Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D is upregulated in osteosarcoma tissues and cell lines. a , b , c EEF1D mRNA and protein expression levels in osteosarcoma cell lines (MNNG/HOS, MG63 and U2OS) and human normal osteoblast cell line (hFOB 1.19) were measured by qRT-PCR and western blotting, respectively. d , e EEF1D expression levels were measured in 20 pairs of osteosarcoma and corresponding adjacent non-tumor tissues. For qRT-PCR, EEF1D expression was normalized to β-actin

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits osteosarcoma cell growth in vitro. a - f Expression levels of EEF1D mRNA and protein in MNNG/HOS, U2OS and MG63 cells were measured after EEF1D siRNA (si-EEF1D) transfection by qRT-PCR and western blotting, respectively. g - i Cell Counting Kit-8 (CCK-8) assays were performed to measure cell proliferation after siRNA transfection. j - o Colony-formation assays were performed for EEF1D-silenced osteosarcoma and control cells. Data are representative of results from three independent experiments. * P < 0.05. For qRT-PCR, EEF1D expression was normalized to β-actin

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, In Vitro, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits osteosarcoma cell cycle G2/M transition. Representative images of the cell cycle assays in MNNG/HOS ( a , b ), U2OS ( d , e ) and MG63 cells ( g , h ) after transfection with nonspecific control siRNA (si-NC) or EEF1D siRNA (si-EEF1D). c , f , i Diagrams showing the results of cell cycle assay in MNNG/HOS, U2OS and MG63 cells

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, Transfection, Control, Cell Cycle Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits Akt-mTOR and Akt-Bad signaling pathways in osteosarcoma cells. a , b MNNG/HOS and U2OS cell extracts were prepared and analyzed using PathScan® intracellular signaling array kit. Images were captured with Odyssey® Infrared Imaging System (LI-COR). c , d Quantification of results from MNNG/HOS and U2OS cells shown in ( a , b ) e , f Western blotting analysis of Akt-mTOR and Akt-Bad signaling pathway molecules in MNNG/HOS and U2OS cells transfected with nonspecific control siRNA (si-NC) or EEF1D siRNA (si-EEF1D). g Western blotting analysis of Akt-mTOR and Akt-Bad signaling pathway molecules, including mTOR, Akt, and Bad, in hFOB 1.19 cells transfected with pcDNA 3.1 or pcDNA 3.1-EEF1D. Quantification of results in ( e , f , g ) were shown in ( h , i and j ) respectively

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, Protein-Protein interactions, Imaging, Western Blot, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: Clinical significance of EEF1D in osteosarcoma patients. a Representative IHC image of EEF1D in non-tumor tissues. b Representative IHC image of EEF1D in osteosarcoma. Original magnification: 50×, 200×

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: Correlation analyses of EEF1D protein expression in relation to clinicopathologic variables of 50 patients with osteosarcoma

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Expressing

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 1. HDAC5 improved pathological symptoms in DHEA-induced polycystic ovary syndrome (PCOS) in mice. A. Brief flow chart of animal experiment. In young female mice, PCOS was induced by dehydroepiandrosterone (DHEA) administration (PCOS group). Some of the PCOS mice received injection of empty adenovirus (PCOS + Ad-vector group), whereas other PCOS mice received adenovirus expressing HDAC5 (PCOS + Ad-HDAC5 group). B. Ovarian morphology in each studied mouse group was determined by H&E staining at the magnification of 40×. Abbreviations: CL — corpus luteum; CF — cystic follicle. C–E. Serum levels of progesterone (C), testosterone (D) and estradiol (E) were detected by ELISA. F–H. mRNA and protein level of HDAC5 in ovary tissue of mice was measured by real-time PCR and Western blotting. Data was expressed as means ± SD (n = 6 in each group).

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Injection, Plasmid Preparation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 2. HDAC5 inhibited angiogenesis in ovaries of DHEA-induced PCOS mice. A. Relative mRNA levels of VEGFA in ova- ries from each group of mice. B–F. Contents of VEGFA (B), PDGFB (C), PDGFD (D), ANGPT1 (E) and ANGPT2 (F) in ovaries from each group of mice were determined by ELISA. G. The positive staining of CD31 was determined by immunohistochemistry. The allotypic serum was used as negative control to determine the specificity of CD31 staining. Magnification: 400×. Data was expressed as means ± SD (n = 6 in each group). Description of experimental groups as in the legend to Fig. 1. #, ## P < 0.05 and P < 0.01, respectively vs. control group; *, ** P < 0.05 and P < 0.01, respectively, vs. PCOS + Ad-vector group.

