Journal: Journal of Hematology & Oncology

Article Title: The LINC00623/NAT10 signaling axis promotes pancreatic cancer progression by remodeling ac4C modification of mRNA

doi: 10.1186/s13045-022-01338-9

Figure Lengend Snippet: NAT10 functions as a downstream mediator of LINC00623by remodeling N4-acetylcytidine (ac4C) modification of mRNA. a Representative images of IHC staining with an anti-NAT10 antibody in PDAC, pancreatic intraepithelial neoplasia (PanIN) or matched adjacent normal pancreatic tissues (NP). b The mRNA level of LINC00623 was positively correlated with the protein level of NAT10 in PDAC tissues ( n = 93). c Kaplan‒Meier survival curve of two groups of patients with PDAC ( n = 93): NAT10 (+), patients with high NAT10 expression; NAT10 (-), patients with low NAT10 expression. The expression of NAT10 was determined by IHC staining. d Schematic diagram of the effects of NAT10 on the stability and translation efficiency of mRNA by catalyzing ac4C modification. e Metagene profile showing the distribution of NAT10 peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were used for the NAT10 RIP assay. f Metagene profile showing the distribution of ac4C peaks across full-length transcripts containing the 5′UTR, CDS, and 3′UTR. BxPC-3 cell lysates were also used for acRIP. g Venn diagram showing the downstream target genes regulated by NAT10 via ac4C modification in BxPC-3 cells. Group 1: The gene set enriched in NAT10 RIP-seq (RIP-seq); Group 2: The set of target genes enriched in parental cells but not in NAT10-silenced cells according to acRIP-seq (acRIP-seq); Group 3: The genes upregulated or downregulated in NAT10-silenced cells compared with control cells (RNA-seq); Group 4: The mRNA transcripts displaying differences in translation efficiency in NAT10-silenced cells (Ribo-seq). h Functional annotation and pathway enrichment analysis of the predicted downstream target genes of NAT10 according to the Metascape database. i The relative mRNA levels of NAT10, KCNN4, LAMB3 and PHGDH were measured by RT-qPCR. j The protein levels of KCNN4, LAMB3 and PHGDH were determined by Western blotting. k RT-qPCR was used to detect the relative enrichment of KCNN4 , LAMB3 and PHGDH mRNAs in NAT10 RIP products. l RT-qPCR was used to detect the relative enrichment of KCNN4, LAMB3 and PHGDH mRNAs in acRIP products. i – l BxPC-3 cells were transfected with NAT10 silencing and control plasmids. Cell lysates were harvested for RIP assays. IgG was used as the isotype control. The values indicate the mean ± SD of three independent experiments. P values are shown as * P < 0.05; ** P < 0.01; *** P < 0.001. Independent Student’s t test

Article Snippet: The sections were then incubated with specific primary antibodies against PCNA (1:200 dilution, ab15497, Abcam, Cambridge, UK), NAT10 (1:500 dilution, ab194297, Abcam), Vimentin (1:500 dilution, ab92547, Abcam), E-cadherin (1:500 dilution, 20874-1-AP, Proteintech), N-cadherin (1:100 dilution, ab76011, Abcam), LAMB3 (1:500 dilution, 26795-1-AP, Proteintech), PHGDH (1:500 dilution, 14719-1-AP, Proteintech) and KCNN4 (1:200 dilution, 23271-1-AP, Proteintech) at 4 °C overnight.

Techniques: Modification, Immunohistochemistry, Expressing, Control, RNA Sequencing, Functional Assay, Quantitative RT-PCR, Western Blot, Transfection

Journal: Molecular Therapy

Article Title: CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient

doi: 10.1016/j.ymthe.2018.07.024

Figure Lengend Snippet: CRISPR/Cas9-Mediated HDR into Intron 2 of LAMB3 Gene in Keratinocytes Derived from a JEB Patient

Article Snippet: PCR primers and 6-carboxyfluorescein (FAM) probes for GAPDH (Hs99999905_m1) or LAMB3 (Hs00989722_m1) were purchased from Applied Biosystems.

Techniques: CRISPR, Derivative Assay

Journal: Molecular Therapy

Article Title: CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient

doi: 10.1016/j.ymthe.2018.07.024

Figure Lengend Snippet: Restored LAMB3 Expression in Gene-Corrected Keratinocytes Selected by In Vitro Adhesion Strategy

Article Snippet: PCR primers and 6-carboxyfluorescein (FAM) probes for GAPDH (Hs99999905_m1) or LAMB3 (Hs00989722_m1) were purchased from Applied Biosystems.

