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Figure 1. Recognition of the 3,849 + 10 kb C>T-induced CFTR Pseudo Exon Requires an SRSF-Dependent ESE (A–C) (A) Diagram of the CFTR splicing reporter. The CFTR minigene of exon 22–23 with a wild-type (WT reporter) or the 3,849 + 10 kb C>T-harboring (C>T reporter) IVS22 with a partial truncation (see STAR Methods for details) is fused with the amino-terminal GST and carboxyl-terminal RFP and GFP. Filled arrow, primer for RT-PCR. (B and C) Representative microscopic images of <t>HEK293</t> cells transfected with the WT or C>F reporter for 24 h (B), and quantiﬁcation of GFP intensity (%) from the total GFP and RFP signal (C). (D) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 24 h. CFTR splicing was detected by primer set oSS25 and oSS27, with the amplicon of 311 bp for the pseudo-exon-inclusion form (22/J/23) and 228 bp for the skipping form (22/23). ACTB was detected by oAM13 and oAM14, serving as a loading control. (E–G) Western blotting of phosphorylated SR proteins pulled down with biotin-conjugated RNA oligonucleotides (#1, #2, or #3 (E), shown in the diagram on the top, or #2 or #2M (G), their sequences shown in (F)) in HEK293 cell lysate. , pull-down product without bait RNA. Mutated ribonucleotides are shown in red. (H) Diagram showing the C>T and C>T(#2M) reporters. The C>T(#2M) reporter harbors point mutations of #2M shown in (F). (I) RT-PCR for HEK293 cells transfected with the WT, C>T, or C>T(#2M) reporter vector for 24 h. CFTR splicing was detected with the primer set oSS25 and oSS27. ACTB was detected by oAM13 and oAM14, serving as a loading control. (J and K) Representative microscopic images (J), and quantiﬁcation analysis of percentage GFP intensity (K), for HEK293 cells transfected with the C>T or C>T(#2M) reporter for 24 h. Scale bars, 50 mm in (B and J). 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively, in (B, D, and J). Each open circle represents the mean GFP intensity (%) of ﬁve random ﬁelds, and the mean ± SD from three independent experiments is shown in (C and K). **p < 0.01 (C and K).

Hek293 Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/puc19/pUC19-E7MR72+(Plasmid+%23103105)/pm32905759-541-0-19
Average 91 stars, based on 2 article reviews

hek293 cells - by Bioz Stars, 2026-09

91/100 stars

Images

1) Product Images from "Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion."

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.

Journal: Cell chemical biology

doi: 10.1016/j.chembiol.2020.08.013

Figure Legend Snippet: Figure 1. Recognition of the 3,849 + 10 kb C>T-induced CFTR Pseudo Exon Requires an SRSF-Dependent ESE (A–C) (A) Diagram of the CFTR splicing reporter. The CFTR minigene of exon 22–23 with a wild-type (WT reporter) or the 3,849 + 10 kb C>T-harboring (C>T reporter) IVS22 with a partial truncation (see STAR Methods for details) is fused with the amino-terminal GST and carboxyl-terminal RFP and GFP. Filled arrow, primer for RT-PCR. (B and C) Representative microscopic images of HEK293 cells transfected with the WT or C>F reporter for 24 h (B), and quantiﬁcation of GFP intensity (%) from the total GFP and RFP signal (C). (D) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 24 h. CFTR splicing was detected by primer set oSS25 and oSS27, with the amplicon of 311 bp for the pseudo-exon-inclusion form (22/J/23) and 228 bp for the skipping form (22/23). ACTB was detected by oAM13 and oAM14, serving as a loading control. (E–G) Western blotting of phosphorylated SR proteins pulled down with biotin-conjugated RNA oligonucleotides (#1, #2, or #3 (E), shown in the diagram on the top, or #2 or #2M (G), their sequences shown in (F)) in HEK293 cell lysate. , pull-down product without bait RNA. Mutated ribonucleotides are shown in red. (H) Diagram showing the C>T and C>T(#2M) reporters. The C>T(#2M) reporter harbors point mutations of #2M shown in (F). (I) RT-PCR for HEK293 cells transfected with the WT, C>T, or C>T(#2M) reporter vector for 24 h. CFTR splicing was detected with the primer set oSS25 and oSS27. ACTB was detected by oAM13 and oAM14, serving as a loading control. (J and K) Representative microscopic images (J), and quantiﬁcation analysis of percentage GFP intensity (K), for HEK293 cells transfected with the C>T or C>T(#2M) reporter for 24 h. Scale bars, 50 mm in (B and J). 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively, in (B, D, and J). Each open circle represents the mean GFP intensity (%) of ﬁve random ﬁelds, and the mean ± SD from three independent experiments is shown in (C and K). **p < 0.01 (C and K).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Amplification, Control, Western Blot

