Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 1. iPSC differentiation towards SMCs. (A) iPSCs were seeded on collagen IV and cultured in DM+P to induce SMC differentiation. Morphology of iPSCs (top left panel) and their SMC differentiated counterparts (top right panel) are shown in bright-field microscopy images, with immunofluorescence staining showing the SMC markers α-SMA (bottom left panel) and calponin (bottom right panel). Scale bar: 50 µm. (B) The SMC marker SMA was induced in a time-dependent manner during SMC differentiation from miPSCs in parallel with the RBP QKI-6 at the mRNA level. (C) Time point protein analysis shows induction of the SMC markers calponin and SM22 in parallel with QKI-6 during miPS-SMC differentiation. The EC marker CD144 and QKI-5, are not induced. (D) Immunofluorescence staining of day 6 miPS-SMCs reveals QKI-6 staining throughout the culture while no CD144 or vWF expression could be observed. Scale bar: 50 µm. (E) Immunofluorescence staining shows co-expression of QKI-6 and SMC markers SM22 and calponin in day 6 miPS-SMCs. Scale bar: 50 µm. (F) qRT-PCR shows that supplemental PDGF-BB is capable of significantly inducing QKI-6 expression at the mRNA level. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Cell Culture, Microscopy, Immunofluorescence, Staining, Marker, Expressing, Quantitative RT-PCR

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 6. QKI-6-derived SMCs exhibit greater contractile ability and, on combination with QKI5-derived ECs, have an increased angiogenic potential in vitro and in vivo. (A) Day 3 miPS-SMCs were transfected with Ex-QKI6 or Ex-mCherry and, after 48 h, were stimulated with KCl. Live imaging was carried out from time of stimulation for 15 min. Transfected cell size was measured at time zero and after 15 min. The percentage change in cell size was calculated. QKI-6-overexpressing cells showed a significantly greater decrease in cell size, indicating their contraction in comparison to mCherry controls. (B) Day 3 miPS-ECs and SMCs were transduced to overexpress Ex-mCherry for both cell types, Ex-QKI-5 for ECs or Ex-QKI-6 for SMCs. After 48 h, cells were subjected to an in vitro Matrigel assay. Combined QKI-5 miPS-ECs and QKI-6 miPS-SMCs produced longer vessels as shown by an increase in the total master segment length (top right panel) and significantly increased levels of vessel branching (bottomright panel). Representative images of mCherry- and QKI-derived vascularcells are shown on the left. Scale bars: 75 µm. (C,D) Cells subjected to the same treatments as in B were also used for an in vivo Matrigel assay. Cells were mixed with Matrigel and injected subcutaneously. The total number of vessel-like structures were quantified after harvest 1 week later. Vessel numbers were calculated per image and averaged for each plug. Plugs containing mCherry- and QKI-induced vascular cells showed significantly increased vessel numbers versus PBS control and there was a significantly increased vessel number in QKI-derived cell plugs versus mCherry-derived cell plugs. Scale bars: 25 µm. (E) In vivo Matrigel plugs were subjected to immunohistochemistry to show direct incorporation of injected cells into the vasculature. Injected cells were labelled with Vybrant (red) and were seen to colocalise with CD144 and SM22 staining within the QKI-derived plugs. Scale bars: 75 µm. Data are mean±s.e.m. (n=3) *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Derivative Assay, In Vitro, In Vivo, Transfection, Imaging, Comparison, Matrigel Assay, Produced, Injection, Control, Immunohistochemistry, Staining

Journal: Materials Today Bio

Article Title: Modulation of endothelial-to-mesenchymal transition via NRP-1 targeting with melittin attenuates pulmonary fibrosis

doi: 10.1016/j.mtbio.2025.102659

Figure Lengend Snippet: MLT attenuates TGF-β1-induced EndMT by inhibiting the TGF-β/Smad and MAPK pathways in HUVECs. (A–D) The mRNA levels of endothelial (CD31, VE-cadherin) and mesenchymal (α-SMA, vimentin) markers were determined by RT-qPCR. (E) EndMT-related protein expression of CD31, VE-cadherin, α-SMA, vimentin, Twist, and Snail was examined by western blot. The original western blot images are provided in Quantitative analysis of CD31, VE-cadherin, α-SMA, vimentin, Twist, and Snail protein levels (relative to β-actin). (L) Immunofluorescence staining of EndMT markers in HUVECs. Scale bar: 50 μm; Blue: DAPI; Green: CD31; Magenta: VE-cadherin; Red: α-SMA; Orange: vimentin. (M) Nuclear transcription factor immunofluorescence during EndMT in HUVECs. Scale bar: 50 μm; Blue: DAPI; Green: Twist; Red: Snail. (N) Protein levels of key components in the TGF-β/Smad and MAPK pathways were assessed by western blot. The original western blot images are provided in Quantitative analysis of Smad4/β-actin, p-Smad2/3/Smad2/3, p-ERK/ERK, p-JNK/JNK and p-P38/P38 protein levels. Data are presented as the mean ± SEM (n = 3); ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Antibodies: VE-cadherin, β-actin and HRP-labeled antibodies were purchased from Boster Company.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Materials Today Bio

