Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Detection of SARS-CoV-2 infected cells using high-content imaging under control conditions. Fluorescent signals: green (anti-SARS-CoV NP mAb), blue (Hoechst). (B) Representative flow cytometry analysis plots showing NP+ cells. (C) Measurement of fluorescent intensity in NP+ cells. (D) Percentage of infected MSCs. (E) The supernatants of infected MSCs were collected and titrated on Vero-E6/TMPRSS2 cells to measure infectious viral particle production using the focus-forming assay. (F) After RNA extraction from infected MSCs, the expression of IDO1 was examined using real-time PCR. The graph displays the expression of IDO1 normalized to GAPDH , with the mock group serving as the control. Data are represented as means ± SEM from three independent biological replicates. For , the Delta condition was available from only two independent donors and was therefore presented descriptively and excluded from the statistical analysis. Statistical comparisons among the remaining groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. One-way ANOVA showed a significant overall difference among groups, F(2, 6) = 6.794, P = 0.0287. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Infection, Imaging, Control, Flow Cytometry, Analysis, Focus Forming Assay, RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative western blot depicting the expression of NRP1 in AD-MSCs (No siRNA), cells transfected with non-targeting siRNA (siControl), or siNRP1 for 24 hours. β-actin was used as a loading control. (B) Graph displaying the quantification of NRP1 normalized to β-actin (n = 3). This experiment represents three independent biological replications. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Transfected cells were visualized using high-content imaging. Fluorescent signals: green for anti-NRP1 mAb, blue for Hoechst. (D) Percentage of NRP1-expressing cells normalized to cells transfected with siControl. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a paired t-test. Next, siRNA-transfected cells were infected with different variants of SARS-CoV-2. (E) SARS-CoV-2-infected cells were detected using high-content imaging. Fluorescent signals: green for anti-SARS-CoV NP mAb, blue for Hoechst. (F) The fluorescent intensity of NP+ cells was measured. All data represent three independent experiments. Statistical analysis among each group of variant was performed using an unpaired t-test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Western Blot, Expressing, Transfection, Control, Analysis, Imaging, Infection, Variant Assay

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Fluorescence intensity of NP-positive UCB-CD34 + HSPCs after infection with WT or Omicron variants of SARS-CoV-2. (B) Infectious viral particle production was determined by the focus-forming assay on Vero-E6/TMPRSS2 cells. (C) HSPC phenotype after infected with SARS-CoV-2 was investigated via the expression of primitive markers through flow cytometry. (D) The representative of HSC phenotype after infected with SARS-CoV-2. (E) Fluorescent intensity of NP-positive UCB-CD34 + HSPCs after infected with heat-inactivated WT and Omicron variant of SARS-CoV-2 compared to active virus. (F) The representative of HSC phenotype after infected with heat-inactivated WT and Omicron compared to active virus. All data are represented as mean ± SEM from three independent biological replicates. Statistical comparisons among Mock, WT, and Omicron in each group were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. For , the unpaired t-test were performed among each group by comparing between Mock vs. WT, Mock vs. Omicron, WT vs. heat-inactivated WT, and Omicron vs. heat-inactivated Omicron. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Fluorescence, Infection, Focus Forming Assay, Expressing, Flow Cytometry, Variant Assay, Virus

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative CFU colonies generated from human UCB-CD34 + HSPCs after exposure to WT or Omicron SARS-CoV-2 compared to Mock. (B) Quantification of burst-forming unit–erythroid (BFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM), and total CFU output (C) Frequency of early erythroid progenitors (CD34 − CD36 ⁺ CD71 ⁺ CD235 − ) following in vitro erythroid differentiation of SARS-CoV-2-exposed CD34 ⁺ cells. (D) CFU output (BFU-E, CFU-GM, and total) in the virus inhibition experiment comparing active versus heat-inactivated virus. (E) Frequency of early erythroid progenitors (CD34 − CD36 ⁺ CD71 ⁺ CD235 − ) in the virus inhibition experiment. The results represent the means and SEM of three independent experiments. For and , the unpaired t-test were performed among each group by comparing of total CFUs, BFU-E, and CFU-GM between Mock vs. WT, Mock vs. Omicron, WT vs. heat-inactivated WT, and Omicron vs. heat-inactivated Omicron. For and , statistical comparisons among Mock, WT, and Omicron in each group were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Generated, In Vitro, Virus, Inhibition

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Detection of SARS-CoV-2 infected cells using high-content imaging under control conditions. Fluorescent signals: green (anti-SARS-CoV NP mAb), blue (Hoechst). (B) Representative flow cytometry analysis plots showing NP+ cells. (C) Measurement of fluorescent intensity in NP+ cells. (D) Percentage of infected MSCs. (E) The supernatants of infected MSCs were collected and titrated on Vero-E6/TMPRSS2 cells to measure infectious viral particle production using the focus-forming assay. (F) After RNA extraction from infected MSCs, the expression of IDO1 was examined using real-time PCR. The graph displays the expression of IDO1 normalized to GAPDH , with the mock group serving as the control. Data are represented as means ± SEM from three independent biological replicates. For , the Delta condition was available from only two independent donors and was therefore presented descriptively and excluded from the statistical analysis. Statistical comparisons among the remaining groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. One-way ANOVA showed a significant overall difference among groups, F(2, 6) = 6.794, P = 0.0287. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: After that, the plate was stained with 1:2500 of rabbit anti-SARS-CoV NP (Sino Biological Inc.) and then anti-rabbit HRP (DAKO) was used as secondary antibody.

Techniques: Infection, Imaging, Control, Flow Cytometry, Analysis, Focus Forming Assay, RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative western blot depicting the expression of NRP1 in AD-MSCs (No siRNA), cells transfected with non-targeting siRNA (siControl), or siNRP1 for 24 hours. β-actin was used as a loading control. (B) Graph displaying the quantification of NRP1 normalized to β-actin (n = 3). This experiment represents three independent biological replications. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Transfected cells were visualized using high-content imaging. Fluorescent signals: green for anti-NRP1 mAb, blue for Hoechst. (D) Percentage of NRP1-expressing cells normalized to cells transfected with siControl. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a paired t-test. Next, siRNA-transfected cells were infected with different variants of SARS-CoV-2. (E) SARS-CoV-2-infected cells were detected using high-content imaging. Fluorescent signals: green for anti-SARS-CoV NP mAb, blue for Hoechst. (F) The fluorescent intensity of NP+ cells was measured. All data represent three independent experiments. Statistical analysis among each group of variant was performed using an unpaired t-test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: After that, the plate was stained with 1:2500 of rabbit anti-SARS-CoV NP (Sino Biological Inc.) and then anti-rabbit HRP (DAKO) was used as secondary antibody.

Techniques: Western Blot, Expressing, Transfection, Control, Analysis, Imaging, Infection, Variant Assay