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Negative Control, Control, Plasmid Preparation

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 3. HDAC5 suppressed oxidative stress in ovaries of DHEA-induced PCOS mice. A. ROS level in each group was mea- sured by flow cytometric analysis using DCFH-DA probe. B–D. The malondialdehyde (MDA) levels (B), catalase (CAT) activity (C) and superoxide dismutase (SOD) activity (D) were measured as described in Methods. Data was expressed as means ± SD (n = 6 in each group). #, ## P < 0.05 and P < 0.01, respectively vs. control group; *, ** P < 0.05 and P < 0.01, respectively, vs. PCOS + Ad-vector group.

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Activity Assay, Control, Plasmid Preparation

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 4. HDAC5 inhibited HIF-1α/VEGF/VEGFR2 signaling in ovaries of DHEA-induced PCOS mice. A–B. Western-blot analysis of the protein levels of HIF-1α, VEGFA, p-VEGFR2 (Tyr 1175) and VEGFR2. C. Real-time PCR analysis of the mRNA levels of HIF-1α and VEGFR2. D. Co-immunoprecipitation was performed to determine the interaction between HDAC5 and VEGFR2 in ovaries of PCOS mice with anti-HDAC5 antibody. E. Acetylation level of VEGFR2 in ovaries from each group of mice was deter- mined by immunoprecipitation using anti-VEGFR2 antibody. F. Brief schematic presentation of the proposed effect of HDAC5 on angiogenesis. Data was expressed as means ± SD (n = 6 in each group). #, ## P < 0.05 and P < 0.01, respectively vs. control group;

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control

Journal: Cell

Article Title: SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

doi: 10.1016/j.cell.2023.09.002

Figure Lengend Snippet: Figure 2. SND1 is a SARS-CoV-2 host dependency factor (A) Western blot analysis of SND1 knockout (KO) and control (CTRL) cell lines (A549ACE2). Actin serves as control. (B) Analysis of dsRNA foci in SND1 KO and CTRL cells compared to wild-type (WT) cells at 8 hpi. Median value indicated by black line. Representative images are shown in Figure S2B. p values determined by one-way ANOVA. (C) RT-qPCR of SARS-CoV-2 RNA levels in SND1 KO and CTRL cells transduced with empty vehicle (+eV) or SND1 (+SND1) as indicated. Quantification relative to 18S rRNA and CTRL at 6 hpi. Values are mean ± SD (n = 4 independent experiments; 3 technical replicates each). p values determined by two-way ANOVA with Dunnett’s test. (D) Time course of SARS-CoV-2-GFP growth in SND1 KO and CTRL cells transduced with empty vehicle (+eV) or SND1 (+SND1) as indicated. Values are mean ± SEM. Representative of 3 independent experiments is shown. (E) Area under the curve (AUC) analysis of 3 independent time course measurements of SARS-CoV-2-GFP growth. Normalization relative to mean of control (CTRL1 +eV). Values are mean ± SD. p values determined by one-way ANOVA with Dunnett’s test. (F) RT-qPCR of M (top) and ORF1a (bottom) RNA levels at 4–12 hpi in SND1 KO and CTRL cells transduced with empty vehicle (+eV), or SND1 (+SND1) as indicated. Quantification relative to 18S rRNA and CTRL at 4 hpi. Values are mean ± SD (n = 2 independent infections). Schematic: primer design to quantify gRNA and sgmRNA expression. p values determined by two-way ANOVA with Dunnett’s test. (G) RT-qPCR of SARS-CoV-2 RNA levels (N) in SND1 KO and CTRL cells transduced with empty vehicle (eV), full-length SND1 (WT), or SND1 deletion mutants as indicated. Quantification relative to 18S rRNA and CTRL. Values are mean ± SD (n = 3 independent infections). p values determined by two-way ANOVA with Dunnett’s test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant. All SARS-CoV-2 infections were performed in A549ACE2 cells at MOI 3 plaque- forming unit (PFU)/cell. See also Figure S2.

