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

Journal: Acta Pharmaceutica Sinica. B

Article Title: Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

doi: 10.1016/j.apsb.2023.03.008

Figure Lengend Snippet: Direct interaction of COX1 with WIPI2. (A) Representative microscopy images of the colocalization of DsRed-WIPI2 and COX1 in AML12 cells. Scale bar = 10 μm. (B) Representative microscopy images of the colocalization of COX1 and WIPI2 using human healthy and NASH liver tissues. Scale bar = 20 μm. (C) Docking model of COX1 and WIPI2 using I-TASSER software. COX1 and WIPI2 are colored gray and green, respectively. Cartoons, sticks, and surface representations were used to profile the binding mode. (D) The co-IP assay was performed using cell lysates from HEK293T cells co-transfected with GFP-WIPI2 and COX1-FLAG plasmids. COX1 was immunoprecipitated from the transfected HEK293T cells using the FLAG antibody and the eluted proteins were subjected to Western blotting to detect WIPI2-GFP and COX1-FLAG. (E) The co-IP assay was performed using cell lysates from HEK293T cells co-transfected with COX1-HIS and WIPI2-FLAG plasmids. WIPI2 was immunoprecipitated from the transfected HEK293T cells using a FLAG antibody, and the eluted proteins were subjected to Western blotting to detect COX1-HIS and WIPI2-FLAG. (F) The co-IP assay was performed using cell lysates from HEK293T cells co-transfected with COX1-FLAG plasmids. COX1 was immunoprecipitated from the transfected HEK293T cells using a FLAG antibody, and the eluted proteins were subjected to Western blotting to detect COX1-FLAG and endogenous WIPI2. Rabbit IgG antibodies were used as negative controls in the Co-IP assay. Protein lysates (10%) were used as input. IP ratios have been normalized to input ratios to account for differences in input protein levels. (G) Surface plasmon resonance results of the binding of recombinant mouse WIPI2 to immobilized recombinant mouse COX1 protein. A gradient of WIPI2 protein was applied to quantify the binding affinity (62.5–1000 nmol/L). The K d (mol/L) value is 2.89 × 10 −6 . (H) The interaction of COX1 with WIPI2 was detected using MST, with 3500 nmol/L as the highest and 100 nmol/L as the lowest COX1 concentration. K d is 3.95 × 10 −8 mol/L.

Article Snippet: To generate AAV8-WIPI2, we used the sequence of mouse Wipi2 coding DNA sequence (CDS) from the National Center for Biotechnology Information (NCBI) (GenBank: NM_178398.4 ).

Techniques: Microscopy, Software, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, SPR Assay, Recombinant, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

doi: 10.1016/j.apsb.2023.03.008

Figure Lengend Snippet: Liver-specific restoration of WIPI2 reduced hepatic lipid accumulation and inflammation in Cox1 Δhepa mice under NASH condition. (A) Western blotting and corresponding quantification of LC3I/II and p62 proteins from the livers of Cox1 Δhepa mice injected with AAV8-GFP or AAV8-WIPI2 viruses ( n = 6–8). (B) Representative microscopic images and quantifications of liver sections stained with Oil Red O and F4/80 in Cox1 Δhepa mice injected with the AAV8-GFP or AAV8-WIPI2 viruses ( n = 6–7). Scale bar = 10 μm. (C) Hepatic TG levels in Cox1 f/f and Cox1 Δhepa mice injected with AAV8-GFP or AAV8-WIPI2 viruses ( n = 6). (D) mRNA levels of key genes of lipid metabolism ( Scd-1 , Ppar-γ , and Cd36 ) and inflammation ( Tnf-α , Il-6 , and Il-1β ) in the livers from Cox1 f/f and Cox1 Δhepa mice injected with the AAV8-GFP or AAV8-WIPI2 viruses ( n = 5–6/group). Values represent means ± SD. Statistical comparisons among groups were analyzed using a one-way ANOVA or Student's t test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. IHC, immunohistochemistry; CDAA, a choline-deficient, l -amino acid–defined, high-fat diet enriched with 1% cholesterol; TG, triglyceride; Scd1, stearoyl-coA desaturase-1; Ppar- γ , peroxisome proliferator-activated receptor gamma; Tnf- α , necrosis factor α ; Il-6, interleukin 6; Il-1 β , interleukin 1 β . The Western blotting bands of LC3I/II were cut and re-arranged because of the loading order. Original images with detailed labeling information can be found in Supporting Information.

Article Snippet: To generate AAV8-WIPI2, we used the sequence of mouse Wipi2 coding DNA sequence (CDS) from the National Center for Biotechnology Information (NCBI) (GenBank: NM_178398.4 ).

Techniques: Western Blot, Injection, Staining, Immunohistochemistry, Labeling