Figure 2. CLK Inhibitor Suppresses Inclusion of the CFTR Pseudo Exon in an ESE-Dependent Manner (A) Percentages of GFP intensity are indicated for HEK293 cells transfected with the WT or C>T reporter for 6 h and treated with the indicated compounds or DMSO (0.1%) for 19 h. (B and C) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 5 h and treated with the indicated compounds (B, TG003; C, SRPIN340) or DMSO (0.1%) for 19 h. CFTR splicing was detected by primers oSS25 and oSS27, and ACTB by oAM13 and oAM14. (D) Diagrams indicating the C>T, C>T-SA, and C>T(#2M)-SA reporters. In the C>T-SA reporter, the pseudo-exonic splice acceptor (SA) is replaced by the conserved SA of exon 23. The C>T(#2M)-SA vector harbors the #2M point mutation in the background of the C>T-SA vector. All three vectors harbor the 3,849 + 10 kb C>T mutation. (E and F) Representative microscopic images (E) and quantiﬁcation of percentages of GFP intensity (F) for HEK293 cells transfected with the WT, C>T, C>T-SA, or C>T(#2M)-SA vector for 5 h and treated with TG003 (30 mM) or DMSO (0.1%) for 19 h. Scale bars, 50 mm in (E). Each open circle represents the mean GFP intensity (%) of ﬁve random ﬁelds, and the mean ± SD from three independent experiments is shown in (A and F). **p < 0.01. 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively in (B, C, and E).

Figure Legend Snippet: Figure 2. CLK Inhibitor Suppresses Inclusion of the CFTR Pseudo Exon in an ESE-Dependent Manner (A) Percentages of GFP intensity are indicated for HEK293 cells transfected with the WT or C>T reporter for 6 h and treated with the indicated compounds or DMSO (0.1%) for 19 h. (B and C) RT-PCR for HEK293 cells transfected with the WT or C>T reporter vector for 5 h and treated with the indicated compounds (B, TG003; C, SRPIN340) or DMSO (0.1%) for 19 h. CFTR splicing was detected by primers oSS25 and oSS27, and ACTB by oAM13 and oAM14. (D) Diagrams indicating the C>T, C>T-SA, and C>T(#2M)-SA reporters. In the C>T-SA reporter, the pseudo-exonic splice acceptor (SA) is replaced by the conserved SA of exon 23. The C>T(#2M)-SA vector harbors the #2M point mutation in the background of the C>T-SA vector. All three vectors harbor the 3,849 + 10 kb C>T mutation. (E and F) Representative microscopic images (E) and quantiﬁcation of percentages of GFP intensity (F) for HEK293 cells transfected with the WT, C>T, C>T-SA, or C>T(#2M)-SA vector for 5 h and treated with TG003 (30 mM) or DMSO (0.1%) for 19 h. Scale bars, 50 mm in (E). Each open circle represents the mean GFP intensity (%) of ﬁve random ﬁelds, and the mean ± SD from three independent experiments is shown in (A and F). **p < 0.01. 22/J/23 and 22/23 stand for the pseudo exon inclusion and skipping products, respectively in (B, C, and E).

Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Mutagenesis

Figure 3. Identiﬁcation of CaNDY as a Highly Potent Agent in Suppressing the 3,849 + 10 kb C>T-Induced Pseudo Exon of CFTR (A) Scatterplot for the recovery rates of CFTR exon 22/23 splicing in the focused library screening of CLK inhibitor analogs. (B) Structure of CaNDY. (C) Sigmoidal dose-response curve for pseudo-exon-suppression activity of CaNDY and TG003 (0.1, 0.3, 1, 3, 10, or 30 mM) in HEK293 cells transfected with the WT or C>T reporter and treated with the compounds for 13 h. (D and E) RT-PCR analysis for HEK293 (D) and Calu-3 cells (E) transfected with the WT or C>T reporter for 5 h and treated with CaNDY (1, 3, or 10 mM) or DMSO (0.1%) for 19 h. Untransfected cells were also analyzed as a negative control (No TF). Exogenous CFTR was detected by oSS25 and oSS27, and endogenous CFTR was detected by oAM619 and oSS30 (yielding a 430-bp product for pseudo exon inclusion and a 346-bp product for pseudo exon skipping). Rates of normal CFTR splicing (% 22/23) were calculated from the band intensities. ND indicates there was no detectable product after 35 cycles of PCR ampliﬁcation. (F) RT-PCR analysis for B lymphocyte GM11860 with CaNDY (1, 3, or 10 mM), DMSO (0.1%), or cycloheximide (CHX) (100 mg/mL) for 8 h. Endogenous CFTR was detected with the primers oSS29 and oSS30, yielding a 340-bp product for pseudo exon inclusion and a 256-bp product for pseudo exon skipping. Identities of the CFTR splicing products were conﬁrmed by Sanger sequencing.

Figure Legend Snippet: Figure 3. Identiﬁcation of CaNDY as a Highly Potent Agent in Suppressing the 3,849 + 10 kb C>T-Induced Pseudo Exon of CFTR (A) Scatterplot for the recovery rates of CFTR exon 22/23 splicing in the focused library screening of CLK inhibitor analogs. (B) Structure of CaNDY. (C) Sigmoidal dose-response curve for pseudo-exon-suppression activity of CaNDY and TG003 (0.1, 0.3, 1, 3, 10, or 30 mM) in HEK293 cells transfected with the WT or C>T reporter and treated with the compounds for 13 h. (D and E) RT-PCR analysis for HEK293 (D) and Calu-3 cells (E) transfected with the WT or C>T reporter for 5 h and treated with CaNDY (1, 3, or 10 mM) or DMSO (0.1%) for 19 h. Untransfected cells were also analyzed as a negative control (No TF). Exogenous CFTR was detected by oSS25 and oSS27, and endogenous CFTR was detected by oAM619 and oSS30 (yielding a 430-bp product for pseudo exon inclusion and a 346-bp product for pseudo exon skipping). Rates of normal CFTR splicing (% 22/23) were calculated from the band intensities. ND indicates there was no detectable product after 35 cycles of PCR ampliﬁcation. (F) RT-PCR analysis for B lymphocyte GM11860 with CaNDY (1, 3, or 10 mM), DMSO (0.1%), or cycloheximide (CHX) (100 mg/mL) for 8 h. Endogenous CFTR was detected with the primers oSS29 and oSS30, yielding a 340-bp product for pseudo exon inclusion and a 256-bp product for pseudo exon skipping. Identities of the CFTR splicing products were conﬁrmed by Sanger sequencing.

Techniques Used: Library Screening, Activity Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing

Figure 4. CaNDY, but Not CFTR Modulator VX-809, Rescues Mature CFTR Expression from the CFTR Gene with the 3,849 + 10 kb C>T mu- tation (A) Diagram of HiBiTWT and HiBiTC>T expression vectors. Upon translation, cleavage at the 2A site releases amino-terminal EGFP from the full-length or truncated CFTR with or without HiBiT-HA, respectively. (B and C) HEK293 cells were transfected with HiBiTWT or HiBiTC>T for 5 h and treated with CaNDY (1 or 3 mM) or DMSO (0.1%) for 48 h. Full-length expression of CFTR was quantiﬁed by relative luminescence units (RLU) generated by the C-terminal HiBiT tag using the Nano-Glo HiBiT Lytic Detection System (B), and expression of EGFP was conﬁrmed by western blotting (C). Columns indicate mean ± SD for independent experiments (n = 3, indicated individually by circles). (D) Structures of CFTRWT-GFP, CFTRC>T-GFP, and CFTRF508d-GFP expression vectors are shown. CFTRWT-GFP and CFTRC>T-GFP vectors harbor a partial IVS22 of wild type or with the 3,849 + 10 kb C>T mutation, respectively, and CFTRF508d-GFP expresses the F508 deletion form of CFTR. In these constructs, mKate2 with the 2A site is fused amino-terminally and AcGFP carboxyl-terminally with the CFTR coding sequence, and inclusion of the pseudo exon results in CFTR truncation without AcGFP translation.