Article Snippet: For detection of negative-sense viral RNA, we performed a denaturation step before adding the split initiator probes: The cells were incubated with pre-heated 90% DMSO at 70 C for 1 h, cooled on ice and washed once with icecold PBS, followed by two washes with PBS at room temperature.64 For combining immunostaining with HCR-based detection, we used anti-J2 antibody (Jena Bioscience, RNT-SCI-10010200, 1:500) or anti-SND1 antibody (Proteintech, 60265-1- Ig, 1:500).

Techniques: Western Blot, Knock-Out, Control, Quantitative RT-PCR, Transduction, Expressing

Journal: Cell

Article Title: SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

doi: 10.1016/j.cell.2023.09.002

Figure Lengend Snippet: Figure 3. SND1 binds negative-sense viral RNA (A) Alignment of strand-separated eCLIP data for SND1 and CNBP8 to SARS-CoV-2 genome. Relative information in IP vs. size-matched input (SMI) is calculated at each position (STAR Methods) and displayed for positive-sense (blue) and negative-sense RNA (magenta). Peaks significantly enriched relative to SMI are indicated in gray. Lower panel displays zoom in to the S to ORF10 region. eCLIP was performed in Huh-7 cells at 24 hpi. (B) IF staining of SND1 and dsRNA in SARS-CoV-2 infected A549ACE2 cells at 8 hpi (MOI = 3 PFU/cell). Representative images are shown. Overlap is quantified by Manders’ co-localization coefficient (n = 8 images). Uninfected cells shown in Fig- ure S3A. (C) HCR RNA-FISH for negative-sense RNA (N) combined with HCR IF for SND1 in SARS-CoV-2 infected A549ACE2 cells at 8 hpi (MOI = 3 PFU/cell). Representative images are shown. Overlap is quantified by Manders’ co-localization coefficient (n = 6 images). Cells were denatured prior to N RNA detection; non-denatured cells shown in Figure S3B. Scale bars, 25 mm. See also Figure S3 and Table S3.

Article Snippet: For detection of negative-sense viral RNA, we performed a denaturation step before adding the split initiator probes: The cells were incubated with pre-heated 90% DMSO at 70 C for 1 h, cooled on ice and washed once with icecold PBS, followed by two washes with PBS at room temperature.64 For combining immunostaining with HCR-based detection, we used anti-J2 antibody (Jena Bioscience, RNT-SCI-10010200, 1:500) or anti-SND1 antibody (Proteintech, 60265-1- Ig, 1:500).

Techniques: Staining, Infection, RNA Detection

Journal: Cell

Article Title: SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

doi: 10.1016/j.cell.2023.09.002

Figure Lengend Snippet: Figure 4. SND1 is required for nascent SARS-CoV-2 RNA synthesis early during infection (A) Outline of SLAM-seq method. (B) Strategy to measure nascent SARS-CoV-2 RNA synthesis in SND1 knockout (KO) and control (CTRL) or wild-type (WT) cells. (C) SLAM-seq analysis at 6 hpi. Average log2 fold changes in total RNA (x axis) and newly synthesized RNA (y axis) are shown for SND1 KO and CTRL cells, relative to WT cells (n = 2 independent infections). (D) Absolute quantification of viral RNA copy numbers by digital droplet PCR. Ratio of sgmRNA (N) relative to gRNA (ORF1a) is shown at 4–12 hpi, in SND1 KO and CTRL cells transduced with empty vehicle (+eV) or SND1 (+SND1) as indicated. Cells infected with SARS-CoV-2 at MOI 3 PFU/cell. Values are mean ± SD (n = 2 independent infections). p values determined by two-way ANOVA with Dunnett’s test. ****p < 0.0001, **p < 0.01, ns = not significant. See also Figure S4.

Article Snippet: For detection of negative-sense viral RNA, we performed a denaturation step before adding the split initiator probes: The cells were incubated with pre-heated 90% DMSO at 70 C for 1 h, cooled on ice and washed once with icecold PBS, followed by two washes with PBS at room temperature.64 For combining immunostaining with HCR-based detection, we used anti-J2 antibody (Jena Bioscience, RNT-SCI-10010200, 1:500) or anti-SND1 antibody (Proteintech, 60265-1- Ig, 1:500).

