Site directed mutagenesis kit

The 5′-flanking region (nucleotides −604 to +5) of S1PR1 was cloned into the pGL3-Basic vector (Promega) by Generay Biotech. The mutant constructs MT1, MT2, and MT3 were generated using a Site-Directed Mutagenesis Kit (Clontech) and sequences were verified. The wild-type and mutant fragments were inserted into a pmirGLO Dual-Luciferase vector (Promega) downstream of the luciferase gene, and the resulting constructs were designated pGL-S1PR1-WT, pGL-S1PR1-MT1, pGL-S1PR1-MT2, and pGL-S1PR1-MT3. Cells were infected with the indicated vector and then transfected with the indicated luciferase constructs, as described in the corresponding figure legend. Cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. According to the manufacturer’s protocol, the cells were assayed 24 h after transfection to determine both firefly and Renilla luciferase activities using a luciferase assay kit (Promega). All transfections were performed in triplicate.

CantonS, elav-GAL4, UAS-PKA-C1, UAS-DsRed, GMR52D11-lexA and P{UAS-CD4-spGFP1–10}3, P{lexAop-CD4-spGFP11}3 were obtained from the Bloomington Stock Center. Pka-C3^NIG.6117R was obtained from the National Institute of Genetics (NIG-Fly), Japan. natalisin-GAL4 was kindly provided by Y. Park and Y-J. Kim and is described in^{25 (link)}. PBacf07226 and PBacPka-C3^f00695 were obtained from the Exelixis Collection at the Harvard Medical School. UAS-PKA-C3 is described in^{21 (link)}. The constitutively inactive PKA-C3 construct was generated by replacing aspartate 397 (D³⁹⁷, see Supp. Figure 3) with alanine by site–directed mutagenesis using the site-directed mutagenesis kit from clontech. D³⁹⁷ is part of the highly conserved YRDLKPEN core sequence of the catalytic loop and mutations in the conserved aspartate have been shown to abolish or dramatically reduce the catalytic activity^{36 (link)–38 (link)}. The construct was tagged with HA. Flies were maintained on standard fly food under a 12:12 h light:dark cycle. Stocks were maintained at 18 °C while crosses and aging flies were maintained at 25 °C. The age of the flies in each experiment is indicated in the figures and figure legends.

The pmirGLO-RTCB-Wt reporter was generated by cloning wild-type putative target region of RTCB to pmirGLO plasmids (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The pmirGLO-RTCB-mut reporter was generated using a site-directed mutagenesis kit (Clontech, San Francisco, CA, USA). Then, 1 × 10⁴ Cells grown in 96-well plates were co-transfected with 50 ng pmirGLO-RTCB-Wt reporter, 50 ng pmirGLO-RTCB-mut reporter, miR34a mimics, or miR-Scr using Lipofectamine 2000 reagent followed by the analysis of luciferase activity using Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland). Firefly luciferase activity normalized against renilla luciferase activity, which was used to represent transfection efficiency, is presented as reporter activity in this study.

Mouse BCL2 cDNA was cloned in pUC19 plasmid. The nucleotides related to each serine (s) residue were replaced with corresponding nucleotides to cause a conserved alteration to alanine (A) using a site-directed mutagenesis Kit (Clontech, San Francisco, CA, USA). Each mutation was identified by cDNA sequencing and then cloned into pCIneo mammalian expression vector (Promega, Madison, WS, USA). pCIneo plasmid containing each BCL2-mutating cDNA was transfected into M-CSF-pretreated OCPs using electroporation. The efficiency of site-directed mutagenesis was verified using western blot analyses.

The 3’-UTR of the human EPLIN mRNA (NM_001114676.1) was amplified from cDNA derived from total RNA of HUVECs, and subcloned into pGL3-promoter vector (Promega, USA) to construct a pGL3-EPLIN-WT reporter plasmid. A mutant reporter (pGL3-EPLIN-mt) with a mutation in the 3’-UTR complementary to the seed sequence of miR-93-5p was generated by using a Site Directed Mutagenesis Kit (Clontech, USA) according to manufacture’s instructions. The PCR primers are listed in Supplementary Table 1. To perform the luciferase reporter assay, HeLa cells were cotransfected with 100 ng of pGL3-EPLIN-WT or pGL3-EPLIN-mt, together with miR-93-5p mimics (50, 100 nM) or control oligos, and pRL-TK (5 ng). Cells were collected 48 h after the transfection, and firefly and renilla luciferase activities were analyzed with the Dual-Luiferase Reporter Assay System (Promega, USA) according to the manufacturer’s instruction.

Flies were maintained in standard cornmeal media at 25 °C. Complementary DNA (cDNA) encoding the full length of TREM2 (NM_018965, RC221132) and TYROBP (NM_198125, RC203771) with Myc-DDK tag were obtained from OriGene Technologies, Inc. These constructs were subcloned into a pJFRC19-13XLexAop2 vector (Addgene #26224). TREM2R47H mutation was introduced by using site-directed mutagenesis kit (Takara Bio Inc.). Transgenic flies were generated by PhiC31 integrase-mediated transgenesis systems (Best Gene Inc.). Transgenic fly lines carrying UAS-Aβ42 and UAS-tau were previously described [43 (link)–45 (link)]. Repo-LexA (#67096), Elav-GAL4 (#458), GMR-GAL4 (#1104), UAS-para RNAi (#31626), and UAS-mcherry RNAi (#35785) were obtained from the Bloomington Stock Center. For RNA sequencing (RNA-seq), around seven-day-old male flies were used. All experiments were performed using age-matched male flies.