Simple Western images illustrating detection of SPOCD1 and siRNA-mediated knockdown efficiency of SPOCD1 in KYSE590 and KYSE70 cells. Error bars indicate the standard deviation. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Subsequent to transfection, cells for conducting functional assays were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), but without antibiotics, for 48 h. To achieve enforced expression of SPOCD1 , the SPOCD1 Human-Tagged Open Reading Frame Clone sequences were inserted into pLenti-C-Myc-DDK-P2A-Puro (product ID, RC218945L3V; OriGene, Rockville, MD, USA), and transduction was performed according to the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Simple Western, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SPOCD1 Enhances Cancer Cell Activities and Serves as a Prognosticator in Esophageal Squamous Cell Carcinoma

doi: 10.21873/cgp.20503

Figure Lengend Snippet: Effects of siRNA-mediated SPOCD1 knockdown on ESCC cell function in vitro. (A) Proliferation of KYSE590 and KYSE70 cells in which SPOCD1 expression was inhibited using an SPOCD1-specific siRNA. (B) Migration wound healing assay in which SPOCD1 expression was inhibited in KYSE590 cells treated with an SPOCD1-specific siRNA. The left panel shows representative images of invasion, and the right panel shows the mean length of migration. (C) Cell invasion assay of KYSE70 cells in which SPOCD1 expression was inhibited by an SPOCD-siRNA. The left panels show representative images of stained invading KYSE70 cells (×200 magnification). The right graph shows the mean numbers of invaded cells in eight randomly selected fields. (D) Efficiency of enforced expression of SPOCD1 in KYSE1440 compared with that in cells transduced with the control vector. (E) Analysis of cell proliferation in KYSE1440 cells transduced with a control vector and si-SPOCD1. Error bars indicate the standard deviation. *p<0.05, **p<0.01, ***p<0.001. Asterisk refers to the comparison of si-SPOCD1 group to both untransfected and si-control groups in plots A and B.

Article Snippet: Subsequent to transfection, cells for conducting functional assays were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), but without antibiotics, for 48 h. To achieve enforced expression of SPOCD1 , the SPOCD1 Human-Tagged Open Reading Frame Clone sequences were inserted into pLenti-C-Myc-DDK-P2A-Puro (product ID, RC218945L3V; OriGene, Rockville, MD, USA), and transduction was performed according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Function Assay, In Vitro, Expressing, Migration, Wound Healing Assay, Invasion Assay, Staining, Transduction, Control, Plasmid Preparation, Standard Deviation, Comparison

Journal: Cancer Genomics & Proteomics

Article Title: SPOCD1 Enhances Cancer Cell Activities and Serves as a Prognosticator in Esophageal Squamous Cell Carcinoma

doi: 10.21873/cgp.20503

Figure Lengend Snippet: Effect of SPOCD1 knockdown on subcutaneous tumor formation in mouse xenograft models of KYSE70 cells. (A) Estimated sizes (mm) of tumors among untransfected, si-Control, and si-SPOCD1 groups (B). Images of all tumor nodules collected from the mice. (C) Average tumor weight comparison among groups. Error bars indicate the standard deviation. *p<0.05, **p<0.01, ***p<0.001. Asterisk refers to the comparison of si-SPOCD1 group to both untransfected and si-control groups in plots A.

Article Snippet: Subsequent to transfection, cells for conducting functional assays were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), but without antibiotics, for 48 h. To achieve enforced expression of SPOCD1 , the SPOCD1 Human-Tagged Open Reading Frame Clone sequences were inserted into pLenti-C-Myc-DDK-P2A-Puro (product ID, RC218945L3V; OriGene, Rockville, MD, USA), and transduction was performed according to the manufacturer’s protocol.

Techniques: Knockdown, Control, Comparison, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SPOCD1 Enhances Cancer Cell Activities and Serves as a Prognosticator in Esophageal Squamous Cell Carcinoma

doi: 10.21873/cgp.20503

Figure Lengend Snippet: Expression of SPOCD1 mRNA in ESCC tissue and its prognostic value. (A) qRT-PCR analysis of SPOCD1 mRNA expression in ESCC tissues and paired normal esophageal mucosa tissues from 164 patients. (B) Kaplan–Meyer analysis of disease-specific survival of 164 patients who underwent radical resection for ESCC. (C) External validation of overall survival data from TGCA. (D) Kaplan–Meyer analysis of disease-specific survival of 164 patients who underwent radical resection for ESCC. (E) Frequencies of the sites of initial recurrence after radical esophagectomy. Error bars indicate standard deviation. *p<0.05, **p<0.01, ***p<0.001, ns: Not significant.

Article Snippet: Subsequent to transfection, cells for conducting functional assays were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), but without antibiotics, for 48 h. To achieve enforced expression of SPOCD1 , the SPOCD1 Human-Tagged Open Reading Frame Clone sequences were inserted into pLenti-C-Myc-DDK-P2A-Puro (product ID, RC218945L3V; OriGene, Rockville, MD, USA), and transduction was performed according to the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Standard Deviation

Journal: Cancer Genomics & Proteomics

Article Title: SPOCD1 Enhances Cancer Cell Activities and Serves as a Prognosticator in Esophageal Squamous Cell Carcinoma

doi: 10.21873/cgp.20503

Figure Lengend Snippet: Analysis of SPOCD1 expression in ESCC tissues and its prognostic value. (A) Representative images of immunohistochemical detection in a TMA. Examples of negative (0) and positive (1+, 2+, and 3+) expression levels are shown (200× and 1,000× magnification). (B) Kaplan–Meyer analysis of disease-specific and disease-free survival of 177 patients stages I-III ESCC who underwent radical resection of the esophagus.

Article Snippet: Subsequent to transfection, cells for conducting functional assays were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), but without antibiotics, for 48 h. To achieve enforced expression of SPOCD1 , the SPOCD1 Human-Tagged Open Reading Frame Clone sequences were inserted into pLenti-C-Myc-DDK-P2A-Puro (product ID, RC218945L3V; OriGene, Rockville, MD, USA), and transduction was performed according to the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemical staining

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: Schema illustrating the role of cellular plasticity in NB immune evasion. This schematic overview highlights how RD3‐loss orchestrates plasticity‐driven tumor immune editing. Therapy‐pressure acquired RD3‐loss prompts EMT induction in the primary tumor, which facilitates migratory and invasive behavior. Concurrently, T u acquires stemness traits and maintains pluripotency, enabling lineage reprogramming and clonal expansion. These phenotypic transitions promote immune escape by downregulating antigen presentation molecules MHC I, MHC II, B2M, and upregulating immune checkpoint molecules PDL1, CD276, resulting in immune inactivation. Enhancing CD24 and GD2 promotes immune suppression by camouflage. The evolving tumors are characterized by reduced infiltration of effector T c CD4, CD8; expansion of immune suppressive subsets Tregs, TAMs, and activation of adenosinergic pathway via CD39, A2AR, CD73. Imposingly, these changes transform the immune landscape from ‘hot’ state to an immune‐silent ‘cold’ state. ↑= upregulated; ↓= downregulated.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: Immunopeptidomics, Activation Assay

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: Clinical therapy pressure acquired RD3 loss associates with stemness acquisition and CSC enrichment in NB (A) ELISA showing reduced RD3 expression in a panel of patient derived post IMCT ex vivo systems when compared with therapy‐naïve NB. Data represent mean ± SD; statistical analysis was performed using two‐way ANOVA with Dunnett's correction (p<0.0001) (B) immunoblots showing RD3‐loss associated rearrangements of key determinants of EMT (E‐cadherin, N‐cadherin, Vimentin, Slug), pluripotency and stemness maintenance (SOX2, OCT3/4, NANOG), dedifferentiation (GFAP, GAP43, NF‐L, B3Tubulin), and metastasis (RONB, MMP9, MMP2, CD54, NOTCH1). GAPDH and/or α‐Tubulin served as loading controls. (C) FACS sorting with appropriate inclusion exclusion gating (total, singlets, live, and ALDH positive side population) strategy to classify CSC subsets based on single, double, and triple positive expression of CD133, CD114, and CD117. A total of 10x10 6 total cells were included across all samples investigated. (D–F) Distribution cris‐cross analysis computing combinations of double positive CSC phenotypes reveals a RD3 −/− dependent dynamic shift in stemness composition of each clone investigated and their CSC distribution (vs. RD3 +/− ). All three combinations of double positive comparison unequivocally identified low CSC enrichment in treatment‐naïve (CHLA‐15 and CHLA‐42) cells, while therapy‐defying PD RD3 −/− cells (e.g., CHLA‐61, SK‐N‐FI) exhibited unparalleled CSC enrichment. (G) Histogram showing RD3‐loss associated enrichment of general CSC‐like ALDH + side population in NB. (H) Compared to Dx RD3 +/+ , in‐depth sorting showing RD3 expression associated frequency distribution of single (CD133 + , CD114 + , CD117 + ); double (CD133 + CD114 + , CD114 + CD117 + , CD133 + CD117 + ) and triple (CD133 + CD114 + CD117 + ) population CSC enrichment in PD‐RD3 −/− . (I) All PD‐ RD3 −/− NB investigated consistently exhibited heightened levels of deadly triple‐positive CSC population compared to the disease at Dx.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Ex Vivo, Western Blot, Comparison

