Pcdh ef1α mcs t2a puro lentivirus vector

The the coding DNA sequences (CDSs) of human L3MBTL2 and CGA were subcloned into the pCDH-EF1α-MCS-T2A-Puro lentivirus vector (System Biosciences: CD527A-1)). The shRNA sequences of L3MBTL2, CGA and their negative control were cloned into the pLVX-shRNA2-BSD lentivirus vector upgraded by pLVX-shRNA2 (Clonetech: 632179) (in which the ZsGreen gene sequence is replaced with a BSD resistance gene sequence). Each of the purified plasmids was packaged into a lentivirus and then transfected into PANC-1, ASPC-1, HepG2 and H1299 cells. The lentivirus packing and transfection methods are same as previously reported (Huang et al., 2022 (link)). Then, positive overexpressed and knockdown cells were selected using 2 μg/mL puromycin or 5 μg/mL blasticidin S for 10 days.

To generate the FAM110A-OE, HIST1H2BK-OE, TSPAN1-OE, shFAM110A, shTSPAN1 and FAM110A-OE coupled with HIST1H2BK-KD stably transfected cell lines, we subcloned the CDSs of FAM110A (transcript variant 1, NM_031424), HIST1H2BK and TSPAN1 into the pCDH-EF1α-MCS-T2A-Puro lentivirus vector (System Biosciences: CD527A-1) and cloned the shRNA sequences of FAM110A and HIST1H2BK into the pLVX-shRNA2-BSD lentivirus vector upgraded by pLVX-shRNA2 (Clonetech:632179). All plasmids were first purified and packaged into lentivirus and then transfected into PANC-1 cells or BXPC-3 cells. After 36 h of transfection, (5 μg/ml) blasticidin S or (2 μg/ml) puromycin was added to the cultured cells to screen the knockdown and overexpressed cells, respectively. After two weeks of screening with puromycin and blasticidin S, all viable cells were considered positive and were further validated by Western blot and qPCR.