Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) Workflow for MutChromSeq. (B) Infection responses, disease scorings, and quantification of fungal biomass in Bowman and six barley EMS M1 lines after inoculation with Bipolaris sorokiniana ND90Pr. Photos were taken at seven days after inoculation. The 1-9 rating scale of Fetch and Steffenson was used to rate the spot blotch disease. Fungal biomass was quantified for Bowman and three EMS M1 lines using quantitative PCR. (C) Gene structure and EMS mutations in Scs6 , a gene encoding a canonical coiled-coiled-type NLR (CNL).

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) The coding sequence of Scs6 was expressed under the maize Ubi promoter and NOS terminator (NOSt), and cloned into the backbone of binary vector pANIC12A . (B) The coding sequence of Scs6 was expressed under the promoter+5’UTR (pMla6+5’ UTR) and 3’UTR+terminator (3’ UTR+tMla6) of Mla6 , which was assembled into binary vector pBract202 . Both constructs use hptII driven by the 35S Cauliflower Mosaic Virus (35S) promoter for plant selection during transformation (shown in purple). Left and right T-DNA borders (LB and RB) are shown with filled grey and black blocks, respectively.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Construct, Virus, Selection, Transformation Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A-B) Representative images of infection responses of Golden Promise (GP), SxGP DH47 (DH47) and derived transgenic Scs6 T1 plants to B. sorokiniana ND90Pr, seven days after inoculation.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Infection, Derivative Assay, Transgenic Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) PCR amplification of Scs6 with genomic DNA and cDNA from Bowman (Bow_DNA and Bow_cDNA), Golden Promise (GP_DNA and GP_cDNA) and three T0 transgenic barley plants (GP_Scs6-1, GP_Scs6-2, and GP_Scs6-3) using primers Scs6-F4+Scs6-R1 . (B) PCR amplification of Scs6 with cDNA derived from Bowman (Bow_cDNA), Golden Promise (GP_cDNA), and five T1 plants derived from each of the two T0 transgenic barley plants (GP_Scs6-4 and GP_Scs6-5) using primers Scs6-F4+Scs6-R1 . * (+) indicated that PCR was positive and (-) indicated that PCR was negative.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Amplification, Transgenic Assay, Derivative Assay

Journal: bioRxiv

Article Title: A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility

doi: 10.1101/2023.12.13.571418

Figure Lengend Snippet: (A) Barley genotypes that express Scs6 (Bowman) or negative control (ND B112) were infiltrated with intercellular washing fluid (IWF) that was isolated from Bowman leaves infected either with wild type B. sorokiniana ND90Pr (IWF ND90Pr ) or mutant B. sorokiniana Δ nps1 (IWF Δ nps1 ), as indicated. (B-C) N. benthamiana plants were transformed transiently, as indicated. Genes were fused in between the 35S promotor sequence and 4xmyc (receptors) or mYFP (AVR A6 without signal peptide) epitope sequences. Twenty-four hours after Agrobacterium -mediated gene delivery, IWF ND90Pr , IWF Δ nps1 or water was infiltrated, as indicated. Cell death phenotypes were assessed and documented at two or four days after agroinfiltration for IWF-triggered cell death or effector-triggered cell death, respectively. Representative pictures of at least six biological replicates (indicated in brackets) are shown and combinations that resulted in cell death are highlighted with a red box. (D) For determination of protein levels of receptor-4xMyc (approx. 114 kDa and AVR A6 -mYFP (39 kDA) in N. benthamiana , leaf tissue was harvested two days post Agrobacterium infiltration.

Article Snippet: The whole coding sequence (CDS) of Scs6 was synthesized by GenScript (Piscataway, NJ) and inserted between the Spe I and Bsr GI restriction sites of the binary vector pANIC12A , producing a new construct pANIC12A- Scs6 with the Scs6 gene driven by a Ubi promoter and stopped by a NOS terminator.