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: Functional characterization of RD3‐dependent clonal expansion, migration, and invasion in NB (A) Representative images from BrdU incorporation assay performed in Dx‐RD3 +/+ (CHLA‐15, CHLA‐42) and PD‐RD3 −/− (CHLA‐20, CHLA‐90, CHLA‐140) depicting RD3‐loss associated clonal enrichment. (B) Histograms of trans well migration assay performed to evaluate RD3 status‐associated motility in a panel of patient‐derived NB clones, including Dx‐RD3 +/+ (n = 2) and PD RD3 −/− (n = 8) NB ex‐ vivo clones. PD‐RD3 −/− clones exhibited enhanced migratory potential compared to RD3 +/+ clones. (C) Quantitative analysis of migratory distance travelled by individual cells measured via live cell time‐lapse imaging and cell tracking algorithm. Consistently, RD3 −/− status at PD corresponded to increased migratory distance travelled compared to RD3 +/+ CHLA‐42 at Dx. (D) Matrigel‐based invasion assay was performed to elucidate RD3‐mediated regulation of invasive potential in a panel of 10 NB clones. Analyzing and quantifying the invaded cells revealed therapy pressure‐driven RD3‐loss enhanced pro‐invasive phenotype in NB. (E) LDTA performed under non‐adherent conditions to elucidate anchorage‐independent growth and stem‐like behavior demonstrated increased sphere‐forming efficiency in RD3 −/− clones compared to Dx‐RD3 +/+ clones. Data are presented as mean ± SEM across biological replicates.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: Functional Assay, Migration, BrdU Incorporation Assay, Derivative Assay, Clone Assay, Ex Vivo, Imaging, Cell Tracking Assay, Invasion Assay

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: RD3 loss associated with activated pluripotency and plasticity in therapy‐defying NB in‐vivo. (A) qPCR analysis of pluripotency and lineage‐defining markers was profiled to assess RD3‐dependent transcriptional modulation in primary (PT, RD3 expressing) and metastatic (MT, RD3‐deficient) tumors developed in mice utilizing SH‐SY5Y NB cells. Top panel shows RD3‐loss‐associated increase in the transactivation of the crucial plasticity determinants KLF4, ZIC3, LIN28, LIF, SMAD4, PI3KR1, NKX3.2, CALCB, and PTPN3 in RD3‐deficient metastatic tumors (vs. PT). Bottom panel shows transcriptional activation of pluripotency core circuitry TFs SOX2, NANOG, and OCT3/4 in RD3‐deficient MT when compared with PT with inherent RD3 presence. (B) Representative microphotographs of SOX2, NANOG, and OCT3/4 IHC affirming the RD3‐associated regulation of the pluripotency triad at the translational level. Tumors derived from PT (RD3 +/+ ) and MT (RD3 −/− ) were assessed for expression of SOX2, NANOG and OCT3/4 proteins. Positivity and cellular localization were validated in normal adrenal and brain tissues. RD3‐deficient MT tumors exhibited elevated levels of all three markers, confirming that RD3‐loss modulates pluripotency translational level. (C) Aperio quantification of IHC for pluripotency‐associated pillars in PT‐RD3 +/+ and MT‐RD3 −/− highlights RD3‐dependent shift in protein abundance and spatial distribution of SOX2, NANOG and OCT3/4. (D) Independent functional ex vivo validation of RD3's role in sustaining pluripotency circuitry. Using metastatic site tumor‐derived MSDAC clones that lack RD3 −/− , a targeted silencing of SOX2 and NANOG was performed to elucidate the definitive interdependence of the core transcription triad. All three TFs showed high constitutive levels of expression RD3 −/− MSDAC. Silencing SOX2 and NANOG in the RD3 −/− MSDAC resulted in downregulation of NANOG and SOX2, respectively. In addition, muting either SOX2 or NANOG exerts partial regulation of OCT3/4. This alternate muting experiment in RD3 −/− MSDAC validated the existence of a self‐reinforcing autoregulatory loop that maintains pluripotency under RD3‐deficient conditions. PT, primary tumor; MT1‐MT‐5 denote MT tumors from five different animals; multiple tumors per animal are designated as MT1‐1, MT‐7 etc.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: In Vivo, Expressing, Activation Assay, Derivative Assay, Quantitative Proteomics, Functional Assay, Ex Vivo, Biomarker Discovery, Clone Assay

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: RD3 engineers NB cell identity and CSC composition. (A) Stable silencing of RD3 in RD3 expressing CHLA‐15, CHLA‐42, SH‐SY5Y (designated as CHLA‐15 RD3 −/− , CHLA‐42 RD3 −/− , SH‐SY5Y RD3 −/− ) and stable restoration of RD3 in RD3‐deficient CHLA‐20, CHLA‐90, CHLA‐140 (designated as CHLA‐20 RD3 +/+ , CHLA‐90 RD3 +/+ , CHLA‐140 RD3 +/+ ) ex‐vivo systems were validated with IF and WB. Representative IF images (top panel) shows RD3 localization and intensity, and immunoblot (bottom panel) confirms RD3 expression status across conditions following stable KO and re‐expression in NB patient derived cells, validating the engineered models generated (B) Ultrastructural and morphological alterations (plasma membrane integrity, nuclear morphology, chromatin organization, organelle distribution, cytoplasm: nuclear ratio) following RD3 KO in RD3 +/+ CHLA‐15 visualized via TEM. RD3 −/− clones displayed smooth intact membranes, homogenous cytoplasm, euchromatin‐rich nuclei, and abundant mitochondria associated with lipids, consistent with lipid‐fueled metabolic phenotype. RD3 +/+ clearly depicted features indicative of relative cellular stress, including irregular membrane, condensed chromatin, granular uneven cytoplasm, suggesting compromised homoeostasis and integrity. Colored arrows denote key features including mitochondrial morphology (dark green), vesicular trafficking (yellow), lipid moieties (brown), lipophagy (light blue), lysosomal contents (purple) and autophagosome (red). Magnification 2000x. (C) FACS sorting with appropriate inclusion exclusion gating (total, singlets, live, and ALDH + side population) strategy to classify CSC subsets based on single, double and triple positive expression of CD133, CD114, and CD117. A total of 1x10 6 total cells were included across all samples investigated. (D) ALDH + cells were computed to identify stem‐like side populations across Dx‐RD3 +/+ , PD‐RD3 −/− , RD3 −/−, and RD3 +/+ ex‐vivo systems. DEAB treated controls were used to define baseline fluorescence. RD3 +/+ cells exhibited a significant decrease of ALDH + , indicative of RD3‐regulated stemness traits in NB, while RD3‐loss promotes enrichment of functionally defined CSC compartments. (E–G) An abundance of single (CD133, CD114, CD117) positive CSC populations across RD3 reverse‐engineered systems. Appropriately matched parental strains were used as controls. Compared to the RD3 −/− CHLA‐20, its RD3 +/+ engineered clone showed a remarkable reduction in all three single CD133 + , CD114 + , CD117 + populations, indicating constrained stemness and lineage fidelity. Conversely, forced silencing of RD3 in CHLA15 showed defined enrichment of these single positive CSC subsets, corroborating the role RD3 as a suppressor of CSC expansion and phenotypic plasticity. (H–J) FACS performed to assess the RD3‐modualted expansion of dual positive CD133 + CD114 + , CD133 + CD117 + , CD114 + CD117 + CSC populations. Forced silencing RD3 −/− cells promoted the expansion and accumulation of dual positive CSC subsets in NB (vs. RD3 +/+ ), mirroring the trends observed in single positive subsets. (K) Triple marker analysis revealed a distinct expansion of CD133 + CD114 + CD117 + aggressive CSC subset in RD3 −/− cells, compared to RD3 +/+ counterpart; and on the flipside forced re‐expression of RD3 in PD‐RD3 −/− resulted in complete negation of this deadly subset expansion. The marked increase in triple‐positive subsets underscores RD3's critical role as a suppressor of CSC expansion and stemness maintenance.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: Expressing, Ex Vivo, Western Blot, Derivative Assay, Generated, Clinical Proteomics, Membrane, Clone Assay, Fluorescence, Marker

