Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: TMR-Lipo-Abs protects mice from CLP-induced polymicrobial sepsis. (A–C) Schematic design of mid-grade (A) or high-grade (B, C) sepsis mouse model induced by cecal ligation and punctuation (CLP) depending on the position of the cecal ligation (top). The survival of CLP mice treated with the indicated therapy was monitored for 10 days (A) or 72 h (B, C); mortality was measured for n = 10 mice per group (bottom). Statistical differences compared with the PBS or TMR-Lipo-Abs-treated mice are indicated (log-rank test). The data are representative of three independent experimental replicates with similar results. (D) Serum cytokine levels. The data shown represent at least three independent experiments ( n ≥ 3), significance was measured by Ordinary one-way ANOVA (TNF- α , ∗∗ P = 0.0010 and ∗∗∗ P = 0.0002, IL-6, ∗∗ P = 0.0057 and ∗∗∗ P = 0.0004 and IL12p40, both ∗∗∗∗ P < 0.0001). n.s = not significant. (E) IB identification and comparison of HMGB1 and PTX3 expression in spleen, lung, and liver cells of CLP mice after different treatment as a DAMP indicator for tissue damage. (F) Representative H&E staining of the lung, spleen, and liver from 3 mice per group. Scale bar, 100 μm.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Ligation, Comparison, Expressing, Staining

Journal: Cell

Article Title: Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

doi: 10.1016/j.cell.2024.03.022

Figure Lengend Snippet: Figure 3. STT3A is required for glycosylation and trafficking of TLR4 to the cell surface (A) Flow cytometry for cell-surface TLR4 in TNFAIP3/ NF-kB-DCK* NALM-6 cells over- expressing exogenous TLR4 (left) and in parental NALM-6 cells with endogenous TLR4 expression (right) after lentiviral CRISPR-Cas9 knockout of indicated genes. PE, phycoerythrin. (B) Immunoblot analysis of whole-cell extracts and cell-surface proteins from TNFAIP3/ NF-kB- DCK* NALM-6 cells after CRISPR-Cas9 gene knockouts with the indicated sgRNAs. Cell surface proteins were captured on streptavidin agarose af- ter surface biotinylation. (C) Immunoblot analysis of cell extracts from the cells in (B) after the extracts were treated with the N- glycosidase PNGase F (PNGase) or endoglycosi- dase H (EndoH) ex vivo. (D) Immunoblot of HEK293T clones that exoge- nously express HA-tagged TLR4 and, where indi- cated, were CRISPR-Cas9 edited to lack STT3A or STT3B. Data shown are representative of three in- dependent clones for each genotype. GLUT1, glucose transporter 1. (E) GFP induction, as measured by FACS, of NALM-6 NF-kB-DCK* reporter cells with knockout of the indicated genes, with or without treatment with the NF-kB agonist LPS at 10 mg/mL for 24 h. (F and G) Immunoblots of RAW264.7 murine macrophage cell line stably transduced with Cas9 and the indicated sgRNAs. In (G), cells were treated, where indicated, with 10 mg/mL LPS for 24 h. (H) NALM6 TNFAIP3/ NF-kB-DCK* reporter cells stably overexpressing either TLR4 or CD16-TLR4 were stimulated or not with LPS, 10 mg/mL for 24 h, followed by FACS for GFP intensity as a marker of NF-kB pathway activity. The left panel shows data from STT3A wild-type cells, while the right panel shows data from STT3A knockout cells. MFI, me- dian fluorescence intensity. See also Figures S1 and S2.

Article Snippet: The sgRNA sequences individually cloned and directly tested for base editing efficiency were as detailed in Table S6. cDNA expression plasmids For experiments where TLR4 was overexpressed, the cDNA for human TLR4 (Addgene #42646) was subcloned into a pDONR vector (F primer: humanTLR4-F, R primer: humanTLR4-R) followed by Gateway cloning to introduce TLR4 into the plenti-Ubc-3XHA-PGKhygro vector.75 For the CD16-TLR4 overexpression experiment, the CD16-TLR4 chimera was created by using overlap PCR to combine the N terminal portion of CD16 (template: Addgene #82896, F primer: 82896-F, R primer: 82896-R) with the C terminal portion of TLR4 with an HA tag appended (template: Addgene #42646, F primer: 42646-F, R primer: 42646-R).

