Pirfp

The following constructs have been described. Human TDP43 cDNA (MGC #3506121; Thermo Fisher Scientific, Waltham, MA) was fused to meGFP45 (link), mKate2 (Evrogen, Moscow, Russia), mTFP1 (Allele Biotechnology, San Diego, CA), and mVenus46 (gift from Prof. T. Nagai); TDP43-GFP, R-TDP43-G, T-TDP43-Y, respectively. The plasmids harboring EGFP-TDP35 and EGFP-TDP25 (gifts from Dr. Y. J. Zhang and Prof. L. Petrucelli) sequence subcloned into the meGFP-C1 vector29 (link) (GFP-TDP35 and GFP-TDP25, respectively). NLS sequences were incorporated into GFP-TDP25 between GFP and TDP25 sequence (GFP-NLS-TDP25 or GFP-NLS^NP-TDP25). FLAG-tagged TDP43 and the CTFs were modified from GFP-tagged plasmids (FLAG-TDP43/35/25/NLS-TDP25). pBOS-H2B-mSECFP (H2B-CFP) and pBOS-H2B-iRFP (H2B-iRFP) was created using pBOS-H2B-GFP47 (link), pRSETb-mSECFP (gift from Prof. T. Nagai), and piRFP (Addgene plasmid #31857). LSSmOrange-DEVD-mKate2 (Addgene plasmid #37132) was used as a sensor for caspase 3-activation. Human ubiquitin sequence (IMAGE #5766897, Thermo Fisher Scientific) subcloned into mCherry-C1 (RFP-Ub). Detailed construction procedures were described in Supplemental Information.

U87-MG cells (ATCC, HTB-14) were infected with lentiviral vectors (from the National RNAi Core Facility, Taiwan) bearing the coding sequences of iRFP (piRFP; Addgene, USA) and GFP (pLKO_AS3w.tGFP; RNAi Core Lab, Taiwan) and then selected for double-fluorescent cells using a flow cytometer (FACSAria III; BD Biosciences). Selected cells were further cultured to yield a stable cell line, U87-L-iRFP. U87-MG and U87-L-iRFP cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin–amphotericin B (Gibco) at 37 °C in 5% CO₂. The double fluorescence of U87-L-iRFP cells was observed by using a confocal microscope (LM-510; Zeiss, Germany). Photos were taken using Zeiss LSM 510 laser confocal microscope (Carl Zeiss, Thornwood, NY) with excitation of GFP at 488 nm laser and excitation of iRFP at 633 nm laser. Plan-Neofluar 20X/0.5Ph2 objective was used and magnification was 200 ×. LSM 510 basic software was used for image capture and analysis.

piRFP was a gift from Vladislav Verkhusha (Addgene plasmid # 31857) [27 (link)]. Using lentiviral vectors (National RNAi Core Facility, Taiwan) carrying the coding sequences of iRFP (piRFP; Addgene, USA) and GFP (pLKO_AS3w. tGFP; RNAi Core Lab, Taiwan), we transfected the 22Rv1 cell line and analyzed the ratio of cells expressing iRFP and GFP fluorescence using flow cytometry (FACSAccuri™ C6 Plus; BD Biosciences). High-speed cell sorting (FACSAriaIII; BD Biosciences) was then used to select for 22Rv1 cells expressing iRFP at an excitation wavelength of 710 nm; these cells were then termed “22Rv1-iRFP-tGFP.” We then observed the fluorescence expression of iRFP and GFP in 22Rv1-iRFP-tGFP via confocal microscopy (Carl Zeiss, Thornwood, NY). 22Rv1-iRFP-tGFP cells of 5 × 10⁵ and 1 × 10⁶ were placed in the agent calibration phantom of FMT to detect whether the iRFP fluorescence signal representing the cells increased with the number of cells. The culturing method for 22Rv1-iRFP-tGFP was the same as for the parental 22Rv1 cells.