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: RD3 impedes cellular plasticity and governs immune editing in NB (A) Representative images from multi‐IF analysis of plasticity‐associated core pluripotency components (SOX2, NANOG, OCT3/4), stemness effectors (ALDH2, CD133), EMT drivers (E‐CAD, N‐CAD, Vimentin), NB maturation determinants (GFAP, GAP43, β‐3‐Tubulin, NF‐H). and metastasis facilitators (MMP9, RON‐β, ICAM1) across a panel of RD3 reverse‐engineered clones (n = 6) and compared to their parental counterparts (n = 6). IF reveals RD3 −/− directed multi‐layered reprogramming towards a stem‐like, mesenchymal phenotype with lineage plasticity and heightened pluripotency in NB. Comparisons of the relative expression for each of these fifteen crucial determinants in all twelve systems were pictured and compared in Figure . (B) BrdU incorporation assay was performed across Dx‐RD3 +/+ CHLA‐15, CHLA‐42; PD‐RD3 −/− CHLA‐20, CHLA‐90, CHLA‐140; RD3‐muted CHLA‐15 RD3 −/− , CHLA‐42 RD3 −/− and RD3‐reinstated CHLA‐20 RD3 +/+ , CHLA‐90 RD3 +/+ , CHLA‐140 RD3 +/+ to elucidate RD3 regulated clonal expansion. RD3 −/− clones showed nuclear BrdU incorporations in higher number of cells compared to Dx‐RD3 +/+ and RD3‐reinstated clones, confirming RD3's potential in restraining hyperplastic state and clonal propagation. (C) RD3‐loss driven plasticity‐regulated immune response in NB ‐immunophenotypic profiling of immune visibility (HLA A/B, HLA DPB1, β2M), checkpoint (PDL1, CD276), surveillance (CD171, GD2) and immune evasive (CD24) determinants profiled in RD3‐engineered clones and its parental counterparts. Representative immunoblots and (D) histograms constructed from densitometric quantification of immunoblots showing expression profiles in PD‐RD3 −/− and RD3‐muted clones depicting downregulation of antigen presentation machinery, upregulation of immune checkpoint and promoted tumor disguise via CD24 compared to RD3‐expressing counterparts, highlighting RD3‐loss driven modulation of tumor‐immune interactions through plasticity‐driven remodeling (E) Representative FACS gating strategy utilized for functional immune assays, cytokine secretion (TNFα and IFNγ; top panel), tumor reactivity assay (CD107a and CD137; middle) and tumor killing assay (Caspase‐3 activity; bottom panel) (F) TNFα and IFNγ secretion in T C CoC with RD3‐expressing and reinstated tumors induced robust cytokine secretion; T C CoC with RD3‐deficient and kncokout clones elicited minimal response (G) Degranulation (CD107a) and sustained activation (CD137) of T C CoC with RD3‐proficient tumors whereas RD3‐silencing reduced T C effector function. (H) Representative images of T C ‐mediated apoptosis (left panel). All CoC systems displayed increased caspase activity, confirming T C ‐mediated cytotoxicity. RD3‐expressing and reinstated tumors showed greater apoptosis of T u compared to RD3‐null clones, indicating an immune evasive phenotype (right panel).

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: Clone Assay, Expressing, BrdU Incorporation Assay, Western Blot, Construct, Immunopeptidomics, Functional Assay, Activity Assay, Activation Assay

Journal: Advanced Science

Article Title: Cellular Identity Crisis: RD3 Loss Fuels Plasticity and Immune Silence in Progressive Neuroblastoma

doi: 10.1002/advs.202519586

Figure Lengend Snippet: RD3‐loss dictates immune defense barrier (A) Multi‐IHF analysis of tumor sections across a panel of five distinct NB models in vivo derived from RD3‐expressing CHLA‐42, RD3‐deficient CHLA‐20, CHLA‐90, and RD3‐reexpressed CHLA‐20 RD3 +/+ and CHLA‐90 RD3 +/+ systems. Tumor sections were stained for curated panel of immune modulatory markers of (i) effector subsets CD4, CD8 (ii) activation CD44, GITR (iii) purinergic immunosuppression axis CD39, CD73, A2AR (iv) immune sensing and antigen presentation CD86, STING, CD206 (v) immune exhaustion CD244.2, and (vi) apoptosis CD95. Representative sections are presented at 20X magnification, and scale bars represent 20 µM. (B) Quantitative analysis of immune marker expression across all experimental conditions comparing RD3‐reinstated, RD3‐expressing and RD3‐null derived tumors. Data is organized into three functional panels reflecting immune activation, suppression and surveillance dynamics. Top panel focusses on functional polarization and activation. RD3 +/+ tumors exhibited increased infiltration of CD4 + and CD8 + effector T c compared to RD3‐null counterparts, indicating improved immune engagement. Frequency of co‐expression of CD44 and GITR on effector subsets (CD8 + GITR + , CD4 + GITR + , CD8 + CD44 + , CD4 + CD44 + , CD8 + CD44 + GITR + , CD4 + CD44 + GITR + ) increased in RD3 +/+ systems reflecting improved tumor immunogenicity and surveillance. Middle panel represents adenopurinergic axis mediated immune suppression and apoptotic regulation. RD3‐null tumors showed elevated co‐expression of CD39, CD73, and A2AR consistent with activation and engagement of immune suppressive and tolerogenic mechanisms promoting immune escape in NB. In contrast, RD3‐reinstated systems promoted CD95 expression on tumors, implicating RD3s’ role in immune‐mediated apoptosis and tumor clearance. The bottom panel shows immune surveillance (CD206, CD86, STING) and exhaustive landscape (CD244.2) in NB tumors. RD3‐reinstated tumors demonstrated reduced expression of immune exhaustive phenotype alongside elevated levels of STING, CD86, and CD206 in immune cells compared to RD3‐null tumors. This reinforces RD3's role in restoring immune visibility and surveillance.

Article Snippet: Re‐expression of RD3 was achieved using RD3 ( NM_183059 ) human tagged ORF Clone in pCMV6‐AC‐GFP (tGFP‐tagged, RD3 transcript variant 1, Origene, MD, USA).

Techniques: In Vivo, Derivative Assay, Expressing, Staining, Activation Assay, Immunopeptidomics, Marker, Functional Assay

Journal: Molecular oncology

Article Title: PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

doi: 10.1016/j.molonc.2014.09.009

Figure Lengend Snippet: Figure 1 e Experimental assays for characterization of PHF21B. (A) Scheme of the PHF21B gene and its promoter-associated CpG islands organized according to the physical location on 22q13.31. This gene contains 13 exons spanning approximately 128 Kb of DNA (UCSC Genome Browser on Human, assembly GRCh37/hg19, http://genome.ucsc.edu) and a highly dense promoter-associated CpG island. This island spans 3336 bp, including the 50 region, with the first two exons of this gene overlapping three alternative transcription start sites: (*) transcript variant 1, NM_138415.4, (**) transcript variant 2, NM_001135862.2, and (***) transcript variant 3, NM_001242450.1. The comprehensive analysis of the PHF21B gene in HNSCC patients included several target regions for PCR-based assays for detection of the DNA copy number (30UTR), gene expression (exons 5e7), and mutations (exons 3, 6, 7, 8, 9, and 11). Changes in DNA methylation of CpG Island 273 were screened for three regions in HNSCC-derived cell lines. (B) DNA copy number changes in the PHF21B gene in matched normal (peripheral blood) and tumoral samples obtained from HNSCC patients. Constitutive losses of PHF21B were detected in cases showing a positive family cancer history (case 04), in case 50 (negative for family cancer history), and in cases 35, 49, and 71 (unknown information). Tumor-specific acquired losses were detected in

Article Snippet: PHF21B overexpression The plasmid containing the human ORF for PHF21B (NM_138415) GFP-tagged was purchased from Origene.

Techniques: Variant Assay, Gene Expression, DNA Methylation Assay, Derivative Assay

Journal: Molecular oncology

Article Title: PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

doi: 10.1016/j.molonc.2014.09.009

Figure Lengend Snippet: Figure 2 e Clonogenesis and migration assays performed for PHF21B. (A) Amino acid sequence alignment between PHF21A and PHF21B reveals conservation of domains important for transcriptional repression. The sequences from the two proteins were aligned using BLASTP. Important functional domains from PHF21A were visualized by UNIPROT: a GLN-rich region is found between residues 4-108; a DNA binding domain A.T Hook is found between 425 and 437; a PHD-type zinc finger domain is found between 488 and 535; a region required for transcriptional repression is found between 486 and 680. (BeH) Subcellular distribution of endogenous and transfected PHF21B. FaDU (B and C) and SCC4 (D)

Article Snippet: PHF21B overexpression The plasmid containing the human ORF for PHF21B (NM_138415) GFP-tagged was purchased from Origene.

Techniques: Migration, Sequencing, Functional Assay, Binding Assay, Transfection

Journal: European Journal of Human Genetics

Article Title: Two novel BMP-2 variants identified in patients with thoracic ossification of the ligamentum flavum

doi: 10.1038/ejhg.2017.2

Figure Lengend Snippet: Analysis of BMP-2 variants in patients with TOLF. (a) Sequences showing two novel variants in BMP-2 (c.C460G:p.R154G, c.G584T:p.R195M), as compared with wild-type sequences. (b and c) Clustal W alignments showing conservation of mutated nucleotide (b) and amino acid (c) sequences.

Article Snippet: The pCMV6-Entry vector carrying the human BMP-2 cDNA ORF and the C-terminal myc-DDK-tag (referred to as WT) was purchased from OriGene Technologies (Beijing, China).

Techniques:

Journal: European Journal of Human Genetics

Article Title: Two novel BMP-2 variants identified in patients with thoracic ossification of the ligamentum flavum

doi: 10.1038/ejhg.2017.2

Figure Lengend Snippet: Functional assays of wide-type and mutant BMP-2 transfected into MC3T3-E1 cells. (a) Exogenous BMP-2 expression as detected using the DDK-tag in different transfected cell lines. (b and c) Expression of osteogenic markers in different transfected cell lines at defined time points: ALP at day 7 (b); OCN and OPN at day 14 (c). (d) Staining for ALP activity at day 7 (upper) and alizarin red staining at day 14 (lower) in different transfected cell lines (scale bars represent 200 μm).

Article Snippet: The pCMV6-Entry vector carrying the human BMP-2 cDNA ORF and the C-terminal myc-DDK-tag (referred to as WT) was purchased from OriGene Technologies (Beijing, China).

