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Hela tet on

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The HeLa Tet-ON is a cell line derived from human cervical cancer cells. It is engineered to express a Tet-ON system, which allows for the inducible expression of genes of interest in the presence of doxycycline.

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Lab products found in correlation

Hela tet off, by Takara Bio (2 mentions) Hek293, by American Type Culture Collection (2 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Effectene, by Qiagen (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Amphotericin b, by Thermo Fisher Scientific (1 mentions) On target plus non targeting control sirna 1, by Horizon Discovery (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Ni nta agarose, by Qiagen (1 mentions) Complete protease inhibitor tablet, by Roche (1 mentions)

7 protocols using hela tet on

1

Cell Line Characterization and Treatment

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HeLa Tet-ON (Clontech), HeLa Tet-OFF (Clontech), and A9D GRN patient fibroblasts (ND40082; obtained from NINDS Human Genetics Resource Center DNA and Cell Line Repository; https://www.coriell.org/1/ninds) were used.
For several experiments, HeLa Tet-ON cells were used for continuity, but doxycycline induction was not used. In these cases, we refer to these cells as “HeLa” in the manuscript to avoid confusion. Cell lines have been further specified in Supplemental Experimental Procedures.
Cycloheximide 50 μg/mL was used for 24 hr in HeLa Tet-ON and 10 μg/mL for indicated time in A9D GRN fibroblasts.
Doxycycline 1 μg/mL was used for indicated time in HeLa Tet-OFF.
Pinarbasi E.S., Karamyshev A.L., Tikhonova E.B., Wu I.H., Hudson H, & Thomas P.J. (2018). Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell reports, 23(10), 2844-2851.
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2

Culturing HeLa, 3T3, and Hippocampal Neurons

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HeLa Tet-ON (referred to within the manuscript as HeLa) cells were used for ease of maintenance and transfection. HeLa Tet-ON (Clontech, Mountain View, CA) cells and 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin at 37 degrees in bank percentage oxygen. Hippocampal neurons were isolated from C57/BL6 rat P2 pups and cultured as previously described in Li et al.41 (link). All animal procedures conformed to the guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center.
Pinarbasi E.S., Cağatay T., Fung H.Y., Li Y.C., Chook Y.M, & Thomas P.J. (2018). Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Scientific Reports, 8, 7083.
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3

Knockout and culture of USP7-deficient cells

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GT1-7 hypothalamic neuronal cells (kindly provided by Pamela Mellon, UCSD)(Wetsel et al., 1991 (link)), HCT116 USP7+/+, USP7+/−, USP7−/− cells (kindly provided by Bert Vogelstein, Johns Hopkins University) (Cummins et al., 2004 (link)), HEK293 (ATCC), HeLa Tet-ON (Clontech), or U2OS (ATCC) cells were grown in DMEM (Invitrogen) supplemented with 10% FBS (Hyclone), 2 mM L-Glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and 0.25 mg/ml amphotericin B (Invitrogen). Plasmid and siRNA transfection were performed for 48-96 hours with Effectene (QIAGEN) or Lipofectamine RNAiMAX (Invitrogen), respectively, according to the manufacturer's protocol.
siRNAs (GE Dharmacon or Sigma) and antibodies used in this study are listed in the supplemental experimental procedures. USP7 knockout GT1-7 cells were generated by cotransfection of plasmids encoding GFP-Cas9 and a gRNA plasmid targeting USP7 (5’-GGTTGCCTCGGAGCGCCAAC-3’). Forty-eight hours after transfection, cells were flow sorted for GFP-positive cells. USP7 disruption was confirmed by western blotting.
Hao Y.H., Fountain MD J.r., Tacer K.F., Xia F., Bi W., Kang S.H., Patel A., Rosenfeld J.A., Caignec C.L., Isidor B., Krantz I.D., Noon S.E., Pfotenhauer J.P., Morgan T.M., Moran R., Pedersen R.C., Saenz M.S., Schaaf C.P, & Potts P.R. (2015). USP7 acts as a molecular rheostat to promote WASH-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder. Molecular cell, 59(6), 956-969.
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4