Figure Legend Snippet: Figure 4. CaNDY, but Not CFTR Modulator VX-809, Rescues Mature CFTR Expression from the CFTR Gene with the 3,849 + 10 kb C>T mu- tation (A) Diagram of HiBiTWT and HiBiTC>T expression vectors. Upon translation, cleavage at the 2A site releases amino-terminal EGFP from the full-length or truncated CFTR with or without HiBiT-HA, respectively. (B and C) HEK293 cells were transfected with HiBiTWT or HiBiTC>T for 5 h and treated with CaNDY (1 or 3 mM) or DMSO (0.1%) for 48 h. Full-length expression of CFTR was quantiﬁed by relative luminescence units (RLU) generated by the C-terminal HiBiT tag using the Nano-Glo HiBiT Lytic Detection System (B), and expression of EGFP was conﬁrmed by western blotting (C). Columns indicate mean ± SD for independent experiments (n = 3, indicated individually by circles). (D) Structures of CFTRWT-GFP, CFTRC>T-GFP, and CFTRF508d-GFP expression vectors are shown. CFTRWT-GFP and CFTRC>T-GFP vectors harbor a partial IVS22 of wild type or with the 3,849 + 10 kb C>T mutation, respectively, and CFTRF508d-GFP expresses the F508 deletion form of CFTR. In these constructs, mKate2 with the 2A site is fused amino-terminally and AcGFP carboxyl-terminally with the CFTR coding sequence, and inclusion of the pseudo exon results in CFTR truncation without AcGFP translation.

Techniques Used: Expressing, Transfection, Generated, Western Blot, Mutagenesis, Construct, Sequencing

Transfection:

Article Title: Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion.
Article Snippet: The entire reaction products were then applied to a LabChip system (Perkin- Elmer, Waltham, MA, USA), and kinase activity was determined by calculation of product rates (P / (P + S) (P, product-derived peak; S, substrate-derived peak). .. HEK293 cells (1.0 3 105 cells in 12-well plates) were transfected with YFP halide sensor vector by Peter Haggie (Addgene #25872, pcDNA3.1 EYFP H148Q/I152L (Galietta et al., 2001)) and CFTRWT-GFP or CFTRC>T-GFP reporter plasmid using FuGENE HD Transfection Reagent (Promega). ..

Plasmid Preparation:

Recombinant:

Article Title: Generation of a genetically-modified induced pluripotent stem cell line harboring an oncogenic gene variant KRAS p.G12V.
Article Snippet: .. Contents lists available at ScienceDirect Stem Cell Research journal homepage: www.elsevier.com/locate/scr https://doi.org/10.1016/j.scr.2023.103105 Received 23 March 2023; Accepted 22 April 2023 Stem Cell Research 69 (2023) 103105 (continued ) Unique stem cell line identifier UMGi014-C-2 clone 64 Inducible/constitutive expression system details N/A Date archived/stock creation date 28th February 2020 Cell line repository/bank Biobank of the University Medical Center Göttingen, https://hpscreg.eu/cell-line/UM Gi014-C-2 Ethical/GMO work approvals Ethics Committee of the University Medical Center Göttingen, approval number: 10/9/15 Addgene/public access repository recombinant DNA sources’ disclaimers (if applicable) N/A ..

Image Search Results

(A) Schematic representation of the SARS-CoV-2 reverse genetics platform used to generate the wild-type (WT) and ΔNsp2 recombinant viruses. The figure was created using BioRender. Overlapping genomic fragments were assembled and recombined into the YAC vector through TAR cloning. YAC DNA was nucleofected into Vero cells to reconstitute the viruses. (B) Viral protein expression following infection of Vero cells with WT or ΔNsp2 recombinant viruses. Cells were infected and harvested as described in the Materials and Methods section. Protein expression was analyzed by Western blot using antibodies against SARS-CoV-2 N (AF488), S(AF647), Nsp3(AF647), Nsp2(AF488) and Nsp1(AF488). Stain-free technology was used as a loading control (see Supplementary Figure 2). The experiment was performed twice, and a representative result is shown. (C–E) Growth kinetics of recombinant viruses in Vero (C), A549- hACE2 (D), and A549-ALI (E) cells. Viral shedding was quantified in cell culture supernatants (C and D) or mucus washes (E). Viral titers are expressed as TCID50/mL (mean ± SD; n = 3–6 per group). The dashed line indicates the limit of detection (LOD). The x-axis represents hours post-infection (hpi). Global growth kinetics were compared using two-way ANOVA. # P < 0.033. Individual time points were analyzed using uncorrected Fisher’s LSD test. ***P < 0.0002; ****P < 0.000