Techniques: Functional Assay, Mutagenesis, Transfection, Expressing, Staining, Activity Assay

Journal: bioRxiv

Article Title: TRIB2 couples UCP1 degradation to thermogenic adaptation and metabolic health

doi: 10.1101/2025.10.26.684708

Figure Lengend Snippet: (A) The UCP1 expression increases in the Trib2 KO mice BAT. (B) The differentiation schematic diagram of the WT1 brown preadipocyte. (C) A decreasing level of TRIB2 expression accompanied by an increasing level of UCP1 expression. The negative correlation (Pearson r : –0.8489) between TRIB2 and UCP1 expression during the differentiation of WT1 brown adipocytes. (D) Knockdown of TRIB2 in WT1 cells increases UCP1 expression. (E) TMRM functional test suggests that knockdown of TRIB2 in WT1 cells increases mitochondrial function. Data A, B, and E were analyzed by student unpaired t-tests. Data C correlation analysis was analyzed by Pearson’s r correlation. Data D was analyzed by two-way ANOVA, followed by Tukey’s post hoc test for multiple comparisons. All data represent the mean ± SD. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: UCP1 Human tagged ORF clone (#RC218901) was purchased from OriGene Technologies and subcloned into EcoRI (#R3101S) and XmaI (#R0180S) sites of pLV-C-GFPSpark Lentivirus vector (#LVCV-35). pCMV-Pam-FL was a gift from Vijaya Ramesh 89 (Addgene plasmid # 42570; http://n2t.net/addgene:42570 ; RRID: Addgene_42570).

Techniques: Expressing, Knockdown, Functional Assay

Journal: bioRxiv

Article Title: TRIB2 couples UCP1 degradation to thermogenic adaptation and metabolic health

doi: 10.1101/2025.10.26.684708

Figure Lengend Snippet: (A) Immunoprecipitated UCP1-GFP successfully co-immunoprecipitated TRIB2-His in 293T cells. (B) TRIB2-GST protein pull-down endogenous UCP1 in BAT. (C) Immunoprecipitated UCP1 with UCP1 antibody co-precipitated TRIB2 in BAT of WT mice. (D) Immunoprecipitated UCP1 with UCP1 antibody co-precipitated TRIB2 in BAT of WT mice, but did not co-precipitate TRIB2 in KO mice. (E) In situ PLA signal indicated a direct interaction between endogenous TRIB2 and UCP1 on the BAT of WT mice. (F) TRIB2-His pseudokinase truncated forms co-precipitate UCP1-GFP, suggesting pseudokinase domains can directly bind to UCP1. FL: Full length; NP: N-terminal + Pseudokinase; P: Pseudokinase only; PC: Pseudokinase + C-terminal.

Article Snippet: UCP1 Human tagged ORF clone (#RC218901) was purchased from OriGene Technologies and subcloned into EcoRI (#R3101S) and XmaI (#R0180S) sites of pLV-C-GFPSpark Lentivirus vector (#LVCV-35). pCMV-Pam-FL was a gift from Vijaya Ramesh 89 (Addgene plasmid # 42570; http://n2t.net/addgene:42570 ; RRID: Addgene_42570).

Techniques: Immunoprecipitation, In Situ

Journal: bioRxiv

Article Title: TRIB2 couples UCP1 degradation to thermogenic adaptation and metabolic health

doi: 10.1101/2025.10.26.684708

Figure Lengend Snippet: (A) A dose-dependent increase in the ubiquitination of UCP1. (B) Co-expressing TRIB2-His and Ubiquitin-HA in 293T cells decreases UCP1 expression. (C) TRIB2 enhances ubiquitination of UCP1. (D) Knockdown of MYCBP2 decreases ubiquitination of UCP1 in WT1 cells. Data C was analyzed by student unpaired t-tests. All data represent the mean ± SD. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: UCP1 Human tagged ORF clone (#RC218901) was purchased from OriGene Technologies and subcloned into EcoRI (#R3101S) and XmaI (#R0180S) sites of pLV-C-GFPSpark Lentivirus vector (#LVCV-35). pCMV-Pam-FL was a gift from Vijaya Ramesh 89 (Addgene plasmid # 42570; http://n2t.net/addgene:42570 ; RRID: Addgene_42570).

Techniques: Ubiquitin Proteomics, Expressing, Knockdown

Journal: bioRxiv

Article Title: TRIB2 couples UCP1 degradation to thermogenic adaptation and metabolic health

doi: 10.1101/2025.10.26.684708

Figure Lengend Snippet: (A)Immunoprecipitated UCP1 with UCP1 antibody did not co-precipitate the TRIB2 reported interacting E3 ligase. (B) The scheme for identifying UCP1-interacting proteins by LC-MS/MS. (C) Immunoprecipitated UCP1 with UCP1 antibody co-precipitates MYCBP2 in BAT of WT mice. (D) Immunoprecipitated UCP1-GFP co-precipitates MYCBP2 in 293T cells. (E&F) In situ PLA signal indicated a direct interaction between endogenous UCP1 and MYCBP2 on the BAT of WT mice, but not on the BAT of KO mice. (G) Overexpressing MYCBP2 in 293T decreases UCP1 expression. (H) Knockdown of MYCBP2 increases UCP1 expression in WT1 cells. The negative correlation (Pearson r : –0.9981) between MYCBP2 and UCP1. (I) TMRM functional test suggests that knockdown of MYCBP2 in WT1 cells increases mitochondrial function. (J) Expressing TRIB2 enhances MYCBP2 to decrease UCP1 expression in 293T cells. Data H was analyzed by two-way ANOVA, followed by Tukey’s post hoc for multiple comparisons and Pearson’s r correlation. Data I was analyzed by student unpaired t-tests. Data J was analyzed by one-way ANOVA, followed by Tukey’s post hoc for multiple comparisons. All data represent the mean ± SD. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: UCP1 Human tagged ORF clone (#RC218901) was purchased from OriGene Technologies and subcloned into EcoRI (#R3101S) and XmaI (#R0180S) sites of pLV-C-GFPSpark Lentivirus vector (#LVCV-35). pCMV-Pam-FL was a gift from Vijaya Ramesh 89 (Addgene plasmid # 42570; http://n2t.net/addgene:42570 ; RRID: Addgene_42570).

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, In Situ, Expressing, Knockdown, Functional Assay

Journal: bioRxiv

Article Title: TRIB2 couples UCP1 degradation to thermogenic adaptation and metabolic health

doi: 10.1101/2025.10.26.684708

Figure Lengend Snippet: Trib2 knockout mice displayed elevated UCP1 expression in brown adipose tissue, leading to enhanced thermogenesis and increased energy expenditure. Mechanistically, TRIB2 binds directly to UCP1 via its pseudokinase domain and promotes its ubiquitination by recruiting the E3 ligase MYCBP2.

Article Snippet: UCP1 Human tagged ORF clone (#RC218901) was purchased from OriGene Technologies and subcloned into EcoRI (#R3101S) and XmaI (#R0180S) sites of pLV-C-GFPSpark Lentivirus vector (#LVCV-35). pCMV-Pam-FL was a gift from Vijaya Ramesh 89 (Addgene plasmid # 42570; http://n2t.net/addgene:42570 ; RRID: Addgene_42570).

Techniques: Knock-Out, Expressing, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Representative confocal imaging of HeLa cells showing localization of H3K9me3-marked heterochromatin (cyan), GFP-tagged PRR14 and mCherry-Lamin A/C (red). (B) A schematic model of H3K9me3/HP1 heterochromatin tethering to the nuclear lamina via PRR14. (C) A schematic representation of the PRR14 modular domain organization. (D) Representative confocal images of live HeLa cells transfected with GFP-tagged PRR14 fragments, as indicated. The N-terminal fragment (1-135) shows localization to heterochromatin, C-terminal fragment (366-585) localizes in nucleoplasm, centrally-located (231-282) fragment identified as a minimal nuclear lamina binding domain (LBD). Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Imaging, Transfection, Binding Assay

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: A series of PRR14 C-terminal truncations were created by introducing stop codons into the N-terminal GFP-tagged PRR14 reading frame. The end points of the C-terminal truncations are indicated. Representative confocal images of live HeLa cells stably expressing wild type mRFP-PRR14 transfected with (A) GFP-tagged truncation mutants. (B) composite mutants with substitutions in the LAVVL sequence required for heterochromatin binding (LAVVLmut). Counterstained with Hoechst.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Stable Transfection, Expressing, Transfection, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: Representative confocal images of HeLa cells transfected with indicated GFP-PRR14 constructs and differentially permeabilized with Triton X-100 (plasma and nuclear membrane) or digitonin (plasma membrane only) to distinguish localization at the inner and outer nuclear periphery. Stained for GFP (red), Lamin A/C (cyan) and DAPI (blue). Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Transfection, Construct, Staining

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) PRR14 map depicting the minimal 231-282 LBD. (B) Protein sequence alignment of the human 231-282 PRR14 LBD with mouse, xenopus, and gecko identified conserved residues: charged (gray) and hydrophobic (orange). Amino acid sequence identity (*) and similarity (.) are indicted. (C) Representative confocal images of HeLa cells transfected with GFP-tagged PRR14 231-282 LBD containing the indicated amino acid substitutions. (D) Box plot demonstrating the proportion of indicated PRR14-GFP proteins at the nuclear lamina, calculated using Lamin A/C signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. Statistical analyses performed using one-way ANOVA test with Dunn’s multiple comparison; **** p<0.0001, ns: not significant. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Sequencing, Transfection

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) GFP-tagged, full length human and mouse PRR14 proteins localize to the nuclear lamina in mouse cells. Human PRR14 localizes to the nuclear lamina in Xenopus cells. (B) GFP-tagged human PRR14 231-282 LBD localizes to the nuclear lamina in xenopus cells. GFP-tagged putative Xenopus PRR14 (see ) 152-203 LBD localizes to the nuclear lamina in HeLa cells. (C) Manual alignment with no gaps of human and mouse, and putative xenopus and gecko LBDs. Amino acid identity (*) and similarity (.) are indicated. Conserved blocks chosen for analyses are indicated by shading. End points of relevant C-terminal deletions analyzed in are indicated. Green arrow indicates retention of nuclear lamina association and red arrows indicate loss of nuclear lamina localization. Deletion into the most conserved region of the human 231-282 LBD resulted in loss of nuclear lamina localization .