Cell Line Maintenance and Transfection Protocols

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HeLa Tet-on (Clontech), HEK293 (American Type Culture Collection, ATCC), HEK293T (ATCC), and T-REx293 IRE1-GFP cells (Li et al., 2010 (link)) were maintained at 37C, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Sigma) supplemented with 4500 mg/L glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, 10% fetal bovine serum (Sigma), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma). T-REx293 IRE1-GFP cells were kindly provided by Dr. Peter Walter (UCSF/HHMI). Transient transfection of plasmids were performed using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Transient gene knockdown was accomplished by transfection of small double-stranded interfering RNAs (siRNA) into cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The siRNAs were synthesized by Dharmacon using the sequence information in the Key resources table. Silencer Negative Control No. 1 siRNA (Ambion) or ON-TARGET plus Non-targeting Control siRNA #1 (Dharmacon) were used as vehicle negative controls.
Wu I.H., Yoon J.S., Yang Q., Liu Y., Skach W, & Thomas P. (2021). A role for the ribosome-associated complex in activation of the IRE1 branch of UPR. Cell reports, 35(10), 109217.
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5

Immortalized TP53-/- Hepatocytes

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Liver progenitor cells from embryonic day 18 fetal livers from TP53−/− mice were isolated and immortalized with MSCV-based retroviruses expressing Myc–IRES-GFP as previously described (Zender et al. 2006 (link)) to generate TP53−/− hepatocytes myc (PHM-1) cells. PHM-1, HuH7, HepG2, Hep3B FLC-4, BNL-1ME, Hc3716-hTERT (Waki et al. 2010 (link)), and HeLa Tet-on (Clontech) cells were grown in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mg/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines have been tested and authenticated by the biosynthesis DNA Identity Testing Center on July 29, 2013.
Shilo A., Ben Hur V., Denichenko P., Stein I., Pikarsky E., Rauch J., Kolch W., Zender L, & Karni R. (2014). Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development. RNA, 20(4), 505-515.
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6

Cell Line Characterization and Treatment

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HeLa Tet-ON (Clontech), HeLa Tet-OFF (Clontech), and A9D GRN patient fibroblasts (ND40082; obtained from NINDS Human Genetics Resource Center DNA and Cell Line Repository; https://www.coriell.org/1/ninds) were used.
For several experiments, HeLa Tet-ON cells were used for continuity, but doxycycline induction was not used. In these cases, we refer to these cells as “HeLa” in the manuscript to avoid confusion. Cell lines have been further specified in Supplemental Experimental Procedures.
Cycloheximide 50 μg/mL was used for 24 hr in HeLa Tet-ON and 10 μg/mL for indicated time in A9D GRN fibroblasts.
Doxycycline 1 μg/mL was used for indicated time in HeLa Tet-OFF.
Pinarbasi E.S., Karamyshev A.L., Tikhonova E.B., Wu I.H., Hudson H, & Thomas P.J. (2018). Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell reports, 23(10), 2844-2851.
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7

Affinity Purification of Tagged Proteins

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HeLa Tet-ON (Clontech) cells were transfected using Lipofectamine 2000 (Invitrogen) on 6-well plates and harvested 18–24 h later. Cells were washed with cold PBS and lysed using buffer A (PBS, 20 mM imidazole, 1% Triton X-100, and a cOmplete protease inhibitor tablet (Roche). Lysates were clarified for 30 min at 13,000 g at 4°C. The supernatant was then incubated with Ni-NTA agarose (Qiagen) for 1 h and washed three times in buffer B (PBS, 20 mM imidazole, and 0.1% Triton X-100). SDS-PAGE loading buffer was added to beads, run on 10% SDS-PAGE gels, and immunoblotted with antibodies.
Safavian D., Kim M.S., Xie H., El-Zeiry M., Palander O., Dai L., Collins R.F., Froese C., Shannon R., Nagata K.I, & Trimble W.S. (2023). Septin-mediated RhoA activation engages the exocyst complex to recruit the cilium transition zone. The Journal of Cell Biology, 222(4), e201911062.
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