293 dual tm null cells  (InvivoGen)

Journal: Cell reports

Article Title: Immune gene diversity and STING1 variants in shaping cancer immunity across different genetic ancestry populations

doi: 10.1016/j.celrep.2025.116882

Figure Lengend Snippet: (A) The K-means clustering algorithm was used to group the cancer cell lines based on their similarity of IFN-β and ISG expression in response to cGAMP treatment (10 μg/mL, 30 min). The STING1 haplotypes of each cell line are indicated using color coding. (B) Heatmap plot illustrates the changes in expression levels of IFN-β and ISGs following cGAMP treatment. The K-means group and STING1 haplotypes of each cancer cell line are represented by color codes at the top of the heatmap. (C) Illustration of the dual gRNA/CRISPR strategy used to knock out an entire STING1 gene allele in the STING1 heterozygous cancer cell lines. (D) rhPCR-based allelic discrimination was used for genotyping the STING1 gene in the collected clones. The normalized reporter signals (Rn) for allele 1 (FAM) and allele 2 (VIC) were plotted on the x and y axes, respectively. The allelic discrimination plot shows three distinct genotype clusters, including individuals homozygous (depicted in red) and heterozygous (shown in green) for the reference allele and those homozygous for the alternate allele (represented in blue). The homogeneous HAQ/HAQ line HARA and WT/WT line H1373 served as genotyping controls. (E) Western blot analysis of p-TBK1 and p-STAT1 was conducted in isogenic SKOV3 cells carrying either WT or HAQ STING1 , following treatment with 10 μg/mL cGAMP. (F) Bubble plot illustrates the results of gene set enrichment analysis (GSEA) for the upregulated genes identified by RNA-seq following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . The IFN gene sets were obtained from Reactome, and the transcription-factor-regulated gene sets were sourced from TRANSFAC. The bubble size corresponds to the −log (adjusted p value), and the color intensity indicates the number of genes within each gene set. (G) Violin plot shows the fold changes of the commonly upregulated genes identified by RNA-seq after treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . (H) The fold changes in expression levels of IFN-β and ISGs, as measured by RT-qPCR, following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 and TOV21G cells carrying WT/HAQ, WT, or HAQ STING1 . (I) The numbers of T cells (Jurkat-CXCR3, top) and NK cells (NK92, bottom) that migrated to the lower compartment, where isogenic SKOV3 cells were pretreated with 5 μg/mL poly(dA:dT) for 4 h. (J) WT or HAQ STING1 cDNA was transduced into SKOV3 or TOV21G cells in which the endogenous STING1 had been knocked out using CRISPR. The expression of STING1 protein was subsequently detected through western blots. (K) The levels of IFN-β expression measured by ELISA following treatment with 10 μg/mL cGAMP for 30 min in SKOV3 and TOV21G cells transduced with either WT or HAQ STING1 . (L) WT or HAQ STING1 cDNA was transduced into HEK293 reporter cells, which lacked endogenous STING1 expression and contained dual reporters. The expression of STING1 protein was subsequently detected through western blots. (M) The activities of ISRE and hIFN-β reporters after treatment with 10 μg/mL cGAMP for 30 min in 293-Dual Null reporter transduced with either WT or HAQ STING1 . Data are represented as means ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: 293-Dual TM Null Cells , InvivoGen , Cat #293d-null.

Techniques: Expressing, CRISPR, Knock-Out, RNase H-dependent PCR, Clone Assay, Western Blot, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transduction

Journal: bioRxiv

Article Title: ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction

doi: 10.1101/2024.09.12.612744

Figure Lengend Snippet: A ) Size exclusion chromatography results of WT and PH1 variant channels purified from HEK cells. Peaks indicate the relative abundance of each channel as oligomers (arrow) or protomers (asterisk). B ) GlowMelt (Biotinum) thermal stability measurement of polycystin protein unfolding. First derivative (slope) of the fluorescence curve (dF/dT) for each channel protein is plotted as a function of temperature (T). Error bars = S.D., N=3 replicates for each channel type. C ) Global resolution maps of PKD2 F629S and R638C structures determined by cryo-EM. Note, pore domain resolution is between 2.8-3.1Å and 2-2.4Å for the F629S and R638C structures, respectively. D ) Overall structure of the variant channels. Left , transmembrane view and structural alignment of the F629S and R638C channels, highlighting the domains within a single channel subunit. Right , external and internal views of the channels demonstrating the domain swapped assembly of the channel.

Article Snippet: HEK 293 PKD2 null cells were electro-transfected with PKD1 sgRNAs (caccGCATAGGTGTGGTTGGCAGC and aaacGCTGCCAACCACACCTATGC) with the All-in-one Cas9 plasmid (Addgene).

Techniques: Size-exclusion Chromatography, Variant Assay, Purification, Fluorescence, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction

doi: 10.1101/2024.09.12.612744

Figure Lengend Snippet: A ) Structural alignment of the variant and WT channels (PDB: 5T4D, Cao et al 2016). Inset, expanded views the pore domains showing the location of the ADPKD-associated variants highlighting the missing pore helix chemical interactions. B ) HOLE analysis (Smart et al 1996) of the PKD2 pore domains highlighting the changes in the internal and external gates. C ) Analysis of the polycystic pore radii plotted along their length. Note the elongated pore restriction at the internal gates (double pointed arrow) of the F629S and R638C compared to the WT channels.

Article Snippet: HEK 293 PKD2 null cells were electro-transfected with PKD1 sgRNAs (caccGCATAGGTGTGGTTGGCAGC and aaacGCTGCCAACCACACCTATGC) with the All-in-one Cas9 plasmid (Addgene).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction

doi: 10.1101/2024.09.12.612744

Figure Lengend Snippet: A ) Super resolution SIM (structured illumination microscopy, Scale bar = 3 μm) images of HEK PKD1 Null :PKD2 Null cells stably expressing ARL13B-GFP (primary cilia reporter) and transiently transfected HA-PKD2 channels immunolabeled with anti-HA antibody (red). B ) Primary cilia length and fluorescence colocalization analysis using the Pearson’s correlation coefficient. C ) Image a voltage clamped HEK cell primary cilium illuminated under fluorescence. D ) Example PKD2 WT and variant single channel records recorded from the cilia and activated by the indicated depolarization steps. E ) Left , single channel current magnitudes comparing WT and variant channels. Unitary conductance was estimated by the slope (γ) of the linear fit. Right , Open probability (Po) plotted as a function of voltage and fit to a Boltzmann equation to estimate the slope (Z) and half-activation voltage (V 1/2 ). Error bars = S.D. and the number of cilia records (N) for each data set is indicated within the parenthesis. P -values indicate results from two tailed, unpaired Student’s t-tests comparing WT and variants channel data sets.

Article Snippet: HEK 293 PKD2 null cells were electro-transfected with PKD1 sgRNAs (caccGCATAGGTGTGGTTGGCAGC and aaacGCTGCCAACCACACCTATGC) with the All-in-one Cas9 plasmid (Addgene).

Techniques: Microscopy, Stable Transfection, Expressing, Transfection, Immunolabeling, Fluorescence, Variant Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: ADPKD variants in the PKD2 pore helix cause structural collapse of the gate and distinct forms of channel dysfunction

doi: 10.1101/2024.09.12.612744

Figure Lengend Snippet:

Article Snippet: HEK 293 PKD2 null cells were electro-transfected with PKD1 sgRNAs (caccGCATAGGTGTGGTTGGCAGC and aaacGCTGCCAACCACACCTATGC) with the All-in-one Cas9 plasmid (Addgene).

Techniques: Variant Assay