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques:

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Map of human PRR14 depicting the 231-282 LBD, del231-282, and IFL to AAA substitution (x) in the conserved core sequence. (B) Representative confocal images of HeLa cells transfected with GFP-tagged constructs (green) depicted in Panel A, Lamin A/C (red), and counterstained with DAPI. (C) Box plot demonstrating the proportion of indicated PRR14-GFP proteins at the nuclear lamina, calculated using Lamin A/C signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. (D) Line signal intensity profiles of corresponding images in panel B indicated by dashed lines. Statistical analyses performed using one-way ANOVA test with Dunn’s multiple comparison; **** p<0.0001, ns: not significant. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Sequencing, Transfection, Construct

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Representative confocal images of murine C2C12 cells transfected with indicated PRR14-GFP constructs similar to experiment shown on . The IFL-AAA and del231-282 constructs colocalization with chromocenters is observed. However, composite mutants incapable of HP1/heterochromatin binding (LAVVLmut) lack chromocenter localization and spread through nucleoplasm. Though, the composite mutants showed some localization to the nuclear periphery, suggesting residual nuclear lamina binding. (B) Representative confocal images of HeLa cells transfected with indicated IFL-AAA and composite IFL-AAA LAVVL-mutant, stained for H3K9me3. Colocalization of the IFL-AAA, but not the composite mutant protein with H3K9me3 heterochromatin is observed. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Transfection, Construct, Binding Assay, Mutagenesis, Staining

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Map, and sequence conservation of region downstream of 231-282 LBD hypothesized to contribute to nuclear lamina binding. Sequence alignment of human and gecko PRR14 identified candidate functional motifs. Amino acid sequence identity (*) and similarity (•) are indicted. Conserved motifs are designated as A through D. (B) Representative confocal images of HeLa cells transfected with indicated N-GFP tagged PRR14 fragments. Independent from the 231-282 LBD, the 283-351 fragment was found to localize to the nuclear lamina as a second independent modular LBD domain. (C) Box plot demonstrating the proportion of indicated PRR14-GFP proteins from panel B at the nuclear lamina, calculated using Lamin A/C signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. (D) Representative confocal images of HeLa cells transfected with indicated N-GFP tagged PRR14 constructs: WT, 231-351 deletion and composite mutant with 231-351 deletion and substitutions in the LAVVL sequence (LAVVLmut) required for heterochromatin binding. Statistical analyses performed using one-way ANOVA test with Dunn’s multiple comparison; *** p<0.001, ns: not significant. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Sequencing, Binding Assay, Functional Assay, Transfection, Construct, Mutagenesis

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: Representative confocal images of HeLa cells transfected with GFP-tagged gecko PRR14 320-371, 320-410, and 320-444 fragments (green), stained for Lamin A/C (red) and DAPI (blue). Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Transfection, Staining

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Map and sequence of the human PRR14 231-282 LBD highlighting in vivo serine/threonine CDK SP/TP phosphorylation sites at positions 242, 266, 270 and 277. Phosphomimetic (PM) and phosphoablation (PA) substitutions are shown. For phosphomimetic substitutions, the four S/T phosphorylation sites were collectively changed to glutamic acid. For phosphoablation, the SP/TP context at all four sites was disrupted using cancer-related mutations (Table 2) (B) Representative confocal images of HeLa cells express the GFP-tagged PRR14 LBD (231-282) phosphomimetic (PM) glutamic acid substitutions resulted in loss of nuclear lamina localization. (C) Box plot demonstrating the proportion of indicated PRR14-GFP proteins from panel B at the nuclear lamina, calculated using Lamin A/C signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. (D) Representative confocal images of C2C12 cells express phosphomimetic (PM) and phosphoablation (PA) GFP-tagged full length PRR14. PM substitutions resulted in loss of PRR14 nuclear lamina association, as seen with the 231-282 LBD. The PA substitutions appeared to result in stronger localization at the nuclear lamina. (E) Box plot demonstrating the proportion of indicated PRR14-GFP proteins from panel D at the nuclear lamina, calculated using Lamin B1 signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. (F) Line signal intensity profiles of corresponding images in panel D indicated by dashed lines. Statistical analyses performed using one-way ANOVA test with Dunn’s multiple comparison; *** p<0.001, ** p<0.01, * p<0.05, ns: not significant. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Sequencing, In Vivo

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: HeLa cells were transfected with GFP-tagged Lamin A or GFP-tagged PRR14. A region of interest at the nuclear lamina was bleached and fluorescent recovery was monitored. (A) Representative confocal images of PRR14-GFP or LaminA/C-GFP after photobleaching and recovery. (B) Graph shows signal monitored over a 5 minute period for comparing PRR14 and Lamin A recovery times. X-axis shows time (s) prior to and after photobleaching. Lamin A was previously reported to have a recovery time of ca. one hour. PRR14 recovered completely within seconds, Data analyses using the EasyFrap software indicated a t1/2 of 6.4 seconds with an R-square value of .95.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Transfection, Software

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: (A) Maps extracted from Pfam comparing human and mouse PRR14 and PRR14L paralogs, and the Drosophila Tantalus protein, showing the relationship with the Tantalus domain. (B) Summary of mutations to test the role of the putative conserved PP2A B56alpha recognition motif L/F/MxxIxE in the Tantalus domain of human PRR14. On the left are shown residues (in bold) in the B56alpha heat repeat that are known to mediate recognition of the substrates encoding the L/F/MxxIxE motif . The Drosophila B56alpha ortholog (dB56-2) shows 100 percent identity with human B56alpha in this region, consistent with the detection of Drosophila Tantalus-Drosophila B56alpha interactions . On the right are shown residues (in bold) in the PRR14 Tantalus domain corresponding to the L/F/MxxIxE B56alpha recognition motif. Shown are positions mutated in B56alpha and PRR14 to test effects on interactions. Amino acid sequence identity (*) is indicted. (C) HeLa cells were cotransfected with human HA-tagged B56alpha and non-fused GFP, or GFP fused to the human PRR14 Tantalus domain. The GFP bait proteins (GFP only, GFP-Tantalus) were collected using anti-GFP beads and analyzed by western for interaction with HA-tagged B56alpha. GFP bait proteins were detected using anti-GFP antibodies and prey proteins were detected with anti-HA antibodies. As shown, GFP-Tantalus pulled down HA-B56alpha, while the control non-fused GFP did not. Amino acid substitutions of non-conserved PRR14 Tantalus residues N483 and K484 (see Panel B) had no effect, while substitution of E500 and E501 with alanine eliminated interaction as predicted . (D) Experimental design was as in Panel C. Substitution of PRR14 Tantalus F495 with alanine inhibited binding, while substitution with leucine increased binding as predicted . (E) Experimental design was as in Panel C. Amino acid substitutions were introduced into the HA-tagged B56alpha subunit that were predicted to have no effect (G216Q) or inhibit binding (R222E and R226E) to the GFP-Tantalus domain . Results were consistent with these predictions. (F) A schematic cartoon showing PP2A interaction with PRR14 Tantalus domain. (G) Representative confocal images of HeLa cells transfected with indicated Tantalus domain amino acid substitutions introduced into full length GFP-tagged human PRR14 (see panel B). (H) Box plot demonstrating the proportion of indicated PRR14-GFP proteins from panel F at the nuclear lamina, calculated using Lamin A/C signal as a mask. Boxes: median, interquartiles range with Tukey whiskers (“+” mean value). n=20 cells per condition. Statistical analyses performed using one-way ANOVA test with Dunn’s multiple comparison; **** p<0.0001, ns: not significant. Scale bars: 5μm.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Sequencing, Western Blot, Binding Assay, Transfection

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: Human and mouse PRR14 and PRR14L Tantalus domain sequences are shown, aligned to positions 145-174 of the 299 amino acid Drosophila Tantalus protein. The 145-174 Drosophila Tantalus region shown is only a subset of the 119-198 Drosophila Tantalus domain, as it was the only region showing significant homology. Human PRR14, PRR14L, and Drosophila Tantalus had been identified as PP2A interactors (see main text) implicating the conserved core sequences (shaded) in mediating PP2A binding. Subsequently, the L/F/MxxIxE motif was identified as a common recognition sequence for the B56alpha regulatory subunit of PP2A (see main text).

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

Article Title: The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting

doi: 10.1101/788356

Figure Lengend Snippet: PRR14 is found to be highly modular protein. Several short evolutionary conserved motifs play key roles in bivalent tethering between heterochromatin and the nuclear lamina, and involved in regulation of association with the nuclear lamina.

Article Snippet: The mouse PRR14 orf was transferred to the N-terminal mGFP vector from OriGene (pCMV6-AN-mGFP, cat# PS100048) using the Origene PrecisionShuttle system.

