trc cloning vector addgene 10878 pplko 1 shvcp  (Addgene inc)

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A-D) H522 ( A, B ) and H661 ( C, D ) parental wild-type (WT), TMEM106B KO and non-targeting (NT) guide RNA transduced cells were inoculated with SARS-CoV-2 S-mNG E 484D at an MOI of 1. RT-qPCR for cell-associated SARS-CoV-2 RNA ( A, C ) and enumeration of mNG positive cells by FACS ( B, D ) at 48 hpi is shown (** P < 0.01; **** P < 0.001 by two-tailed unpaired t -test).

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: ( A-F ) WT and TMEM106B KO H522 ( A-C ) and H661 ( D-F ) cells stably transduced with an empty vector (EV), full-length (FL) TMEM106B, TMEM106BΔCTD, or TMEM106BΔNTD were inoculated with SARS-CoV-2-mNG S E484D ( A, D ) or VSV-GFP-SARS-CoV-2 S E484D ( B, E ) at an MOI of 1. RT- qPCR analysis of cell-associated SARS-CoV-2 RNA at 72 hpi ( A, D ) and flow cytometric quantification for GFP positive cells at 48 hpi ( B, E ). Western blot analysis of indicated cell lysates using an anti-HA antibody ( C, F ). Beta-actin was used as loading control. Data in A, B, D, E show the mean of three independent biological replicates, error bars show the SEM. For statistical analysis, TMEM106B complemented cells were compared to EV-transduced cells. (non-significant (ns), ** P < 0.01, **** P < 0.0001 by one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 TMEM106B KO cells stably transduced with an empty vector (EV), full length TMEM106B (FL), TMEM106BΔCTD, TMEM106BΔNTD as well as H522 cells stably expressing ACE2 were inoculated with SARS-CoV-2-mNG S E484D or WT SARS-CoV-2-mNG at an MOI of 1 at 16°C. At 1 hpi, cells were fixed and analyzed for viral RNA (green) using RNAScope as detailed in Materials and Methods. Nuclear DAPI staining is shown in blue. (B) Quantification of the data shown in A. For S E484D infections, all data was compared to EV, for S-WT infections H522-ACE2 OE data was compared to H522-TMEM106BΔNTD (* P < 0.05; **** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons). Scale bar=10μM.

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Expressing, RNAscope, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 TMEM106B KO cell derivatives stably transduced with an empty vector (EV), and HA-tagged full length TMEM106B (FL), TMEM106BΔCTD, TMEM106BΔNTD were subjected to immunofluorescence microscopy. Detection of TMEM106B variants (anti-HA, green), Rab7a (lysosome marker, in red), wheat germ agglutinin (membrane, cyan), and cellular nuclei (DAPI, blue). Scale bar=10μM. (B, C) BHK-21 cells stably expressing CD71-TMEM106B-CTD-HA (CD71-CTD-HA) chimeric protein were subjected to western blotting ( B ) or surface staining and flow cytometry ( C ) using an anti-HA antibody. (D, E) BHK21-CD71-TMEM106B-CTD-HA cells were inoculated with SARS-CoV-2 mNG S E484D at a MOI of 1. Infected cells were subjected to FACS to enumerate mNG positive cells at 48 h post-infection (D) or RT-qPCR analysis to analyze cell-associated SARS-CoV-2 RNA at 48 h post infection (E). ( F ) BHK21-CD71-TMEM106B-CTD cells were infected as in (D, E) but in the presence of 50 mM ammonium chloride as in . Cell-associated SARS-CoV-2 RNA levels were assessed by RT-qPCR at 48 h post infection. ( G, H ) SARS-CoV-2 S E484D (MOI=2 equivalent) was pre-incubated with the indicated concentrations of Fc-TMEM106B-LD in SF-DMEM at 37°C for 30 min. Fc decoy-virus mixture was added on H522 (G) and H661 (H) cells and incubated at 37°C for 2h. Virus inoculum was removed and cells were replenished with complete culture media. Cell-associated viral RNA was analyzed by RT-qPCR at 72 h post infection. Data in E-H show the average of three independent replicates with error bars displaying the SEM. ** P < 0.01; *** P < 0.001; *** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons.

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Immunofluorescence, Microscopy, Marker, Membrane, Expressing, Western Blot, Staining, Flow Cytometry, Infection, Quantitative RT-PCR, Incubation, Virus, Two Tailed Test

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 and H661 WT cells were inoculated with WT VSV-GFP-SARS-CoV-2 S (WT) or a derivative bearing the S E484D substitution at an MOI of 1. GFP positive cells were enumerated by flow cytometry at 48 hpi. (B) VSV-GFP-SARS-CoV-2 S E484D was serially passaged in H522 cells for 8 rounds as explained in Materials and Methods. Aliquots of virus from each passage was titered on H522 and H522-TMEM106B KO cells using 1, 5, 25 or 125 µl of the inoculum. Data show the percentage of GFP positive cells at the indicated inoculum for all 8 passages. (C) Aliquots of virus from (B) was titered on H661 cells. (D) Five different virus clones were plaque purified from Passage 7 and sequenced. Table shows the observed substitutions and their location within S. (E-G) H522-ACE2 ( E ), H522 ( F ), and H661 ( G ) cells were inoculated with parental VSV-GFP-SARS-CoV-2 S E484D or the five different plaque purified virus clones from (D) at an MOI of 0.1. H522-ACE2 cells were fixed at 16 hpi, and H522 and H661 were fixed at 48 hpi for enumeration of GFP positive cells by flow cytometry. Data show the mean from three independent experiments, error bars show the SEM. For D, E, and F, all five clones were compared to the parental virus (nonsignificant (ns), * P < 0.05 by one-way ANOVA with Dunnett’s correction for multiple comparisons).

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Flow Cytometry, Virus, Clone Assay, Purification

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet:

Article Snippet: Briefly, lentiviruses were produced in HEK293T cells by polyethyleneimine (Polysciences)-mediated transfection using a pLV-EF1a-IRES-Puro (Addgene Plasmid #85132)-based vector expressing TMEM106B derivatives, psPAX2 as packaging plasmid (Addgene #12260) and VSV-G expressing envelope plasmid (Addgene #12259).

Techniques: Cloning