Techniques: Expressing, In Vitro

Journal: Molecular Therapy

Article Title: CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient

doi: 10.1016/j.ymthe.2018.07.024

Figure Lengend Snippet: Molecular Characterization of LAMB3-Corrected Clones

Article Snippet: PCR primers and 6-carboxyfluorescein (FAM) probes for GAPDH (Hs99999905_m1) or LAMB3 (Hs00989722_m1) were purchased from Applied Biosystems.

Techniques: Clone Assay

Journal: Molecular Therapy

Article Title: CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient

doi: 10.1016/j.ymthe.2018.07.024

Figure Lengend Snippet: Phenotypic Analysis of LAMB3-Corrected Clones

Article Snippet: PCR primers and 6-carboxyfluorescein (FAM) probes for GAPDH (Hs99999905_m1) or LAMB3 (Hs00989722_m1) were purchased from Applied Biosystems.

Techniques: Clone Assay

Journal: Molecular Therapy

Article Title: CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient

doi: 10.1016/j.ymthe.2018.07.024

Figure Lengend Snippet: In Vivo Skin Regeneration from LAMB3-Corrected Clones

Article Snippet: PCR primers and 6-carboxyfluorescein (FAM) probes for GAPDH (Hs99999905_m1) or LAMB3 (Hs00989722_m1) were purchased from Applied Biosystems.

Techniques: In Vivo, Clone Assay

Journal: Stem Cell Reports

Article Title: Long-Term Stability and Safety of Transgenic Cultured Epidermal Stem Cells in Gene Therapy of Junctional Epidermolysis Bullosa

doi: 10.1016/j.stemcr.2013.11.001

Figure Lengend Snippet: Regeneration of a Functional Transgenic Epidermis (A and B) In situ hybridization with a vector-specific probe on 20-μm-thick skin sections shows the homogeneous expression of laminin 332-β3 transcripts in all epidermal layers (B, arrowheads). Sections from normal skin were used as a control (A). Dotted lines indicate the basal lamina. Asterisks mark the stratum corneum. Scale bars, 10 μm. (C–F) Light microscopy of 0.5 μm sections from a skin biopsy of the upper leg of a healthy donor (C and E) and Claudio (D and F) were stained with toluidine blue. In both cases, normal-looking epidermis (Ep) and dermis with well-organized collagen bundles (c) are evident. Asterisks mark the stratum corneum, which is thicker in the regenerated epidermis. Scale bars, 10 μm. (G and H) Transmission electron microscopy of 70 nm skin sections shows that basement membranes (arrowheads) and hemidesmosomes (arrows) are clearly evident in both control (G) and transgenic (H) skin. Scale bars, 1 μm.

Article Snippet: IF was performed on 7 μm skin sections as previously described ( ) using laminin 332-β3 6F12 monoclonal antibody (mAb; Acris Antibodies), 332-γ2 D4B5 mAb (Chemicon), 332-α3 BM165 mAb (a gift from Patricia Rousselle, IBCP), 332-α6 450-30A mAb and 332-β4 450-9D mAb (Thermo Fisher Scientific), rabbit purified anti-p63α immunoglobulin G (IgG; PRIMM) , K10 and K14 guinea pig antisera (Progen), K9 sc-58743 mAb (Santa Cruz Biotechnology), elastin MAB2503 mAb (Millipore), and human involucrin mAb (Leica Biosystems).

Techniques: Functional Assay, Transgenic Assay, In Situ Hybridization, Plasmid Preparation, Expressing, Light Microscopy, Staining, Transmission Assay, Electron Microscopy

Journal: Stem Cell Reports

Article Title: Long-Term Stability and Safety of Transgenic Cultured Epidermal Stem Cells in Gene Therapy of Junctional Epidermolysis Bullosa

doi: 10.1016/j.stemcr.2013.11.001

Figure Lengend Snippet: Expression of LAM332 and α6β4 Integrins (A–J) IF analysis of laminin 332-β3 (A and B), 332-γ2 (C and D) 332-α3 (E and F), α6 integrin (G and H), and β4 integrin (I and J) in control (WT) and transgenic (Claudio) skin sections. The transgenic epidermis expresses normal amounts of laminin 332 and α6β4 integrins properly located at the epidermal-dermal junction. Scale bars, 40 μm.