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic representation of the SARS-CoV-2 reverse genetics platform used to generate the wild-type (WT) and ΔNsp2 recombinant viruses. The figure was created using BioRender. Overlapping genomic fragments were assembled and recombined into the YAC vector through TAR cloning. YAC DNA was nucleofected into Vero cells to reconstitute the viruses. (B) Viral protein expression following infection of Vero cells with WT or ΔNsp2 recombinant viruses. Cells were infected and harvested as described in the Materials and Methods section. Protein expression was analyzed by Western blot using antibodies against SARS-CoV-2 N (AF488), S(AF647), Nsp3(AF647), Nsp2(AF488) and Nsp1(AF488). Stain-free technology was used as a loading control (see Supplementary Figure 2). The experiment was performed twice, and a representative result is shown. (C–E) Growth kinetics of recombinant viruses in Vero (C), A549- hACE2 (D), and A549-ALI (E) cells. Viral shedding was quantified in cell culture supernatants (C and D) or mucus washes (E). Viral titers are expressed as TCID50/mL (mean ± SD; n = 3–6 per group). The dashed line indicates the limit of detection (LOD). The x-axis represents hours post-infection (hpi). Global growth kinetics were compared using two-way ANOVA. # P < 0.033. Individual time points were analyzed using uncorrected Fisher’s LSD test. ***P < 0.0002; ****P < 0.000

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Recombinant, Plasmid Preparation, Cloning, Expressing, Infection, Western Blot, Staining, Control, Cell Culture

(A) Schematic overview of the mouse infection protocol. This figure was created using BioRender. (B) Survival curves of K18- hACE2 mice following infection with wild-type or ΔNsp2 recombinant SARS-CoV-2. Survival is expressed as the percentage of mice not reaching the ethical endpoint at each day post-infection (DPI) (n = 7 per group). Statistical analysis was performed using the Mantel–Cox (log-rank) test. *P < 0.033. (C) Clinical disease scores following infection. Disease scores represent the cumulative score of individual parameters including body weight, activity, coat condition, and respiratory symptoms (each scored from 0 to 3). A total score of 4 corresponded to the ethical endpoint, as euthanasia was required when two or more parameters reached a score ≥2 (mean ± SD; n = 7 per group). (D) Body weight changes following infection, expressed as the percentage of initial body weight at each DPI (mean ± SD; n = 7 per group). For panels C and D, differences over the entire infection course were analyzed using mixed-effects models. # P < 0.033. Individual time points were compared using uncorrected Fisher’s LSD tests. (E) Infectious viral loads in lung tissues. Recombinant SARS-CoV-2 titers were determined in lung homogenates using the 50% tissue culture infectious dose (TCID₅₀) assay and normalized to total protein content (mean ± SD; n = 5 per group). (F) SARS-CoV-2 E gene RNA levels in lung tissues, quantified by RT-ddPCR and normalized to mouse Gapdh mRNA copy number (mean ± SD; n = 5 per group). For panels E and F, data across the infection course were analyzed using two-way ANOVA. ## P < 0.0021. Groups at individual time points were compared using uncorrected Fisher’s LSD tests. ****P < 0.0001.