Techniques: Glycoproteomics, Flow Cytometry, Expressing, CRISPR, Knock-Out, Western Blot, Ex Vivo, Clone Assay, Stable Transfection, Transduction, Marker, Activity Assay

Journal: Cell

Article Title: Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

doi: 10.1016/j.cell.2024.03.022

Figure Lengend Snippet: Figure 4. Regulation of TLR4 and NF-kB by OST-A requires STT3A catalytic activity and can be inhibited with drug-like molecules (A) Immunoblot of HEK293T cells stably infected to express HA-tagged TLR4 and the indicated HiBIT- tagged STT3A proteins after CRISPR-based editing with the indicated sgRNAs. (B) Immunoblot of TNFAIP3/ NF-kB-DCK* NALM-6 cells overexpressing TLR4-HA and treated with NGI-1. (C) Immunoblot of RAW264.7 cells treated with NGI-1 and/or LPS. (D) GFP induction, as measured by FACS, of TNFAIP3/ NF-kB-DCK* reporter cells treated with the indicated concentration of NGI-1 for 48 h, fol- lowed by the addition of 10 mg/mL LPS for 24 h. GFP values were normalized to values for cells not treated with LPS or NGI-1. (E) Kill curves of TNFAIP3/ NF-kB-DCK* reporter cells that were pretreated, where indicated, with NGI-1 for 24 h and then treated with 10 mM BVdU in the presence or absence of 10 mg/mL LPS. Data were normalized to cells unexposed to BVdU and LPS. n = 3. Error bars represent standard deviation. (F) Immunoblot analysis of whole-cell extracts and cell-surface proteins from TNFAIP3/ NF-kB-DCK* NALM-6 cells after CRISPR-Cas9 gene knockouts with the indicated sgRNAs. Cell surface proteins were captured on streptavidin agarose after surface biotinylation. Where indicated, cells were treated with NGI-1 (1 or 10 mM) or tunicamycin, 1 mM for 24 h. Mobility shifts in proteins between the whole-cell fraction and the surface fraction may be due to different loading buffers used for these fractions (see STAR Methods). (G) Structures of NGI-1 and NGI-235. (H and I) Anti-HA immunoblot analysis (H) and anti- TLR4 FACS (I) to detect cell-surface TLR4 of TNFAIP3/ NF-kB-DCK* NALM-6 cells expressing exogenous TLR4-HA and treated with the indicated concentrations of either NGI-1 or NGI-235 for 24 h. GLUT1, glucose transporter 1; MFI, median fluores- cence intensity. See also Figures S3 and S4.

Article Snippet: The sgRNA sequences individually cloned and directly tested for base editing efficiency were as detailed in Table S6. cDNA expression plasmids For experiments where TLR4 was overexpressed, the cDNA for human TLR4 (Addgene #42646) was subcloned into a pDONR vector (F primer: humanTLR4-F, R primer: humanTLR4-R) followed by Gateway cloning to introduce TLR4 into the plenti-Ubc-3XHA-PGKhygro vector.75 For the CD16-TLR4 overexpression experiment, the CD16-TLR4 chimera was created by using overlap PCR to combine the N terminal portion of CD16 (template: Addgene #82896, F primer: 82896-F, R primer: 82896-R) with the C terminal portion of TLR4 with an HA tag appended (template: Addgene #42646, F primer: 42646-F, R primer: 42646-R).

Techniques: Activity Assay, Western Blot, Stable Transfection, Infection, CRISPR, Concentration Assay, Standard Deviation, Expressing

Journal: Cell

Article Title: Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

doi: 10.1016/j.cell.2024.03.022

Figure Lengend Snippet: Figure 5. Base-editor screens identify muta- tions in STT3A that render it resistant to NGI-1 (A) Workflow of base-editor screen done in TNFAIP3/ NF-kB-DCK* NALM-6 cells, high- lighting the dual approach using a library of guides tiling STT3A that were co-introduced with either an A / G base editor or a C / T base editor. (B and C) Enrichment (y axis) of STT3A sgRNAs (placed on the x axis according to their position along the STT3A gene [or arbitrary number for control guides]) recovered from the top 1% GFP+ cells in the NGI-1-treated arm, as compared with the top 1% GFP+ cells in the DMSO-treated arm, for the STT3A A / G base-editor screen (B) and STT3A C / T base-editor screen (C). (D and E) NGS of genomic DNA after introduction of the top-scoring sgRNA in the A / G base-editor screen (D) or C / T base-editor screen (E) into the screening cell line, TNFAIP3/ NF-kB-DCK* NALM-6 cells. The pie chart shows percentage of NGS reads that contained substitutions, and the nucleotide bar graph shows the frequency of indi- vidual substitutions at the indicated nucleotides, along with canonical amino acid sequence above and validated mutated amino acid sequence below. Note that the sgRNAs in both panels align with the com- plementary DNA strand, so substitutions introduced are T / C and G / A in this orientation. A naturally occurring A/G SNP (rs2241502) is present in panel (D) as well. Analysis done with CRISPResso2. (F and G) Anti-TLR4 FACS (F) to detect cell-surface TLR4 and anti-HA immunoblot analysis (G) of STT3A/ HEK293T cells expressing exogenous TLR4-HA that were rescued with the indicated STT3A variants and treated with NGI-1 for 24 h. Error bars represent standard deviation. See also Figure S5.