Techniques: Infection, Knock-Out, Control, Synthesized, Transduction

Journal: Cell

Article Title: SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

doi: 10.1016/j.cell.2023.09.002

Figure Lengend Snippet: Figure 5. SND1 interacts with RTC compo- nents involved in viral RNA biogenesis (A) Strategy to globally identify SND1 protein- protein interactome changes upon SARS-CoV-2 infection. (B) Fold change correlation plot displaying SND1 interacting proteins enriched over IgG control in SARS-CoV-2 infected (SCoV-2, y axis) and unin- fected (mock, x axis) A549ACE2 cells (n = 2 inde- pendent experiments). Candidates with a fold change > 1.5 or < 0.66 and FDR < 0.2 in infected relative to uninfected cells are displayed. Proteins with a substantial SND1 interaction change (ab- solute log2 fold change > 1, FDR < 0.05) are highlighted in red and blue. (C) Computational slices of representative elec- tron tomograms of SND1 knockout (KO) and control (CTRL) cells infected with SARS-CoV-2. Zoom in to region containing DMVs is shown at different magnifications. Left: scale bars, 1 mm; center: scale bars, 250 nm; right: scale bars, 100 nm. (D) Quantification of cross-sectional DMV area in SND1 KO and CTRL cells. Box: 25th and 75th

Article Snippet: For detection of negative-sense viral RNA, we performed a denaturation step before adding the split initiator probes: The cells were incubated with pre-heated 90% DMSO at 70 C for 1 h, cooled on ice and washed once with icecold PBS, followed by two washes with PBS at room temperature.64 For combining immunostaining with HCR-based detection, we used anti-J2 antibody (Jena Bioscience, RNT-SCI-10010200, 1:500) or anti-SND1 antibody (Proteintech, 60265-1- Ig, 1:500).

Techniques: Infection, Control, Knock-Out

Journal: Cell

Article Title: SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

doi: 10.1016/j.cell.2023.09.002

Figure Lengend Snippet: Figure 6. NSP9 is covalently linked to SARS-CoV-2 RNA and SND1 modulates NSP9 occupancy and its covalent linkage to viral RNA (A) Schematic of covalent RNA immunoprecipita- tion (cRIP) to map covalent RNA-protein linkages formed in the absence of UV-crosslinking. (B) Alignment of strand-separated NSP9 cRIP data to SARS-CoV-2 genome. Relative informa- tion in IP vs. SMI is calculated at each position (STAR Methods) and displayed for positive-sense (blue) and negative-sense (magenta) RNA. Zoom- in views of 50 and the 30 end of the viral RNA genome are shown for representative experiment in A549ACE2 cells at 8 hpi. NSP9 peaks significantly enriched relative to SMI (log2 fold change > 2, p < 0.05, one-sided Fisher test) are indicated. RT stops are denoted in black (relative information in IP vs. SMI). Green and red boxes indicate regions of interest. Scale: colored numbers relate to normalized coverage (IP vs. SMI); black numbers relate to normalized RT stops (IP vs. SMI). (C) Zoom-in view of NSP9 cRIP signal and RT stops (relative information in IP vs. SMI) in negative-sense RNA at 50 and 30 end of the viral genome. Gray boxes indicate TRS-L region and poly(A/U) tail. Sequence corresponds to positive strand. (D) SND1-dependent changes in NSP9 binding on peak regions relative to non-peak regions in negative-sense RNA in A549ACE2 cells (SND1 KO vs. CTRL) at 12 hpi. Color scale reflects localiza- tion of peaks. Peaks with strongest NSP9 binding change are annotated. Initiation site-proximal peaks are encircled. (E) As in (D) but for positive-sense RNA. *Actual p value of peak (5–55 nt) was computed as p < 4.9 3 10324; for visualization this p value was set to p = 1.0 3 10100. (F) SND1-dependent changes in covalent NSP9-RNA linkages across the SARS-CoV-2 genome in positive and negative-sense RNA. Representative experiment in A549ACE2 cells (SND1 KO vs. CTRL) at 12 hpi is shown. Actual p value of nucleotide 1 was computed as p < 4.9 3 10324; for visualization this p value was set to p = 1.0 3 10100. See also Figure S6 and Tables S3, S5, and S6.

Article Snippet: For detection of negative-sense viral RNA, we performed a denaturation step before adding the split initiator probes: The cells were incubated with pre-heated 90% DMSO at 70 C for 1 h, cooled on ice and washed once with icecold PBS, followed by two washes with PBS at room temperature.64 For combining immunostaining with HCR-based detection, we used anti-J2 antibody (Jena Bioscience, RNT-SCI-10010200, 1:500) or anti-SND1 antibody (Proteintech, 60265-1- Ig, 1:500).

Techniques: Sequencing, Binding Assay