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic overview of the mouse infection protocol. This figure was created using BioRender. (B) Survival curves of K18- hACE2 mice following infection with wild-type or ΔNsp2 recombinant SARS-CoV-2. Survival is expressed as the percentage of mice not reaching the ethical endpoint at each day post-infection (DPI) (n = 7 per group). Statistical analysis was performed using the Mantel–Cox (log-rank) test. *P < 0.033. (C) Clinical disease scores following infection. Disease scores represent the cumulative score of individual parameters including body weight, activity, coat condition, and respiratory symptoms (each scored from 0 to 3). A total score of 4 corresponded to the ethical endpoint, as euthanasia was required when two or more parameters reached a score ≥2 (mean ± SD; n = 7 per group). (D) Body weight changes following infection, expressed as the percentage of initial body weight at each DPI (mean ± SD; n = 7 per group). For panels C and D, differences over the entire infection course were analyzed using mixed-effects models. # P < 0.033. Individual time points were compared using uncorrected Fisher’s LSD tests. (E) Infectious viral loads in lung tissues. Recombinant SARS-CoV-2 titers were determined in lung homogenates using the 50% tissue culture infectious dose (TCID₅₀) assay and normalized to total protein content (mean ± SD; n = 5 per group). (F) SARS-CoV-2 E gene RNA levels in lung tissues, quantified by RT-ddPCR and normalized to mouse Gapdh mRNA copy number (mean ± SD; n = 5 per group). For panels E and F, data across the infection course were analyzed using two-way ANOVA. ## P < 0.0021. Groups at individual time points were compared using uncorrected Fisher’s LSD tests. ****P < 0.0001.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Infection, Recombinant, Activity Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Cytokine, chemokine, and interferon profiles in lung tissues following mock or rSARS-CoV-2 infection. Mediator concentrations were normalized to total protein content, and values were scaled from 0 to 10 for each mediator to facilitate visualization in the heatmap (mean; n = 5 per group). Differences between groups at the same time point for the mediator profile were assessed using two-way ANOVA (**P < 0.0021), followed by Fisher’s LSD test for individual mediators. (B) Carstairs staining of lung sections from mock-, wild-type–, or ΔNsp2-infected K18- hACE2 mice. Whole lung sections were scanned, and representative images from each group are shown. (C) Histological assessment of lung inflammation in perivascular, peribronchial, and parenchymal regions. Whole lung sections were independently scored for inflammation (scale 0–5) in each anatomical compartment (mean; n = 4–5 per group). Global inflammation across time points was analyzed using Fisher’s LSD test. (C) *P < 0.033; ****P < 0.0001

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Infection, Staining

(A) Systemic inflammatory response in plasma from mock-, wild-type–, or ΔNsp2-infected mice. Chemokines and interferons were quantified in Triton X-100–inactivated, platelet-depleted plasma and expressed as pg/mL (mean; n = 4–5 per group). Mean mediator levels were compared between groups at the same time point using uncorrected Fisher’s LSD test. (B) Quantification of circulating leukocytes. Citrate-treated whole blood was subjected to red blood cell lysis, and leukocytes were stained and analyzed by flow cytometry. Results are expressed as CD45⁺ cells per 100 µL of whole blood. (C–I) Abundance of circulating T helper (Th) cells (C), cytotoxic T lymphocytes (CTLs) (D), B cells (E), natural killer (NK) cells (F), monocytes (Mo) (G), neutrophils (Neutro) (H), and PD-L1–expressing neutrophils (I). Data are presented as percentages of CD45⁺ cells (mean ± SD; n = 4–5 per group). For all panels, comparisons across the infection course were performed using two-way ANOVA. # P < 0.033. Group comparisons at individual time points were assessed using uncorrected Fisher’s LSD test. *P < 0.033; **P < 0.0021; ***P < 0.0002; ****P < 0.0001.

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Systemic inflammatory response in plasma from mock-, wild-type–, or ΔNsp2-infected mice. Chemokines and interferons were quantified in Triton X-100–inactivated, platelet-depleted plasma and expressed as pg/mL (mean; n = 4–5 per group). Mean mediator levels were compared between groups at the same time point using uncorrected Fisher’s LSD test. (B) Quantification of circulating leukocytes. Citrate-treated whole blood was subjected to red blood cell lysis, and leukocytes were stained and analyzed by flow cytometry. Results are expressed as CD45⁺ cells per 100 µL of whole blood. (C–I) Abundance of circulating T helper (Th) cells (C), cytotoxic T lymphocytes (CTLs) (D), B cells (E), natural killer (NK) cells (F), monocytes (Mo) (G), neutrophils (Neutro) (H), and PD-L1–expressing neutrophils (I). Data are presented as percentages of CD45⁺ cells (mean ± SD; n = 4–5 per group). For all panels, comparisons across the infection course were performed using two-way ANOVA. # P < 0.033. Group comparisons at individual time points were assessed using uncorrected Fisher’s LSD test. *P < 0.033; **P < 0.0021; ***P < 0.0002; ****P < 0.0001.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Clinical Proteomics, Infection, Red Blood Cell Lysis, Staining, Flow Cytometry, Expressing