Techniques:

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a ConSurf analysis of hSPNS2 protein. b Pairwise Alignment Scores for hSPNS2 protein and DNA sequences with putative homologs arranged according to the degree of sequence identity. c S1P levels in blood (n = 13, 15), lymph fluid (n = 10, 12), perfused liver, lung, kidney, heart (n = 3, 3). d Glucose levels in blood (n = 10,14), lymph fluid (n = 7, 13), perfused liver (n = 7, 5), lung (n = 7, 10), kidney (n = 5, 5), heart (n = 5, 5). e Glucose levels in urine (n = 7, 6), feces (n = 10, 8), tibialis anterior muscle (n = 5, 5), and gonadal adipose tissue (n = 4, 5). f Body weights (n = 20, 20). Body composition expressed as percentage of fat mass (n = 12, 12). g Food consumption, energy balance, energy expenditure, and locomotor activity (Beam breaks/h) determined with the PhenoMaster (n = 9,10; N = 3). h Blood hemoglobin concentration (n = 17, 9) and percentage of glycosylated hemoglobin, HbA1c (n = 7, 7). i Oral glucose tolerance test (GTT) and area under the curve (AUC) (n = 5, 5; N = 2). j Plasma levels of fasting insulin (n = 14, 14), glucagon (n = 8, 8) and thyroid hormone triiodothyronine (T3). Data are means ± s.e.m. k – n PET-CT imaging analysis of 2-FDG in Spns2 –/– mice. PET tracer 2-FDG (270–550 µCi) was gavaged prior to anesthetization of WT or Spns2 –/– mice for PET-CT scans. (n = 3, 3, N = 2). k , l At 60 min, tracer activity of target organs was quantified in volumes of interest (VOI). Data are percentage of whole-body activity for the right kidney (RK), heart, and bladder (Bl). l Coronal sections (0.4 mm thick) of PET images are presented according to a spectral scale for tracer activity, from red (highest), to green (intermediate), to blue (lowest). m The distribution of 2-FDG in representative WT at the specified times following gavage is shown to demonstrate the assessment of gastric emptying and intestinal absorption. St, stomach; In, intestines. n Representative images of the VOI in bladder to determine % of urinary excretion. o % of urinary excretion, gastric emptying and intestinal absorption. Data are means ± s.d. Two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Sequencing, Activity Assay, Concentration Assay, Clinical Proteomics, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Proliferation of Spns2 overexpressing (SPNS2-OE1) or Spns2 deleted cells (SPNS2-KO1) and controls cells. 10 5 cells were cultured in medium containing 4.5 g/L glucose without or with 500 nM S1P as indicated and cell numbers measured after 72 h (n = 3). b – f Cells were cultured in medium without or with 4.5 g/L glucose and/or 500 nM S1P as indicated. b , c Migration of cells in wound healing assays 24 h after creating a gap in a confluent monolayer and change to media containing glucose or S1P as indicated. b Representative images at 0 or 24 h after initiation of migration and ( c ) percentages of wound closures determined in cell migration assays in the presence of aphidicolin (n = 4). d – f ECIS measurements of the resistance of the indicated cells. d Representative continuous resistance measurements. e normalized endpoint resistance of the indicated cells cultured for 118 h. Resistance was normalized to the value measured at 12 h. f Normalized endpoint resistance of the indicated cells cultured for 118 h in the absence or presence of glucose and/or S1P (n = 3). g FITC-dextran leakage from the indicated cell monolayers cultured in the present of glucose (n = 3, 6). h FITC-dextran leakage from the indicated cell monolayers in the absence or presence of glucose and/or S1P (n = 4). a , c , e – h Data are means ± s.d. ns, not significant; One-way analysis of variance test followed by Sidak's multiple comparisons test or ( h ) Welch’s ANOVA multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Cell Culture, Migration

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Molecular dynamics simulations of the inward-facing open conformation of hSPNS2 (PDB ID 8EX4) show that S1P can slither up as its hydrocarbon tail becomes vertically aligned from its kinked structure. b Changes in the vertical distance of the S1P phosphate headgroup from its original location near S232 of SPNS2. c The upward translocation of S1P causes noticeable structural changes at the extracellular vestibule of SPNS2. d The root-mean-squared deviation (RMSD) of the SPNS2 structure as S1P slithers upward during the unbiased simulation. The set of hydrophobic residues shown in Fig. 4a pose a barrier for the upward movement illustrated by the free-energy plot shown in Supplementary Fig. . Once the phosphate group makes it past these residues, further upward movement is relatively easier, and the hydrophobic residues close in to obstruct its downward movement. e – g Snapshots of the representative, inward-facing open structures of SPNS2-S1P complexes at various times during the MD simulation in which S1P moves upward. Transient glucose-binding pockets were obtained by molecular docking of glucose into 2000 frames extracted from the 1000 ns simulation. h – j Close-up views of the glucose-binding pockets are shown in ( e – g ). Residues near the most stable glucose-binding pockets are indicated. Lower panels highlight residues within 3.5 Å of glucose in the top (site 1), central (sites 2 and 3), and bottom (site 4) glucose-binding pockets and H-bonds are indicated with dotted lines. k Glucose molecule hydrogen bonds with S1P and nearby protein residues before transitioning of glucose to the intracellular side (during 150–300 ns in the distance plot shown in m ). l Trace the glucose pathway as it enters the intracellular side from the extracellular side. m Changes in the vertical distance of glucose from its initial position at the central site 3 binding pocket as it traverses through the channel and passes into the intracellular side. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Purification, Two Tailed Test, Scintillation Proximity Assay, Binding Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Western Blot, Plasmid Preparation, Expressing, Fluorescence, Comparison, Functional Assay, Liposomes, Control

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: Introgression of the BN Shroom3 gene onto FHH background improves glomerular and overall kidney function. ( A ) At 14 wk of age, both homozygous and heterozygous FHH.BN14a congenic animals showed a significantly lower degree of albuminuria compared to FHH ( n = 4, 7, 8, and 3, respectively). ( B ) Both heterozygous and homozygous FHH.BN14a demonstrated significantly improved glomerular permeability (Palb) compared to FHH. ( n = 4 animals/115 glomeruli, 3 animals/76 glomeruli, and 2 animals/70 glomeruli, respectively. Palb in BN was obtained from previously published data .) ( C ) FHH.BN14a kidney showed a decreased presence of glomerular sclerosis compared to FHH at 14 wk of age. A minimum of 30 glomeruli from three kidneys for each strain were scored for a percentage of sclerosis using a scale from 0 (no sclerosis) to 4 (complete sclerosis). ( D ) Representative trichrome-stained images of glomeruli from FHH and FHH.BN14a are shown. Fibrotic tissues are indicated by blue stain. Scale bars = 50 µm. ( E ) Electron microscopic images of glomeruli showed podocyte foot process fusion (indicated by arrow) in FHH compared to FHH.BN14a animals at 18 wk of age. Scale bars = 500 µm. (CL) Capillary lumen, (*) P < 0.05 vs. FHH, (#) P < 0.05 vs. BN.

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: Permeability, Staining

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: FHH Shroom3 is defective and contributes to glomerular dysfunction. ( A ) Schematic representation of the rat Shroom3 protein is shown. Vertical lines represent the amino acid variants found in FHH Shroom3 . Variants predicted to be damaging by PolyPhen-2 are shown in red. ( B ) Co-injection of shroom3 + tp53 morpholino (MO) with full-length BN, but not FHH, Shroom3 mRNA rescued the edema phenotype. ([***] P < 0.001 vs. MO and MO + FHHmRNA) and ( C ) cell death induced by shroom3 + tp53 MO ([*] P < 0.05 vs. uninjected control). ( D ) Representative fluorescence images of individual dorsal aorta at 1, 24, and 48 h following 70-kDa dextran injection are shown. ( E ) Co-injection with BN but not FHH Shroom3 mRNA rescued the dextran leakage induced by knockdown of endogenous shroom3 in zebrafish. ( n = 35, 26, 23, and 22, respectively. [*] P < 0.05 vs. uninjected and MO + BNmRNA.)

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: Injection, Control, Fluorescence, Knockdown

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: The G1073S variant disrupts the function of the FHH Shroom3 gene. ( A ) Schematic of the different recombinant Shroom3 cDNAs, where a specific region of the BN Shroom3 sequence was replaced by FHH. ( B ) Co-injection of shroom3 + tp53 MO with Shroom3∆641–3044 or Shroom3∆4117–5966 mRNA, but not Shroom3∆3044–4117 mRNA, rescued dextran leakage induced by the MO ( n = 23, 9, 18, 28, and 15, respectively). ( C ) Schematic of Shroom3 single-amino acid mutants created by site-directed mutagenesis. ( D ) Co-injection of ∆Y1291C or ∆A1356V restored normal glomerular permeability, while ∆G1073S failed to exhibit functional rescue ( n = 17, 12, 17, 23, and 21, respectively). (*) P < 0.05, (**) P < 0.001 vs. uninjected.