Techniques: Negative Control, Isolation, Infection, Mutagenesis, Transformation Assay, Sequencing

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: The expression of HMGCL was downregulated in lung cancer. (A) Representative images (with 4-fold and 10-fold magnification) of HMGCL expression examined by IHC staining were shown. Scale bar, 50 µm. Details about the IHC protocol were provided in the “Materials and Methods” section. (B-C) The H scores for HMGCL protein expression in a tissue array (84 noncancerous tissues and 84 cancerous tissues) were calculated and statistically analyzed, and the correlation between HMGCL expression and survival was analyzed. The tissue array was examined by IHC staining, and the H score was calculated with the Vectra 2 system. (D) The correlation between the expression of HMGCL and survival in lung cancer was determined via the Human Protein Atlas database ( https://www.proteinatlas.org/ENSG00000117305-HMGCL/pathology/lung+cancer ). (E) The correlation between the HMGCL mRNA level and survival in lung cancer was determined via the Kaplan-Meier Plotter database (kmplot.com/analysis/index.php?p=service). (F) Western blotting was performed to examine HMGCL protein expression in lung tissues derived from KP mice treated with 1 × 10 9 PFU of Ad-Cre or Ad-GFP virus per mouse. Details about the KP mouse model are provided in the “Materials and Methods” section. (G) HMGCL protein levels in the normal cell line HBE and cancer cell lines (H520, A549, H1299, H157, 95C, 95D and H358) were examined by western blotting. **, P <0.01.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Expressing, Immunohistochemistry, Western Blot, Derivative Assay, Virus

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: HMGCL inhibited the growth of lung cancer cells. (A) Overexpression of HMGCL (Flag-HMGCL) in A549 and H1299 cells was confirmed by western blot analysis. The CDS of HMGCL was inserted into the lentiviral vector pLvx. The lentivirus was packaged in 293T cells and purified (12000 × g, 4 °C). A549 and H1299 cells were incubated with the lentivirus for 8 hours and selected with puromycin (2 µg/ml). (B) Knockdown of HMGCL expression in A549 and H1299 cells was confirmed by western blot analysis. The HMGCL shRNA oligo was inserted into the lentiviral vector pLKO.1. The lentivirus was packaged in 293T cells and purified (12000 × g, 4 °C). A549 and H1299 cells were incubated with the lentivirus for 8 hours and selected with puromycin (2 µg/ml). (C-D) Measurement of the content of β-HB in A549 and H1299 cells with HMGCL overexpression or knockdown. (E) A CCK-8 assay was performed to examine the effects of HMGCL knockdown and β-HB on the growth of A549 and H1299 cells. The working concentration of β-HB was 10 mM. (F-G) A soft agar assay was performed to examine the effects of HMGCL knockdown and β-HB on the anchorage-independent growth of A549 and H1299 cells. β-HB (10 mM) was added to the soft agar. (H) The level of phosphorylated p70S6K (p-Thr389) was evaluated in A549 and H1299 cells overexpressing HMGCL. *, P <0.05; **, P <0.01.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Purification, Incubation, Knockdown, Expressing, shRNA, CCK-8 Assay, Concentration Assay, Soft Agar Assay

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: HMGCL inhibited tumorigenesis. (A-B) The effects of HMGCL overexpression on the tumorigenicity of H1299 and A549 cells were evaluated. A total of 10 6 cells (control cells or cells overexpressing HMGCL) were injected subcutaneously into nude mice. Six weeks later, tumors were harvested and weighed. (C) Western blotting was performed to evaluate HMGCL protein levels in the tumors. Exo, exogenous; endo, endogenous. (D) Measurement of the β-HB content in the tumors. (E) Map of the pSECC vector. This vector contained the sgRNA cassette and expressed Cas9 as well as Cre. NLS, the nuclear localized sequence. (F-G) IHC analysis was performed to examine the levels of HMGCL protein. The expression of HMGCL was scored and statistically analyzed. Lentivirus expressing the sgRNA targeting HMGCL was used to treat the mice (10 9 PFU per mouse). Twelve weeks later, the lungs were harvested, and the expression of HMGCL was evaluated using IHC staining. (H-I) HE staining was performed to evaluate tumorigenesis in the lungs harvested as described in . The number of tumors was statistically analyzed. *, P <0.05; **, P <0.01.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Over Expression, Control, Injection, Western Blot, Plasmid Preparation, Sequencing, Expressing, Immunohistochemistry, Staining