(A) Dimension reduction of the transcriptome. Transcript counts were normalized using DESeq2 to stabilize variance, and Uniform Manifold Approximation and Projection (UMAP) was applied to visualize sample clustering and distances between conditions (B–D) Differential gene expression between ΔNsp2 and wild-type SARS-CoV-2-infected mice. Differential expression analysis was performed using DESeq2 from raw transcript counts. Results are presented as log2 fold change (log2FC) for ΔNsp2 relative to wild-type infection at 3 days post-infection (DPI) (B), 5 DPI (C), and 7 DPI (D) (mean; n = 4–5 per group). The y-axis represents the adjusted p-value (padj), corrected for multiple testing using a false discovery rate (FDR) threshold of 0.05. Transcripts with a padj below 0.05 and an absolute log 2 (FC) above 1.5 were colorized in red (up-regulated) or blue (down-regulated).

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Dimension reduction of the transcriptome. Transcript counts were normalized using DESeq2 to stabilize variance, and Uniform Manifold Approximation and Projection (UMAP) was applied to visualize sample clustering and distances between conditions (B–D) Differential gene expression between ΔNsp2 and wild-type SARS-CoV-2-infected mice. Differential expression analysis was performed using DESeq2 from raw transcript counts. Results are presented as log2 fold change (log2FC) for ΔNsp2 relative to wild-type infection at 3 days post-infection (DPI) (B), 5 DPI (C), and 7 DPI (D) (mean; n = 4–5 per group). The y-axis represents the adjusted p-value (padj), corrected for multiple testing using a false discovery rate (FDR) threshold of 0.05. Transcripts with a padj below 0.05 and an absolute log 2 (FC) above 1.5 were colorized in red (up-regulated) or blue (down-regulated).

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Gene Expression, Infection, Quantitative Proteomics

Functional enrichment was performed using Gene Set Enrichment Analysis (GSEA) with the MSigDB M2 curated gene sets. DESeq2 Wald statistics from the ΔNsp2 versus wild-type comparison at each DPI were used as input for GSEA. The top 50 most differentially enriched pathways across all time points are shown in the heatmap. Pathways were hierarchically clustered based on enrichment patterns, and colors represent normalized enrichment scores (NES), with red indicating pathways enriched (upregulated) and blue indicating pathways depleted (downregulated) in ΔNsp2-infected mice relative to wild-type infection (n = 4–5 per group). The 4 major clusters were highlighted with dotted frame.

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: Functional enrichment was performed using Gene Set Enrichment Analysis (GSEA) with the MSigDB M2 curated gene sets. DESeq2 Wald statistics from the ΔNsp2 versus wild-type comparison at each DPI were used as input for GSEA. The top 50 most differentially enriched pathways across all time points are shown in the heatmap. Pathways were hierarchically clustered based on enrichment patterns, and colors represent normalized enrichment scores (NES), with red indicating pathways enriched (upregulated) and blue indicating pathways depleted (downregulated) in ΔNsp2-infected mice relative to wild-type infection (n = 4–5 per group). The 4 major clusters were highlighted with dotted frame.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Functional Assay, Comparison, Infection

(A) Schematic representation of Cross-linking and Immunoprecipitation Sequencing (CLIP-seq) procedure. The figure was created using BioRender. (B) Functional enrichment of transcript interacting with Nsp2. Genes with peak identified by both peak caller algorythms were analysed using Metascape. Term with a P adjusted value for false discovery rate (FDR) below 0.05 were retained. One representative term per cluster were presented and the top 15 are shown. Dot size represents the number of gene detected within the term (Gene Count), color scale represents the P adjusted value (padj) and x-axis represents the enrichment score.

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic representation of Cross-linking and Immunoprecipitation Sequencing (CLIP-seq) procedure. The figure was created using BioRender. (B) Functional enrichment of transcript interacting with Nsp2. Genes with peak identified by both peak caller algorythms were analysed using Metascape. Term with a P adjusted value for false discovery rate (FDR) below 0.05 were retained. One representative term per cluster were presented and the top 15 are shown. Dot size represents the number of gene detected within the term (Gene Count), color scale represents the P adjusted value (padj) and x-axis represents the enrichment score.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Immunoprecipitation, Sequencing, Functional Assay

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1) Product Images from "Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion."

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