Article Title: Modulation of endothelial-to-mesenchymal transition via NRP-1 targeting with melittin attenuates pulmonary fibrosis

doi: 10.1016/j.mtbio.2025.102659

Figure Lengend Snippet: Nanoparticle delivery of MLT potentiates anti-PF efficacy. (A) Experimental timeline of BLM-induced PF and therapeutic intervention. Mice received intratracheal BLM on day 0, followed by intraperitoneal administration of pLNPs, MLT-L (0.5 mg/kg), MLT-H (2 mg/kg), M-pLNPs (0.5 mg/kg as MLT) every other day from day 7 to day 21. (B) Representative macroscopic morphological images of lung tissues from each treatment group. (C) Histopathological analyses of lung tissues. Scale bar: 20 μm. (D) Quantitative analysis of fibrosis severity by Ashcroft scoring. (E) Quantitative assessment of Masson staining. (F) Representative Micro-CT images of lung tissue at three anatomical planes: sagittal, coronal, and transverse, demonstrating the spatial distribution of fibrotic lesions. Scale bar: 15 mm. (G) Immunofluorescence staining of key therapeutic targets in lung tissues. Scale bar: 50 μm. Blue: DAPI; Red: CD31; Yellow: VE-cadherin; Magenta: α-SMA; Green: COL-I; Cyan: Vimentin. (H) Immunofluorescence staining images showing the expression and localization of nuclear transcription factors Twist and Snail in lung tissues. Scale bar: 50 μm. Blue: DAPI; Red: Twist; Magenta: Snail. (I–N) Quantitative analysis of pulmonary function parameters including respiratory system resistance (Rrs) (I), elastance (Ers) (J), compliance (Crs) (K), forced vital capacity (FVC) (L) and forced expiratory volume at 0.2 s (FEV0.2) (M), and static compliance (Cst) (N). Data are presented as the mean ± SEM (n = 5); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.

Article Snippet: Antibodies: VE-cadherin, β-actin and HRP-labeled antibodies were purchased from Boster Company.

Techniques: Staining, Micro-CT, Immunofluorescence, Biomarker Discovery, Expressing

Journal: Frontiers in Pharmacology

Article Title: Positive Effect of Andrographolide Induced Autophagy on Random-Pattern Skin Flaps Survival

doi: 10.3389/fphar.2021.653035

Figure Lengend Snippet: The upregulation of angiogenesis in the skin flaps via Andro (A,C) IHC of Cadherin5 and VEGF in both groups of ischemic skin flaps (original magnification, ×200; scan bar, 50 μm). (B,D) The total absorbance of Cadherin5 and VEGF in IHC. (E) The results of immunoblotting i.e., the expressions of cadherin 5, MMP9, VEGF in the control as well as Andro groups. (F–H) The quantification of Cadherin5, MMP9, and VEGF expressions in the flaps by measuring their optical densities. The obtained data were presented as means ± SEM. Significance: * p -value < 0.05 and ** p -value < 0.01, vs. control group (n = 6 per group).

Article Snippet: Diaminobenzidine (DAB) developer, pentobarbital sodium, and the H&E Kit were procured from Solarbio Science and Technology (Beijing, China), while Cdh5-Rabbit Monoclonal Antibodies were provided via Boster Biological Technology, China.

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Positive Effect of Andrographolide Induced Autophagy on Random-Pattern Skin Flaps Survival

doi: 10.3389/fphar.2021.653035

Figure Lengend Snippet: 3MA reversed the effects of Andro on random-pattern skin flap viability (A) The immunofluorescence data of LC3II in the ischemic skin flap (scan bar, 50 μm). (B) T he histogram representing the percentage of the LC3II-positive cells. (C,E) The immunoblotting of the autophagy associated proteins VPS34, p62, LC3II, Beclin 1, and CTSD; angiogenesis associated proteins VEGF, cadherin5, and MMP9; the oxidative stress-related proteins i.e., SOD1, HO1, and eNOS and the apoptosis-associated proteins i.e., Bax, CYC, and CASP3. (D,F) The quantification of autophagy-, angiogenesis, and oxidative stress-related proteins by measuring their optical densities, as previously mentioned. The obtained data were presented as means ± SEM. Significance: * p -value < 0.05 and ** p -value < 0.01, vs. control group (n = 6 per group).