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: HMGCL protein levels were decreased upon treatment with TNFα. (A-B) Treatment with TNFα decreased HMGCL protein levels in a dose- and time-dependent manner. In (A), A549 and H1299 cells were treated with 0, 20, or 50 ng/ml TNFα for 3 hours, and western blotting was performed to evaluate HMGCL protein expression. In (B), A549 and H1299 cells were treated with 10 ng/ml TNFα for 6, 12, and 24 hours, and western blotting was performed to evaluate HMGCL protein expression. (C) Treatment with TNFα exerted only minor effects on HMGCL mRNA levels. A549 and H1299 cells were treated with 10 ng/ml TNFα for 24 hours, and HMGCL mRNA levels were examined by qPCR. IL8 was used as the positive control. (D) The HMGCL protein level was increased upon treatment with ACHP (ACHP Hydrochloride, 20 nM), an inhibitor of the IKK complex. A549 and H1299 cells were treated with ACHP (20 nM) for 24 hours, and the expression of HMGCL and phosphorylated IKKβ was evaluated by western blotting. (E-F) The half-life of the HMGCL protein was evaluated. A549 and H1299 cells were treated with CHX in the presence or absence of TNFα for different durations, and the HMGCL protein levels were evaluated and quantified. **, P <0.01.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Western Blot, Expressing, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: IKKβ phosphorylated HMGCL. (A) Immunoprecipitation was performed to examine the interaction between exogenous IKKβ (HA-IKKβ) and HMGCL (Flag-HMGCL). Details about the immunoprecipitation protocol are provided in the “Materials and Methods” section. (B) The interaction between IKKβ and the GST-HMGCL fusion protein was examined using a GST pulldown assay. Details about the GST pulldown assay protocol are provided in the “Materials and Methods” section. (C) Immunoprecipitation was performed to examine the interaction between endogenous IKKβ and HMGCL in A549 and H1299 cells. A549 and H1299 cells were lysed with IP lysis buffer. After centrifugation, an anti-IKKβ antibody was added to the supernatant for immunoprecipitation overnight. Binding of IKKβ and HMGCL was examined by western blotting. (D) IKKβ upregulated the phosphorylation of HMGCL. The expression vectors (Flag-HMGCL and HA-IKKβ) were cotransfected into A549 and H1299 cells. Exogenous HMGCL protein was enriched by immunoprecipitation, and phosphorylation of HMGCL was detected using a pan-phospho-Ser/Thr antibody. (E-F) Mapping the Ser/Thr residue phosphorylated by IKKβ. An HMGCL truncation (containing only amino acids 1-250) was constructed, and the phosphorylation of full-length and truncated HMGCL was examined (E). The Ser/Thr residues in the deleted region of HMGCL (amino acids 251-325) were sequentially mutated to A (alanine), and the phosphorylation of the HMGCL truncation (amino acids 251-325) was examined (F). (G) The effects of wild-type and mutant (S258A) HMGCL on the anchorage-independent growth of H1299 and A549 cells were evaluated by a soft agar assay. Colonies were counted and statistically analyzed. **, P <0.01.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Immunoprecipitation, GST Pulldown Assay, Lysis, Centrifugation, Binding Assay, Western Blot, Phospho-proteomics, Expressing, Residue, Construct, Mutagenesis, Soft Agar Assay