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: Variant Assay, Recombinant, Sequencing, Injection, Mutagenesis, Permeability, Functional Assay

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: The G1073S variant decreases the actin-binding affinity of SHROOM3 protein. ( A ) FLAG-tagged BN, FHH, or ∆G1073S SHROOM3 proteins were overexpressed in HEK293 cells, followed by immunoprecipitation against FLAG. Immunoprecipitated lysates were immunoblotted using antibodies against FLAG, ROCK1, and ACTB. A representative Western blot of immunoprecipitated lysate is provided. ( B ) Quantification of the Western blot showed that FHH SHROOM3 and ∆G1073S mutant had significantly reduced actin-binding affinity compared to BN SHROOM3. ( n = 3 per group. [*] P < 0.05 vs. BN.) ( C ) ROCK1-binding affinity was not different among the three alleles ( n = 3 per group).

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: Variant Assay, Binding Assay, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: rs181194611 (p.P1244L) associated with nondiabetic ESKD impairs SHROOM3 function in vivo. ( A ) Proline at the amino acid position 1244 in SHROOM3 is evolutionarily conserved. ( B ) Representative images of dorsal aorta at 1, 24, and 48 h following 70-kDa dextran injection are shown. ( C ) Co-injection of nonmutated human SHROOM3 mRNA restored normal glomerular permeability, while ∆P1244L failed to show functional rescue. (*) P < 0.05 vs. uninjected.

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: In Vivo, Injection, Permeability, Functional Assay

Journal: Genome Research

Article Title: Shroom3 contributes to the maintenance of the glomerular filtration barrier integrity

doi: 10.1101/gr.182881.114

Figure Lengend Snippet: Podocyte-specific disruption of shroom3 causes increased glomerular permeability and podocyte effacement in zebrafish. ( A ) Specific transgenic (TG) lines were crossed to obtain embryos expressing podocin:Gal4 and podocin:Gal4;UAS:shrm3DN . The control and mutants were injected with 70-kDa FITC-labeled dextran at 4.5–5 d post-fertilization (dpf) and analyzed for dextran clearance at 24 and 48 h post-injection (hpi). (DA) Dorsal aorta. ( B ) Representative fluorescence images of individual dorsal aorta at 1, 24, and 48 hpi are shown. ( C ) podocin:Gal4;UAS:shrm3DN mutants had significantly decreased FITC signal at 24 and 48 hpi compared to control, indicating that the glomerular filtration barrier was disrupted. ( n = 20 and 25, respectively. [***] P < 0.001.) ( D ) Electron micrograph revealed intact podocyte foot processes (indicated by arrowhead) in podocin:Gal4 animals. Foot process effacement (indicated by arrow) was observed in the podocin:Gal4;UAS:shrm3DN mutants. Scale bars = 500 µm. (CL) Capillary lumen. ( E ) podocin:Gal4;UAS:shrm3DN had a significantly reduced number of foot processes contacting glomerular basement membrane (GBM) and increased foot process diameter compared to the control. ( n = 3 per group. [**] P = 0.01, [***] P = 0.0008.)

Article Snippet: To test the human alleles, we used the commercially available myc-ddk-tagged human SHROOM3 cDNA ORF (Origene), and the mutant allele was generated using site-directed mutagenesis.

Techniques: Disruption, Permeability, Transgenic Assay, Expressing, Control, Injection, Labeling, Fluorescence, Filtration, Membrane

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: In silico analysis of rod-specific photoreceptor cGMP phosphodiesterase (PDE6D) expression in hepatocellular carcinoma (HCC). ( A ) Oncomine TM human cancer microarray database analysis of six patient datasets depicting PDE6D mRNA expression levels in HCCs and non-tumorous livers (* p < 0.05 vs. non-tumorous livers). ( B , C ) Oncomine TM human cancer microarray database analysis of PDE6D expression as detected in large-scale RNA profiling studies comparing diverse carcinomas of different origins (* p < 0.05 vs. average expression).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: In Silico, Expressing, Microarray

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: PDE6D expression in HCC in vivo and in vitro. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis of HCC patient samples and paired non-tumorous liver tissues (* p < 0.05). ( B ) Detection of PDE6D mRNA in human HCC cells (PLC, Hep3B, HepG2) after qRT-PCR amplification using gel electrophoresis (left panel) and relative PDE6D mRNA levels (qRT-PCR) in human HCC cell lines (PLC, Hep3B, HepG2) compared with primary human hepatocytes derived from different donors (#1–3) (right panel) (* p < 0.05 vs. hepatocytes). ( C ) Exemplary Western blot image (left panel) and summarized densitometric quantification (right panel) of PDE6D protein levels in HCC cells (PLC, Hep3B, HepG2, Huh-7) compared with hepatocytes derived from different donors (#1–2) (* p < 0.05 vs. hepatocytes).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Quantitative RT-PCR, Amplification, Nucleic Acid Electrophoresis, Derivative Assay, Western Blot

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Effects of PDE6D knockdown on HCC proliferation and clonogenicity. Prior to functional experiments, HCC cell lines (PLC, Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis (* p < 0.05 vs. control). ( B ) PDE6D protein levels as quantified by Western blot analysis. The left panel depicts an exemplary Western blot image, and the right panel depicts the summarized densitometric quantification (* p < 0.05 vs. control). ( C ) Real-time cell proliferation (xCELLigence). Exemplary proliferation curves (left panel) and quantified “slopes” (summarizing the proliferative ability) (right panel) are shown (* p < 0.05 vs. control). ( D ) Relative (to mean) PDE6D as correlated to CyclinD1 mRNA expression levels (qRT-PCR) in human HCC patient tissue samples. ( E , F ) Anchorage-dependent clonogenic assay (an exemplary image (Hep3B) is depicted in the left panel of ( E )). Quantification of colony number (right panel of ( E )) and sizes ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Functional Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Clonogenic Assay

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and function of PDE6D in sorafenib resistance. ( A ) PDE6D mRNA levels as quantified by qRT-PCR analysis in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B and HepG2 cell clones (* p < 0.05). ( B ) Exemplary image (left panel) and summarized densitometric quantification (right panel) of Western blot analysis of PDE6D levels in non-resistant (“non-resist.”) as compared to sorafenib-resistant (“resistant”) Hep3B cells (rel DM: relative (PDE6D/Actin) densitometry) (* p < 0.05 vs. non-resist.). ( C , D ) Prior to functional experiments, resistant cells (Hep3B) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). ( C ) Depicts relative proliferation (cell numbers) and ( D ) depicts exemplary clonogenic assays. ( E , F ) Forced overexpression of PDE6D protein (PDE6D-OE) in HCC cells (e.g., PLC) was performed by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (an empty control vector without the PDE6D ORF was used as control treatment). ( E ) Depicts Western blot analysis depicting the overexpressed Myc-DDK-tagged PDE6D protein after PDE6D-OE as well as endogenous (arrow) PDE6D in both PDE6D-OE and control-treated cells. ( F ) Depicts exemplary images (representing 8 replicate values of 2 independent experiments) of cells cultured in 6-wells for 72 h (100,000 cells were initially seeded per 6-well) and treated with different doses of sorafenib (0, 4, 8 µM).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Western Blot, Functional Assay, Transfection, Over Expression, Plasmid Preparation, Cell Culture

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: TGF-â-mediated regulation of PDE6D and the effect of PDE6D on HCC cell migration. ( A – C ) Quantitative RT-PCR analysis of VIMENTIN, SNAIL, S100A4 ( A ) and KRAS ( B ) mRNA levels in HCC cells (PLC) that were stimulated with different doses of recombinant human TGF-â1 protein for 72–96 hours (* p < 0.05 vs. control). ( C , D ) Quantitative RT-PCR revealing mRNA levels ( C ) as well as protein levels as quantified by Western blot analysis (including a representative Western blot image) (the densitometric values represent two independent Western blot analysis) ( D ) of PDE6D expression in HCC cells (PLC) that were treated with different doses of recombinant human TGF-â1 for 72–96 hours. ( D ) also depicts co-treatment with 15 µM of the TGF-â-receptor-1 (TGFBR1) inhibitor LY2157299 (“galunisertib”) (* p < 0.05 vs. control). ( E , F ) Prior to Boyden chamber experiments, non-resistant HCC cells (PLC, Hep3B) ( E ) and sorafenib-resistant Hep3B cells ( F ) were transfected with si-RNA-pools against PDE6D (“PDE6D”) or the according control- si-RNA-pool (“Control”). Migration (migrating cells per visual field) as measured by Boyden chamber migration assay (duration of migration: 4 hours) is depicted as absolute cell counts ( E ) or as normalized migration ( F ) (* p < 0.05).

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Migration, Quantitative RT-PCR, Recombinant, Western Blot, Expressing, Transfection

Journal: Cancers

Article Title: The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression

doi: 10.3390/cancers11030398

Figure Lengend Snippet: Expression and cellular localization of PDE6D in HCC in vivo and in vitro. ( A ) PDE6D staining (exemplary images) of non-tumorous liver tissues (left side) and HCC tissues (right side) deposited on the Human Protein Atlas database. ( B ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues ( N = 117) as compared with corresponding non-tumorous liver tissues ( N = 127) (Fisher’s exact P < 0.001). ( C , D ) Tissue microarray analysis of PDE6D expression levels in human HCC tissues correlated with tumor grading (Fisher’s exact P = 0.030) ( C ) and tumor stages ( D ). ( E ) Exemplary immunohistological images of PDE6D protein expression in human HCC samples and corresponding non-tumorous liver tissues applying a tissue microarray revealing nuclear staining next to cytoplasmatic staining patterns (paired samples of two different patients (#1, #2) are depicted). ( F , G ) Tissue microarray analysis comparing tumor stages ( F ) and ERK activation (p-ERK) ( G ) in human HCC tissues with (“yes”) and without (“no”) cytoplasmatic localization pattern of PDE6D. ( H ) In silico-based analysis of importin-á-dependent nuclear localization signals (NLS, red letters ) using the “cNLS Mapper” predicted bipartite NLS in both isoforms of PDE6D (score for both isoforms was 5.3). A legend depicts that higher scores indicate stronger NLS activity and defines major localizations in dependence of each score. ( I , J ) Exemplary immunofluorescence ( I ) and Western blot analysis ( J ) depicting nuclear localization of PDE6D (I) and expression of PDE6D in both nuclear and cytoplasmatic fractions ( J ) of HCC cell lysates.