Article Snippet: The sgRNA sequences individually cloned and directly tested for base editing efficiency were as detailed in Table S6. cDNA expression plasmids For experiments where TLR4 was overexpressed, the cDNA for human TLR4 (Addgene #42646) was subcloned into a pDONR vector (F primer: humanTLR4-F, R primer: humanTLR4-R) followed by Gateway cloning to introduce TLR4 into the plenti-Ubc-3XHA-PGKhygro vector.75 For the CD16-TLR4 overexpression experiment, the CD16-TLR4 chimera was created by using overlap PCR to combine the N terminal portion of CD16 (template: Addgene #82896, F primer: 82896-F, R primer: 82896-R) with the C terminal portion of TLR4 with an HA tag appended (template: Addgene #42646, F primer: 42646-F, R primer: 42646-R).

Techniques: Control, Sequencing, Western Blot, Expressing, Standard Deviation

Journal: Cell

Article Title: Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

doi: 10.1016/j.cell.2024.03.022

Figure Lengend Snippet: Figure 6. Cryo-EM structure of OST-A with NGI-1 bound (A) OST-A structure is shown in ribbon representa- tion. Subunits are colored individually and labeled. Bound NGI-1 is shown as orange spheres. Bound LLO (Dol-PP-GlcNAc2Man9Glc3) is shown as black sticks. The inset shows a close-up view of NGI-1 and LLO binding sites, with STT3A shown in ribbon. NGI-1 is shown in orange sticks, and bound LLO is shown in black sticks. The dolichyl tail and the glycan moiety of the LLO are indicated and labeled. Manganese (II) ion is shown as a pink sphere. (B) Close-up view of the bound LLO in the presence of NGI-1. OST subunits are shown in ribbon represen- tation with subunits colored as in (A). NGI-1 and LLO are shown as the inset in (A), with the dolichyl tail and glycan parts indicated and labeled. The water mole- cule is shown as a red sphere and the manganese (II) ion as a pink sphere. EM density is shown as a blue mesh. (C) Ribbon representation of STT3A with bound LLO and NGI-1. The three clusters identified by the base- editor screen are indicated in boxes and labeled. NGI-1 and LLO are shown as in (A). Transmembrane helix 9 (TM9) is represented as a green cylinder and labeled. (D) Close-up of NGI-1 binding to the STT3 subunit. Residues involved in binding are shown as sticks. EM density, NGI-1, LLO, water, and manganese (II) ion are shown as in (B) and labeled. (E) Anti-TLR4 FACS to detect cell-surface TLR4 after 24 h of NGI-1 treatment of STT3A/ 293T cells exogenously expressing TLR4-HA and the indicated STT3A variants. Error bars represent standard devi- ation. See also Figures S6 and S7.

Article Snippet: The sgRNA sequences individually cloned and directly tested for base editing efficiency were as detailed in Table S6. cDNA expression plasmids For experiments where TLR4 was overexpressed, the cDNA for human TLR4 (Addgene #42646) was subcloned into a pDONR vector (F primer: humanTLR4-F, R primer: humanTLR4-R) followed by Gateway cloning to introduce TLR4 into the plenti-Ubc-3XHA-PGKhygro vector.75 For the CD16-TLR4 overexpression experiment, the CD16-TLR4 chimera was created by using overlap PCR to combine the N terminal portion of CD16 (template: Addgene #82896, F primer: 82896-F, R primer: 82896-R) with the C terminal portion of TLR4 with an HA tag appended (template: Addgene #42646, F primer: 42646-F, R primer: 42646-R).

Techniques: Cryo-EM Sample Prep, Labeling, Binding Assay, Glycoproteomics, Expressing