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: NEDD4 promoted the ubiquitination of HMGCL. (A) A549 cells were treated with chloroquine (50 µM) for different durations (0, 12 hours and 24 hours) and the levels of HMGCL protein were examined by western blotting. LC3 acted as a positive control. (B) The prediction of the E3 ligase for HMGCL using the UbiBrowser database (UbiBrowser.bio-it.cn/ubibrowser). The scores of the top 4 E3 ligases are indicated. (C) The interaction between exogenous HMGCL and NEDD4 was evaluated using immunoprecipitation. Details about the immunoprecipitation protocol are provided in the “Materials and Methods” section. (D) A GST pulldown assay was performed to examine the binding between endogenous HMGCL in 293T cells and the GST-NEDD4 fusion protein. Details about the GST pulldown protocol are provided in the “Materials and Methods” section. (E) The interaction between endogenous HMGCL and NEDD4 was evaluated using immunoprecipitation. H1299 and A549 cells were lysed with IP lysis buffer. After centrifugation, an anti-NEDD4 antibody was added to the supernatant for immunoprecipitation overnight. The binding of NEDD4 and HMGCL was examined by western blotting. (F) A ubiquitination assay was performed to examine the effects of NEDD4 on the ubiquitination of HMGCL. Details about the ubiquitination assay protocol are provided in the “Materials and Methods” section. 48 hours after transfection, the cells were incubated with MG132 for another 8 hours. Then, the cells were lysed with IP lysis buffer, and immunoprecipitation was performed with an anti-HMGCL antibody. (G) A ubiquitination assay was performed to determine the K48- or K63-linked ubiquitination of HMGCL by NEDD4. Wild-type, K48-only (linkage-specific ubiquitin) or K63-only (linkage-specific ubiquitin) ubiquitin expression vectors were cotransfected with the NEDD4 expression vector. Details about the ubiquitination assay are provided in the “Materials and Methods” section. (H) A ubiquitination assay was performed to examine the effects of NEDD4 on the ubiquitination of wild-type and mutant (S258A) HMGCL. Details about the ubiquitination assay protocol are provided in the “Materials and Methods” section. The cells were lysed with IP lysis buffer, and immunoprecipitation was performed with an anti-Flag antibody. (I) The levels of wild-type and mutant HMGCL protein upon treatment with TNFα were examined using western blotting. (J) The levels of HMGCL protein and p-IKKβ in 5 lung cancer tissues (C) and paired noncancerous tissues (N) were examined by western blotting.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Ubiquitin Proteomics, Western Blot, Positive Control, Immunoprecipitation, GST Pulldown Assay, Binding Assay, Lysis, Centrifugation, Transfection, Incubation, Expressing, Plasmid Preparation, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: Working model. The working model for the regulation of HMGCL by IKKβ and NEDD4. TNFα induced the activation of IKKβ, which phosphorylated HMGCL at Ser258 and promoted the degradation of HMGCL.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Activation Assay

Journal: International Journal of Biological Sciences

Article Title: Phosphorylation by IKKβ Promotes the Degradation of HMGCL via NEDD4 in Lung Cancer

doi: 10.7150/ijbs.82015

Figure Lengend Snippet: The correlation between the expression of HMGCL and clinical features of patients with lung cancer.

Article Snippet: The coding sequence (CDS) of wild-type HMGCL was synthesized by Sangon Biotech and inserted into the pLVX-IRES-puro vector.

Techniques: Expressing

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) mScarlet expression in the 6 indicated KOs, rescued with their respective V5-tagged protein-coding sequence (CDS), or with an empty vector. Each data point is an individual clone (n=3 clones per KO). (B) Hygromycin resistance after rescue. One representative clone is shown for each KO. (C) Dazl expression after rescue (n=3 rescued clones per KO). (D) Western blot analysis of DAZL and the indicated V5-tagged CDS. (E) RT-qPCR assays showing the expression changes of Dazl , differentiation ( Fgf5 ) and pluripotency ( Prdm14, Pou5f1 ) markers upon spontaneous differentiation after withdrawal of LIF. (F) MeDIP assay showing the relative levels of 5mC at the Dazl promoter for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (G) LUMA assay showing the changes in the global level of DNA methylation for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO). (H and I) LC-MS/MS assay showing the changes in the global level of 5mdC/C (H) and 5hmdC/C (I) for 6 candidates KO, compared to the parental DASH mES cells (n=3 clones per KO).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