Article Snippet: Overexpression of PDE6D protein in HCC cells was induced by transfection of a human PDE6D open reading frame (ORF) Myc-DDK-tagged plasmid vector (pCMV6-Entry cDNA vector system) from OriGene (Rockville, MD, USA) (CAT#: RC203172) (an according empty control vector without the PDE6D ORF was used as control treatment).

Techniques: Expressing, In Vivo, In Vitro, Staining, Microarray, Activation Assay, In Silico, Activity Assay, Immunofluorescence, Western Blot

Journal: Human molecular genetics

Article Title: Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

doi: 10.1093/hmg/ddad034

Figure Lengend Snippet: Figure 1. Pedigree of variable panocular malformations (VPM) and genetic analyses. (A) VPM pedigree. Arrow indicates the proband. Hash marks indicate patients subjected to systematic molecular analyses. Asterisks indicate individuals who underwent Sanger sequencing. (B) Fundus photographs and optical coherence tomography images of seven VPM patients. Images of the right eye of V-4 were not obtained because of low transparency by aftercataract. Arrows, PFV; arrowheads, areal atrophy of the retina and choroid; asterisk, excavation of coloboma or pit; F, fovea. The fovea was dislocated to near the optic disc in III-2 (both eyes), IV-2 (right eye) and V-2 (both eyes), hypoplastic in IV-2 (right eye), IV-3 (left eye), V-2 (both eyes) and V-3 (right eye), and associated with areal atrophy of the retina and choroid (ectopic coloboma) in V-3 (left eye) and V-4 (left eye). Myelinated nerve fiber was seen in IV-3 (left eye). The optic nerve head was anomalous with pit in III-2 (right eye) and IV-2 (left eye), coloboma in IV-2 (right eye), IV3 (left eye) and V-4 (left eye), and hypoplasia in V-2 (left eye). (C) The INTS15 variant identified in seven VPM patients. A protein-altering SNV was identified in the last exon.

Article Snippet: Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased.

Techniques: Sequencing, Tomography, Variant Assay

Journal: Human molecular genetics

Article Title: Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

doi: 10.1093/hmg/ddad034

Figure Lengend Snippet: Figure 2. Genotypes and phenotypes of knockout mice. (A) Genotypes of offspring from Ints15 exon 6 +/- intercrosses (a) and Ints15 exon 2 +/- intercrosses (b). (B) Fundus (a) and histology (b) of the optic nerve head (OPNH) in wild type mice (WT). (C) Fundus (a) and histology (b) of heterozygous Ints15 exon 6 +/- mice. PFV on OPNH is indicated with arrows. (D) Fundus (a, e) and histology (b-d, f-h) of homozygous Ints15 exon 6 -/- mice. a-c, PFV on OPNH (arrows) and glial proliferation around vessels in the retina (arrowheads) are evident compared with WT (d). e, f, h, Local chorioretinal atrophy, characterized by defects of the choroid (arrowheads) and deformity of the outer retina layers with glial proliferation (arrow), is evident compared with WT (g). (E) Fundus (a), anterior segment (b, d, g), and histology (c, e, f, h) of heterozygous Ints15 exon 2 +/- mice. a-c, PFV radiating from OPNH to the lens (arrows) contributes to the formation of cataract (arrowhead). d-f, Corneal opacity, iris anomaly (arrows), cataract, PFV behind the lens that drags the retina (asterisk), and ectopic lens tissue (arrowhead) are shown. g, h, Microphthalmos with malformation of whole ocular tissues. C, cornea; L, lens; R, retina; Li, lid, ON, optic nerve. Bb, Cb, Db, Df, Dg, Ec, Ee, Ef, Eh, HE staining; Dc, Dd, Dh, anti-glial fibrillary acidic protein immunohistochemistry; brown bar, 100 μm; blue bar, 200 μm.

Article Snippet: Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased.

Techniques: Knock-Out, Staining, Immunohistochemistry

Journal: Human molecular genetics

Article Title: Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

doi: 10.1093/hmg/ddad034

Figure Lengend Snippet: Figure 3. Expression and subcellular localization of INTS15 and its role in cell survival. (A) In situ hybridization of Ints15 in E8.0 mouse embryos. a, Stereomicroscopy of whole mount in situ hybridization. b, Sections of the whole mount specimen: transvers plates of the head and neck; coronal plates of the chest and abdomen. Arrows, eye (optic cup); T, telencephalon; D, diencephalon, NT; neural tube, E; esophagus; L, lung; Li, liver; I, intestine, FL, forelimb, HL, hindlimb. Scale bar, 500 μm. (B) Immunofluorescence staining of Ints15/INTS15 in E14.5 mouse embryos and cell lines. The nuclei were counterstained with DAPI. Scale bars, 10 μm. (C) HeLa cells transfected with pU6-shINTS15-GFP or control pU6-shRNA-GFP were immunostained for M30 to visualize apoptotic cells. Staurosporine was used as a positive control. Scale bar for higher magnification, 5 μm; scale bars for the rest, 15 μm. (D) The ratios of M30-positive apoptotic cells in 50 transfected GFP-positive cells. HeLa cells transfected with the indicated constructs were analyzed as in C. ∗, P < 0.01 (unpaired two-tailed t-test); n = 3.

Article Snippet: Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased.

Techniques: Expressing, In Situ Hybridization, Immunofluorescence, Staining, Transfection, Control, shRNA, Positive Control, Construct, Two Tailed Test

Journal: Human molecular genetics

Article Title: Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

doi: 10.1093/hmg/ddad034

Figure Lengend Snippet: Figure 4. INTS15 binds to Integrator, is critical for snRNA processing. (A) 293T cells overexpressing Flag-INTS5 or GFP were subjected to anti-Flag immunoprecipitation (IP), followed by immunoblotting with the indicated antibodies. (B) Integrator was immunoprecipitated from HeLa cell nuclear extract using anti-INTS5 antibody, followed by immunoblotting with the indicated antibodies. (C) RNA was prepared from HeLa cells transfected with INTS15 siRNA or control siRNA, and qRT-PCR was performed using primer sets detecting both mature and precursor snRNAs (total) and those detecting only the latter. ∗, P < 0.05 (paired t-test), error bars, S.D.; n = 6. (D) Gene set enrichment analysis of INTS15-regulated genes. 700 transcripts found to be downregulated by 50% or more in INTS15 siRNA-treated HeLa cells were subjected to gene set enrichment analysis using DAVID v6.5. Top annotation terms in the functional annotation chart are shown along with the fractions of genes associated with respective terms. ∗P < 0.05, modified Fisher exact test. (E) Missplicing events induced by INTS15 knockdown in HeLa cells. A total of 2,777 missplicing events were identified by RNA-seq using a threshold level of P < 0.01 and categorized into five types. (F) Representative missplicing events associated with INTS15 knockdown. RT-PCR analysis was carried out to validate the findings obtained by RNA-seq data (Fig. 4E and Supplementary Material, Data S4). Primers sets (Supplementary Material, Data S7) were designed so that they amplify splice variants in question simultaneously, and semiquantitative RT-PCR products were analyzed on agarose gels. Ensembl transcript names and exon structures examined are shown.

Article Snippet: Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased.

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Quantitative RT-PCR, Functional Assay, Modification, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Human molecular genetics

Article Title: Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

doi: 10.1093/hmg/ddad034

Figure Lengend Snippet: Figure 5. INTS15 regulates genes associated with brain and eye development. (A, B) Heatmaps showing log2(FPKM) of 200 genes that are associated with brain and eye development and most highly expressed in control HeLa cells. Also shown are the results of RT-PCR, carried out to validate RNA-seq data. (C) Human iPSCs-derived retinal tissue (R) including RGCs and adjacent brain-like tissue (B), treated with INTS15 siRNA or control siRNA, were stained for TUJ1, a marker of axon and dendrite. Scale bar, 500 μm. (D) qRT-PCR analysis of developing brain markers, BF1 and FGF8, and β-actin in microdissected brain-like tissue surrounded by a red dashed line. ∗, P < 0.05 (unpaired two-tailed t-test); error bars, S.D.; n = 3. KD, knockdown. (E) qRT- PCR analysis of RGC markers, BRN3B and ISLET1, and β-actin in microdissected retinal tissue surrounded by a blue dashed line. ∗, P < 0.05 (unpaired two-tailed t-test); error bars, S.D.; n = 4.

Article Snippet: Human INTS15 (NM_024067) ORF clone with C-terminal Myc-DKK tag (Origene #RC219786; pCMV C7orf26-Flag) and negative control GFP expressing clones (TaKaRa Bio #Z2470N; pAcGFP1-C1) were purchased.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Derivative Assay, Staining, Marker, Quantitative RT-PCR, Two Tailed Test, Knockdown