Article Snippet: Diaminobenzidine (DAB) developer, pentobarbital sodium, and the H&E Kit were procured from Solarbio Science and Technology (Beijing, China), while Cdh5-Rabbit Monoclonal Antibodies were provided via Boster Biological Technology, China.

Techniques: Immunofluorescence, Western Blot, Control

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet: Establishment of PCBP1 KO cells (A) Schematic diagram of the selection of PCBP1 KO monoclonal clones and experimental design at the cellular level. (B) A targeting diagram of representative sgRNAs on the PCBP1 locus. The red bases indicate the PAM sequence. (C) The cleavage efficiency of sg95 and sg97. The pools of PK-15 cells separately transfected with PX330-sg95/sg97 were amplified using specific primers, followed by Sanger sequencing. The nested peaks in the chromatogram near the cleavage site of the Cas9 endonuclease indicated the efficiency of the sgRNAs. The red arrow indicates the cleavage site of the Cas9 protein. The bases in the purple rectangle are PAM sequences. The bases in the orange rectangle are crRNA sequences. (D) Selection of positive PCBP1 KO clones. The targeting regions on the PCBP1 loci of different clones were amplified, and then the amplicons were cut by T7 endonuclease. The black arrow indicates the band size of the amplicons. Red asterisks indicate the fragments cut by T7 endonuclease. c15, c16, c24, c25, c27, and c28 were selected from the cell pool transfected with PX330-sg97 and are indicated in blue font. c40, c41, c42, c45, and c46 were selected from the cell pool transfected with PX330-sg95 and indicated in red font. The expected sizes of both cleavage fragments in c15, c16, c24, c25, and c27 were approximately 170 and 260 bp, respectively. The expected sizes of both cleavage fragments in c40, c41, c42, c45, and c46 were approximately 150 and 285 bp, respectively. (E) Representative image of PCBP1 protein levels in c15, c27, and c40. The total-cell lysates of c15, c27, c40, and WT cells were separated using SDS‒PAGE, followed by immunoblotting with anti-PCBP1 antibody. GAPDH was used as an internal control. M, protein marker (10–180 kDa). (F) Gray intensity analysis of PCBP1 using Fiji software. The PCBP1 band intensity was normalized to that of GAPDH in the same sample. Every sample was measured three times by Fiji. Bars are presented as the mean ± SEM, and data were analyzed using Student’s t- test using GraphPad Prism 8.0. ∗∗∗∗ p < 0.0001; ns, no significance; n = 3.

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Selection, Clone Assay, Sequencing, Transfection, Amplification, Western Blot, Control, Marker, Software

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet: Viral proliferation kinetics in PCBP1-deficient/KO PK-15 cells (A) The replication kinetics of CSFV in PCBP1 KO/deficient PK-15 cells. WT, c15, and c40 cell cultures were infected with CSFV (MOI = 0.1). At the indicated time after infection, samples were collected, and RT‒qPCR was performed to detect the CSFV genome copy number. (B) Representative images from immunofluorescence assays (IFA) detecting CSFV in CSFV-infected WT，C15, and C40 cells. Cells were seeded in a 24-well plate. After attachment, cell cultures were infected with CSFV (MOI = 1) for 48 h, and then the monolayer was fixed for staining with E2-specific antibody (PAb). Scale bar, 50 μm. (C) The extracellular CSFV particle copy number in WT, c15, and c40 cells at various hpi. WT, c15, and c40 cells were infected with CSFV (MOI = 1). At the indicated time after infection, the culture medium was collected and subjected to RT‒qPCR. (D) CSFV genome copy numbers in PCBP1-overexpressing WT, c15, and c40 cells. WT, c15, and c40 cells were transfected with pcDNA3.1-HA-PCBP1-His using polyethyleneimine (PEI). At 48 h post-transfection, cell cultures were infected with CSFV (MOI = 1) for 48 h, and then the monolayer was collected for RT‒qPCR. (E) The replication kinetics of PEDV in PCBP1 KO/deficient PK-15 cells. WT, c15, and c40 cell cultures were infected with PEDV (MOI = 0.01). At the indicated time after infection, samples were collected and RT‒qPCR was performed to detect the PEDV genome copy number. (F) The replication kinetics of PRV in PCBP1 KO/deficient PK-15 cells. WT, c15, and c40 cell cultures were infected with PRV (MOI = 0.01). At the indicated time after infection, samples were collected, and RT‒qPCR was performed to detect the CSFV genome copy number. Bars are presented as the mean ± SEM, and data were analyzed using Student’s t- test using GraphPad Prism 8.0. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no significance; n = 3.