Article Snippet: IF was performed on 7 μm skin sections as previously described ( ) using laminin 332-β3 6F12 monoclonal antibody (mAb; Acris Antibodies), 332-γ2 D4B5 mAb (Chemicon), 332-α3 BM165 mAb (a gift from Patricia Rousselle, IBCP), 332-α6 450-30A mAb and 332-β4 450-9D mAb (Thermo Fisher Scientific), rabbit purified anti-p63α immunoglobulin G (IgG; PRIMM) , K10 and K14 guinea pig antisera (Progen), K9 sc-58743 mAb (Santa Cruz Biotechnology), elastin MAB2503 mAb (Millipore), and human involucrin mAb (Leica Biosystems).

Techniques: Expressing, Transgenic Assay

Journal: International journal of molecular sciences

Article Title: TNF Induces Laminin-332-Encoding Genes in Endothelial Cells and Laminin-332 Promotes an Atherogenic Endothelial Phenotype.

doi: 10.3390/ijms25168699

Figure Lengend Snippet: Figure 10. LN332-encoding genes’ transcripts are elevated and correlate with TNF in human carotid atherosclerotic lesions. Gene expression of LAMA3 (a), LAMB3 (b), and LAMC2 (c) in human carotid atherosclerotic tissues and adjacent macroscopically intact tissues (n = 32). Pearson’s correlation of LAMA3 (d), LAMB3 (e), and LAMC2 (f) with TNF in human carotid atherosclerotic lesions (n = 32). Solid line indicates Pearson’s correlation coefficient (r), and dashed line indicates 95% confidence band of the best-fit line. Data are acquired from human carotid atheroma gene expression (accession number, GSE43292). p-value smaller than 0.05 is considered statistically significant.

Article Snippet: AMAb91123, Atlas antibodies, Bromma, Sweden) Anti-LAMB3 (1:5000, catnr.

Techniques: Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-455-5p suppresses PDZK1IP1 to promote the motility of oral squamous cell carcinoma and accelerate clinical cancer invasion by regulating partial epithelial-to-mesenchymal transition

doi: 10.1186/s13046-023-02597-1

Figure Lengend Snippet: MiR-455-5p and PDZK1IP1 regulate collective migration and partial EMT in OSCC cells. A Upper panel: HSC-3 control cells cultured in 2.5D culture medium. Lower panel: HSC-3 cells transfected with pLe-miR-455-5p in 2.5D culture medium. B Upper panel: SAS control cells exhibited higher migrative ability and collective cell movement in 2.5D cell culture. Lower panel: SAS cells transfected with PDZK1IP1 overexpressed plasmid demonstrated low migrative ability and loss collective pattern of cell movement. (Red circle indicated tumor cluster at time point of 0-h, blue circle indicated tumor cluster at 8 h later and green circle indicated tumor cluster at 17 h later). C GSEA software analysis pointed out KEGG_FOCAL_ADHESIN and KEGG_ECM_RECEPTOR_INTERACTION gene sets. D RNA sequencing dataset from SAS cells with PDZK1IP1 overexpression in 2.5D collagen-coated culture system was evaluated by online tool: metascape. Bar graph demonstrated GO enrichment analysis. E LAMB3, PDPN, LAMC2 or PDZK1IP1 mRNA expression of OEC-M1 cells transfected with pLe-miR-455-5p (or controls) or shMAP17 (or controls) in 2D culture system. F LAMB3 , PDPN , LAMC2 or PDZK1IP1 mRNA expression of OEC-M1 cells transfected with shMAP17 or control in 2.5D collagen-coated culture system. G LAMB3, PDPN, LAMC2 or PDZK1IP1 mRNA expression of SAS cells transfected with MAP17 overexpression plasmids or control in 2.5D collagen-coated culture system. H–M Correlations between PDZK1IP1 expression and VIM ( H ), SNAI2 ( I ), PDPN ( J ) , partial EMT score ( K ) , SPRR1B ( L ) and S100A9 ( M ) expression in the NKCU-OrCA-40TN data set. N . PDZK1IP1 expression in patients with and without risk factors for recurrence (perineural invasion, lymphovascular invasion, and extracapsular extension) in the NKCU-OrCA-40TN data set. O Partial EMT scores of patients with and without risk factors for recurrence in the NKCU-OrCA-40TN data set. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test. Correlation p value was determined through Pearson correlation analysis

Article Snippet: In addition, in the 2.5D collagen-coated culture system, the OEC-M1 cells with PDZK1IP1 knockdown had higher LAMB3 , PDPN and LAMC2 mRNA expression levels (Fig. F), and the LAMB3 , LAMC2 mRNA expression levels of the SAS cells with PDKZI1P1 overexpression were decreased (Fig. G).

