Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: (A) Summary of gene deletion vs. gene overexpression approaches. (B) Rationale for the present study. (C) Experimental outline of the present study. (D) HEK293 and Jurkat cells were inoculated with different volumes of Ebola or rabies pseudovirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This revealed that Jurkat cells are largely refractory to Ebola or rabies pseudovirus entry, relative to HEK293 cells. (E) Construction of a clonal Jurkat cell line, known as “Jurkat C6”, stably expressing a degron-tagged CRISPRa construct ( left ). Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence or absence of TMP (1 μM) for 3 days, followed by flow cytometry to detect hCD19 expression ( right ). (F) At each of the indicated points of the genome-wide CRISPRa screen, the cell population was challenged with Ebola or rabies pseudovirus, and cell infectivity was evaluated by flow cytometry. Upon successive rounds of the screen, the cell population became progressively more susceptible to infection to either Ebola or rabies pseudovirus, respectively. (G) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for Ebola pseudovirus entry. (H) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for rabies pseudovirus entry.

Article Snippet: Afterwards, cells were centrifuged at 1000g for 10 minutes, resuspended in FACS buffer, and flow cytometric measurements were performed on a CytoFLEX S flow cytometry (Beckman Coulter).

Techniques: Over Expression, Marker, Mutagenesis, Flow Cytometry, Infection, Stable Transfection, Expressing, Construct, Transduction, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) Plasmids used to pseudotype non-replicating lentiviruses with either Ebola or rabies envelope proteins. EBOV-GP: glycoprotein of Ebola virus, Makona variant. RABV-GP N2C: glycoprotein of rabies virus, N2C variant. B) HEK293 and Jurkat cells were inoculated with different volumes of VSV envelope protein-pseudotyped lentivirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This served as a positive control to confirm that Jurkat cells and HEK293 cells are both susceptible to VSV pseudovirus entry. C) Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence of different TMP concentrations (0-4 μM) for 2-3 days, followed by flow cytometry to detect human CD19 expression.

Article Snippet: Afterwards, cells were centrifuged at 1000g for 10 minutes, resuspended in FACS buffer, and flow cytometric measurements were performed on a CytoFLEX S flow cytometry (Beckman Coulter).

Techniques: Virus, Variant Assay, Marker, Mutagenesis, Flow Cytometry, Infection, Positive Control, Transduction, Expressing

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. B) L-SIGN or DC-SIGN were expressed in Jurkat or primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of primary T cells to authentic Ebola and Sudan virus infection.

Article Snippet: Afterwards, cells were centrifuged at 1000g for 10 minutes, resuspended in FACS buffer, and flow cytometric measurements were performed on a CytoFLEX S flow cytometry (Beckman Coulter).

Techniques: Expressing, Control, Flow Cytometry, Infection, Mutagenesis, Virus

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. As positive controls, flow cytometry was used to confirm successful delivery of the sgRNA construct as part of the CRISPRa workflow (as denoted by BFP expression) and that NGFR was expressed (upon cDNA expression). B) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. Cells expressing the highest levels of L-SIGN and DC-SIGN were preferentially infected by Ebola pseudovirus. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. On days 0, 1, and 2 post-infection, flow cytometry was performed to determine the percentage of infected cells and qPCR was performed on cell culture supernatants to quantify viral genome replication. This revealed that L-SIGN or DC-SIGN expression enabled authentic Ebola and Sudan virus entry into primary human T cells, but viral genome replication was impaired, perhaps reflective of cell-intrinsic restriction factors.

Article Snippet: Afterwards, cells were centrifuged at 1000g for 10 minutes, resuspended in FACS buffer, and flow cytometric measurements were performed on a CytoFLEX S flow cytometry (Beckman Coulter).

Techniques: Expressing, Control, Flow Cytometry, Infection, Construct, Mutagenesis, Virus, Cell Culture

Journal: Redox Biology

Article Title: Upregulation of PPTC7 in brain microvascular endothelial cell aggravates diabetic cognitive dysfunction by impairing PHB2 mediated mitochondrial function

doi: 10.1016/j.redox.2025.103991

Figure Lengend Snippet: PPTC7 mediates HGHF-induced oxidative stress, MMP reduction, and apoptosis in HBMECs. (A, B) Flow cytometry assay of DCFH-DA and statistical graph for detecting ROS levels in HBMECs with PPTC7 knockdown under NG and HGHF conditions. n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (C, D) Flow cytometry assay of MitoSOX and statistical graph for determining mitochondrial ROS levels in HBMECs with PPTC7 knockdown under NG and HGHF conditions. n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (E, F) Confocal images and statistical graph of ROS immunofluorescence in HBMECs with PPTC7 knockdown under NG and HGHF conditions. Scale bars, 40 μm, n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (G, H) Confocal images and statistical graph of mitochondrial ROS immunofluorescence in HBMECs with PPTC7 knockdown under NG and HGHF conditions. Scale bars, 50 μm, n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (I, J) Flow cytometry assay of TMRE and statistical graph for determining mitochondrial membrane potential (MMP) in HBMECs with PPTC7 knockdown under NG and HGHF conditions. n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (K, L) Representative images and statistical data of JC-1 staining in HBMEC transfected with shNC or shPPTC7 plasmid for detecting MMP under NG and HGHF conditions. Scale bars, 50 μm, n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (M, N) Representative images and statistical graph of TUNEL staining for detecting apoptosis in HBMECs with PPTC7 knockdown under NG and HGHF conditions. Scale bars, 100 μm, n = 3 biological replicates for each group, one-way ANOVA with Tukey's multiple comparison test. (O, P) Western blot images and densitometric quantification of anti-apoptotic protein BCL2, pro-apoptotic proteins BAX, and cleaved caspase-3 in HBMECs transfected with shNC or shPPTC7 plasmid under NG and HGHF conditions. n = 3 biological replicates for each group, two-way ANOVA with Tukey's multiple comparison test. The data are presented as means ± SEMs.

Article Snippet: Hoechst 33342 (0.5 μg/mL, Beyotime) was added for 5 min. After three times washes with warm 1 × Phosphate-Buffered Saline (PBS, 37 °C) to fully remove the unbound probes, The fluorescence signals were detected within 30 min using an Olympus optical microscope (Olympus, Japan) or Beckman CytoFLEX Flow Cytometry (FCM, Beckman Coulter).

Techniques: Flow Cytometry, Knockdown, Comparison, Immunofluorescence, Membrane, Staining, Transfection, Plasmid Preparation, TUNEL Assay, Western Blot