Techniques: Expressing, Sequencing, Plasmid Preparation, Clone Assay, Western Blot, Quantitative RT-PCR, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, compared to the parental DASH mES cells (n=3 clones per KO). (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Spop KO, Mcm3ap KO, Kdm5c KO, Zbtb14 KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Spop KO, Mcm3ap KO, Kdm5c KO, and Zbtb14 KO, transfected with either an empty plasmid or their respective CDS, compared to the parental DASH mES cells (n=3, clones per KO). (D) Western blot analysis of V5, ZSCAN4, and MERVL-Gag in 4 candidates KO, transfected with an empty plasmid or their respective V5-tagged CDS. (E) Model for role of novel epigenetic factors ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D in the regulation of germline genes, and 2 cell-like cell state.

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

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

Journal: bioRxiv

Article Title: A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

doi: 10.1101/2021.05.03.442415

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression of 2C-specific genes in DASH mES cells cultured in 2i, Bend3 KO, and Kmt2d KO. (B) Western blot analysis of ZSCAN4 and MERVL-Gag for Bend3 KO, Kmt2d KO, and parental DASH mES cells cultured in Serum or 2i. (C) RT-qPCR analysis of the expression of 2C-specific genes for Bend3 KO (n=3, clones), transfected with an empty plasmid or their respective CDS, compared to the parental DASH mES cells. (D) Western blot analysis of ZSCAN4 and MERVL-Gag for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A). (E) RT-qPCR analysis of the expression of 2C-specific genes for Kdm5c KO clones, transfected with an empty plasmid, or a wild-type Kdm5c CDS, or a catalytically dead Kdm5c CDS (H514A).

Article Snippet: For rescue experiments, the coding sequence (CDS) of candidates was synthesized (for Bend3, Spop, and Zbtb14) (GenScript), amplified from cDNA ( Kdm5c ), or obtained from colleagues (for Mcm3ap , kindly shared by Prof. N. Sakaguchi, ).

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

Journal: JCI Insight

Article Title: Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15

doi: 10.1172/jci.insight.96560

Figure Lengend Snippet: (A) TRANSFAC analysis of the human rhodopsin promoter identifies transcription factors (TFs) predicted to bind the rhodopsin regulatory motif hRHO-cis (–88 to –58 from the transcription start site, TSS; Figure 2A and refs. 12, 13) including the TF KLF15 (orange arrow, minus strand). (B) Immunofluorescence analysis of Klf15 in C57BL6/J retina shows its absence in photoreceptors in the outer nuclear layer (ONL) and expression in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). Scale bar: 50 μm. (C) qPCR of mRNA (2–ΔCt) shows that Klf15 is not expressed in porcine rods. Porcine rods transduced with AAV8-hGNAT1-eGFP (1 × 1012 genome copies [gc]) and FACS sorted show lack of expression of Klf15. For comparison the retina-specific cone-rod homeobox (Crx) and rod-specific neural retina leucine zipper (Nrl) TFs are shown. (D) Gel mobility shift titrations of hKLF15 and artificial ZF6-DB TF with the hRHO 65-bp oligonucleotide. In the saturation-binding experiments the nanomolar concentration of specific binding data were plotted against nanomolar increasing concentration of DNA ligand. KLF15 and the synthetic TF ZF6-DB show similar binding affinity for the target sequence (12, 13). (E) qPCR ChIP analysis of the human rhodopsin TSS region, after the transfection of hKLF15 in HEK293 cells, shows enrichment of binding in the Rho promoter region compared with eGFP-transfected cells. Data are shown as the mean ± SEM. **P < 0.01 by 2-tailed Student’s t test. n = 3 independent experiments.

Article Snippet: The human KLF15 coding DNA sequence (CDS) and the murine KLF15 CDS were synthesized by Eurofins MWG.