Techniques: Migration, Control, Cell Culture, Transfection, Plasmid Preparation, Software, RNA Sequencing, Over Expression, Expressing

Journal: Nature Communications

Article Title: Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells

doi: 10.1038/s41467-021-22779-9

Figure Lengend Snippet: a Immunofluorescence analysis of FOXM1 expression in 7-μm-thick skin sections prepared from normal skin (WT) and LAMB3-deficient JEB patient (JEB-1). DAPI (blue) stains nuclei. Dotted line marks the epidermal–dermal junction. Scale bars, 20 μm. Representative image of what observed in skin biopsy derived from five independent healthy donors and five sections randomly taken from JEB-1 skin biopsy. b Western analysis of total cell extracts from normal keratinocytes (WT), JEB, LAMB3-transduced, JEB cultures immunostained with indicated antibodies (representative images of n = 6). c Western analysis of total cell extracts from normal keratinocytes (WT), JEB, YAP-transduced, JEB cultures immunostained with indicated antibodies (representative images of n = 3). d Western analysis of total cell extracts from normal keratinocytes (WT), JEB, FOXM1-transduced, JEB cultures immunostained with indicated antibodies. Endogenous FOXM1 is not visible in WT extract due to the presence of high levels of enforced FOXM1 in FOXM1-transduced cells (representative images of n = 4). e Representative images of FOXM1 localization (green) and Pan-Keratin (red) in JEB, LAMB3-, and YAP-transduced JEB. DAPI (blue) stains nuclei. Scale bar 20 μm (representative images of n = 3). f Representative images of YAP localization (green) in normal (WT), JEB, LAMB3-, and FOXM1-transduced JEB. Colonies are indicated with white dotted lines. Scale bar 20 μm (representative images of n = 4).

Article Snippet: Accordingly with IHC guidelines, Molmed S.p.A, Milan, Italy produced a GMP-grad master cell bank of a high-titer packaging clone (GP+envAm12-LAMB3 cells), The packaging cells ere cultured in DMEM supplemented with10% irradiated fetal bovine serum, penicillin/streptomycin (50 IU/ml), glutamine (2 mM), and subjected to quality and safety tests under GMP conditions.

Techniques: Immunofluorescence, Expressing, Derivative Assay, Western Blot

Journal: Nature Communications

Article Title: Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells

doi: 10.1038/s41467-021-22779-9

Figure Lengend Snippet: a Calculation of cumulative cell doublings in JEB, LAMB3-corrected, YAP-transduced, FOXM1-transduced JEB cultures derived from one representative experiment. Number of cell doublings was calculated as described in “Methods”. b CFE of keratinocyte cultures at passage 5, 7, 9, and 15 initiated from the control JEB patient and after LAMB3, YAP, or FOXM1 gene addiction. The number of cells per dish plated in the CFE is indicated between brackets and colonies were stained with Rhodamine B 12 days later. c Clonal analysis of normal keratinocytes (WT), untransduced, LAMB3-corrected, and FOXM1-transduced clonogenic JEB (see “Methods”). The percentage of holoclones, meroclones, and paraclones is indicated in red, light blue, and gray columns, respectively ( n = 157 clones analyzed). d Top: adhesion of confluent cultured epidermal sheets prepared from normal keratinocytes (WT), JEB, LAMB3-corrected, YAP-transduced, FOXM1-transduced JEB29 cultures. The mere transportation of the flask from the incubator to the hood caused the spontaneous detachment of JEB and JEB-YAP and JEB-FOXM1 cultures, while WT and LAMB3-corrected keratinocytes remained firmly attached to the substrate. Bottom: CFE performed on the above cultures. The CFE was plated at a density of 4000 cells per dish, and colonies were stained with Rhodamine B 12 days later.

Article Snippet: Accordingly with IHC guidelines, Molmed S.p.A, Milan, Italy produced a GMP-grad master cell bank of a high-titer packaging clone (GP+envAm12-LAMB3 cells), The packaging cells ere cultured in DMEM supplemented with10% irradiated fetal bovine serum, penicillin/streptomycin (50 IU/ml), glutamine (2 mM), and subjected to quality and safety tests under GMP conditions.

Techniques: Derivative Assay, Staining, Clone Assay, Cell Culture