Site directed mutagenesis kit

All plasmids were transfected with Lipofectamine 2000 (Life Technologies-Invitrogen, USA) according to the manufacturer’s instructions. H1.2, H1.3 and H1.4 cDNAs were amplified and cloned into the p3× FLAG-CMV-10 vector (Addgene, USA). Full-length H1.2 and various fragments (N-terminal domain, 1–35 aa; globular domain, 36–112 aa; C-terminal domain, 113–213 aa; ΔC1, 1–179 aa; ΔC2, 1–112+180–213 aa; ΔC, 1–112 aa) were cloned into the pEGFP-C2, pGEX-4T3 or pET28a vectors (Addgene, USA). The full-length FLAG-ATM expression construct was purchased from Addgene, USA. GST-ATM fragments (F1, 1–247 aa; F2, 250–522 aa; F3, 523–769 aa; F4, 722–1102 aa; F5, 1098–1371 aa; F6, 1245–1435 aa; F7, 1239–1770 aa; F8, 1764–2138 aa; F9, 2141–2428 aa; F10, 2427–2841 aa; F11, 2842–3056 aa; F12, 2682–3012 aa) were provided by Dr. Fabrizio d’Adda di Fagagna (FIRC Institute of Molecular Oncology Foundation, Italy). Site-specific mutations of H1.2 (T126/146/165A, T126/146/165E, E115A, S173A, S188A) were generated using a site-directed mutagenesis kit (Vazyme, China).

Control human lens epithelia cells (HLEpiCs) from an unaffected subject were obtained from American Type Culture Collection (ATCC) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). The cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere.
Recombinant human GJA8 was amplified from genomic DNA and inserted into the pEGFP-N1 vector. The mutant GJA8 gene was obtained using a Site-Directed Mutagenesis Kit (Vazyme, China). Recombinant plasmids containing the normal or mutant gene were transfected into HLEpiCs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocols.

SIRT7 and PCAF cDNAs were cloned into p3×FLAG-CMV-10, pET-28a, and pGEX-4T1 plasmids. All indicated mutations were generated using a site-directed mutagenesis kit (Vazyme). All expression constructs were verified by DNA sequencing (Shanghai Sangon Biotech Company). siRNAs (Shanghai GenePharma Company) were used to silence target genes by transient transfection with lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The RNAi oligonucleotide sequences were as follows:
NC siRNA: 5′-UUCUCCGAACGUGUCACGU-3′;
SIRT7 siRNA 1#: 5′-TAGCCATTTGTCCTTGAGGAA-3′;
SIRT7 siRNA 2#: 5′-GAACGGAACTCGGGTTATT-3′.

LT-171-861 was originally designed and synthesized by China Pharmaceutical University. Sorafenib (Cat. #3009) and AC220 (Cat. #5793) were purchased from ApexBio Company (Houston, TX). The human leukemia cell lines MV4-11, MOLM13, and murine pro-B cell line BaF3 were purchased from Cobioer company (Nanjing, China). Human leukemia cell line THP-1, U937 and human marrow stromal cell line HS-5 were originally obtained from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). MV4-11 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Grand Island, NY) containing 10% FBS (Grand Island, NY). MOLM13, THP-1, and U937 cells were cultured in RPMI 1640 medium (Grand Island, NY) with 10% FBS. Murine pre-B cell line BaF3 was cultured in RPMI 1640 (Grand Island, NY) containing 10% FBS and 3 ng/mL interleukin (IL)-3 (R&D, MN, USA). HEK‑293T and HS-5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Grand Island, NY) supplemented with 10% FBS. Plasmids for construction of transformed BaF3-FLT3-wt and BaF3-FLT3-ITD were obtained from Dr. Bailiang He from The University of Hong Kong. A site-directed mutagenesis kit (Vazyme Biotech Co., Ltd, Nanjing, China) was used to generate plasmids for the construction of transformed BaF3-FLT3-ITD-F691L, BaF3-FLT3-ITD-N676D, BaF3‑FLT3-ITD-Y842C and BaF3-FLT3-D835Y.

The pQE-30 plasmid and Escherichia coli BL21(DE3) were purchased from Novagen Inc. (Madison, WI, USA) for T7 RNAP heterologous expression. The site-directed mutagenesis kit and dsRNA quantitative kit were supplied by Vazyme Co., Ltd. (Nanjing, China). ATP, GTP, CTP, UTP, pyrophosphatase, and murine RNase inhibitor were purchased from Vazyme Co., Ltd. (Nanjing, China). MgCl₂, glycerol, dithiothreitol (DTT), and other chemicals were purchased from Aladdin Industrial Inc. (Shanghai, China) and other commercial sources.