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Infection, Immunofluorescence, Staining, Transfection

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet: The effect of PCBP1 deficiency on the CSFV life cycle and IFN pathway (A) Cells were infected with CSFV at a MOI of 10 at 4°C for 1 h and then shifted to 37°C for 0 (binding), 1 (entry), and 24 h (replication). Samples were collected and lysed to determine the number of CSFV RNA units using RT‒qPCR. (B) The relative mRNA fold change of IFN pathway genes and downstream effectors in PCBP1 KO cells. (C) The relative mRNA fold change in IFN pathway genes and downstream effectors in CSFV-infected PCBP1 KO cells compared with cells infected with PEDV-infected at 48 hpi. (D) The predicted interactors of PCBP1. The thickness of the gray line represents combined scores. (E) The relative mRNA fold change of predicted genes in PCBP1 KO cells. (F) The relative mRNA fold change in predicted genes in CSFV-infected PCBP1 KO cells compared with cells infected with PEDV at 48 hpi. Bars are the mean ± SEM, and the data were analyzed using Student’s t- test using GraphPad Prism 8.0. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no significance; n = 3.

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Infection, Binding Assay

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet: Production of PCBP1-deficient pigs (A) Schematic diagram of the production of PCBP1-deficient pigs using somatic cell nuclear transfer (SCNT) and the experimental design of ex vivo viral challenge. (B) Cell viability of PCBP1-deficient PFFs. PCBP1-deficient PFFs and WT cells were seeded in 96-well plates. After attachment, CCK8 working solution was added to the culture medium. The absorbance at 450 nm was detected after incubation for 2 h. (C) Photograph of the F 0 generation of PCBP1-deficient piglets. (D) T-cloning and Sanger sequencing of the PCBP1 alleles of F 0 piglets. The targeting region on the PCBP1 locus was amplified, and then the amplicons were ligated into T vectors. Seven colonies were selected for sequencing. PAM sites are highlighted in red. Indels are shown in yellow. #590, #592, #594, and #596 were the codes used for the individual litters of the surrogate. +/+, WT pig; −/+, heterozygous PCBP1-deficient pigs. Bars show the mean ± SEM and the data were analyzed using Student’s t- test using GraphPad Prism 8.0. ns, no significance; n = 3.

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Ex Vivo, Incubation, Cloning, Sequencing, Amplification

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet: Growth of CSFV in primary fibroblasts isolated from PCBP1-deficient pigs (A) Representative IFA images of CSFV in CSFV-infected fibroblasts. Primary fibroblasts isolated from the WT and F 0 - and F 1 -generation were seeded in a 24-well plate. After attachment, cell cultures were infected with CSFV (MOI = 1) for 48 h. The monolayer was fixed and probed with E2-specific antibody (PAb), and the supernatants were collected to determine the CSFV copy number using RT‒qPCR. Scale bar, 50 μm. (B) Intracellular CSFV particles in primary fibroblasts isolated from the F 0 - and F 1 -generation PCBP1-deficient pigs. The isolated primary fibroblasts were seeded in a siz-well plate. The cell cultures were infected with CSFV (MOI = 1) for 48 h. The cell pellets were collected to determine the intracellular CSFV particle number. (C) Extracellular CSFV particles in primary fibroblasts isolated from the F 0 - and F 1 -generation PCBP1-deficient pigs. Primary cells isolated from pig tails were seeded in a 24-well plate. The cell cultures were infected with CSFV (MOI = 1) for 48 h, and the supernatants were collected to determine the extracellular CSFV copy number. Bars show the mean ± SEM, and the data were analyzed using Student’s t- test using GraphPad Prism 8.0. ∗ p < 0.05; ∗∗ p < 0.01; n = 3.

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Isolation, Infection

Journal: iScience

Article Title: Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing

doi: 10.1016/j.isci.2022.105268

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-PCBP1 antibody , BOSTER , Cat# A02636-1.

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, Lysis, Western Blot, Protease Inhibitor, CCK-8 Assay, Sequencing, Software