Techniques: Immunofluorescence, Expressing, Transduction, Comparison, Mobility Shift, Binding Assay, Concentration Assay, Sequencing, Transfection

Journal: JCI Insight

Article Title: Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15

doi: 10.1172/jci.insight.96560

Figure Lengend Snippet: (A) Alignment of human, porcine, and murine rhodopsin (Rho) proximal promoter around the hRHO-cis. In red, the sequence recognized by KLF15 retrieved by TRANSFAC analysis (Figure 1A and Supplemental Table 1). (B) qPCR of mRNA levels (2–ΔΔCt) of adult porcine retina injected subretinally with AAV8-hGNAT1-hKLF15 (n = 6) or AAV8-hGNAT1-eGFP (n = 6) at a vector dose of 2 × 1010 genome copies (gc) 15 days after vector delivery shows significant repression of the Rho transcript; Gnat1, guanine nucleotide–binding protein 1; Arr3, arrestin 3. Data are shown as the mean ± SEM. ***P < 0.001; 2-tailed Student’s t test. (C) Western Blot analysis of porcine retinae injected with AAV8-hGNAT1-hKLF15 and AAV8-hGNAT1-eGFP shows the decrease in Rho protein consequent to KLF15 expression. (D) Rho (cyan) and KLF15 (red) immunofluorescence confocal analysis shows expression of hKLF15 in the outer nuclear layer (ONL) of injected retina (coinjected with AAV8-hGNAT1-eGFP, green) toward the nuclear interior of rod photoreceptor nuclei (euchromatin; see ref. 33), the collapse of the Rho-deprived outer-segment (OS), and partial retention of Rho in the cytoplasm. (E) Histological confocal immunofluorescence analysis of Gnat1 (red), which marks the soma of rods, confirmed rod-specific expression of hKLF15 upon transduction with AAV8-hGNAT1-hKLF15. Scale bars: 50 μm. (F) Venn diagrams showing pairwise intersection of differentially expressed genes (DEGs) between hKLF15 and ZF6-DB (12, 13). An adjusted P value (false discovery rate < 0.1), without filtering on fold change levels, resulted in 156 and 19 DEGs, in hKLF15- and ZF6-DB–treated retinae, respectively (12, 13). (G) Transcriptional activation and repression concordances among log fold changes of the genes in common between ZF6-DB and hKLF15.

Article Snippet: The human KLF15 coding DNA sequence (CDS) and the murine KLF15 CDS were synthesized by Eurofins MWG.

Techniques: Sequencing, Injection, Plasmid Preparation, Binding Assay, Western Blot, Expressing, Immunofluorescence, Transduction, Activation Assay

Journal: JCI Insight

Article Title: Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15

doi: 10.1172/jci.insight.96560

Figure Lengend Snippet: (A) Electroretinography (ERG) traces from a representative mouse injected with AAV carrying hKLF15, mKlf15, or eGFP measured at increasing luminances (cd⋅s/m2). (B) ERG analysis on P347S mice subretinally injected at postnatal day 14 (P14) with AAV8-hGNAT1-hKLF15 (n = 12), AAV8-hGNAT1-mKlf15 n = 9), AAV8-hGNAT1-eGFP (n = 14), or not injected (n = 6) and analyzed at P30. Retinal responses in both scotopic (dim light) and photopic (bright light) showed that both a- and b-wave amplitudes, evoked by increasing light intensities, were more preserved in hKLF15- and mKlf15-injected eyes compared with eGFP control eyes. (C) Immunofluorescence staining of P347S mouse retina, injected at P14 with AAV8-hGNAT1-hKLF15, AAV8-hGNAT1-mKlf15, or AAV8-hGNAT1-eGFP and analyzed at P30. hKLF15- and mKlf15-treated retinae show KLF15-positive expression toward the periphery of rod photoreceptor nuclei, an inverted pattern compared with pig (Figure 2D and ref. 33), and higher preservation of the outer nuclear layer (ONL) compared with eGFP controls. INL, inner nuclear layer. (D) qPCR of mRNA levels (2–ΔCt normalized to the mGnat1 gene) demonstrates that hKLF15 and mKLF15 downregulate human RHO-P347S expression without changing the endogenous wild-type murine rhodopsin transcript.

Article Snippet: The human KLF15 coding DNA sequence (CDS) and the murine KLF15 CDS were synthesized by Eurofins MWG.

Techniques: Injection, Control, Immunofluorescence, Staining, Expressing, Preserving