Sf solution

For the induced dimerization experiments, 0.3 × 10⁶ WT MNT1 cells were nucleofected with 200 ng of pEGFP-N3-OCA7, 500 ng AKAP1-FRB-BFP2, and 400 ng pmCherry-C2-Rab32-CC/AA-FKBP or pmCherry-C2-Rab38-CC/AA-FKBP plasmids in 20 μl Lonza SF solution using Nucleofector 4D program DS-137, transferred to glass bottom microscopy dishes, and incubated for 24 h with KGM Gold media. Dishes were first imaged in the absence of dimerization reagent. Then, the media were replaced with fresh media containing 100 nM rapalog (Takara A/C heterodimerizer), and cells were incubated for 1 h to achieve efficient dimerization. After incubating with rapalog, dishes of cells were imaged again to test for OCA7-EGFP recruitment to Rab32/38-labeled mitochondria. Colocalization analysis was performed in Slidebook6.

To obtain co-knock of V-ATPase and SOX11, Z138 SOX11^IND+/SOX11^IND- cells were transfected using an Amaxa 4D-Nucleofector (Lonza, Basel, Switzerland). Cells, 2.5 million were resuspended in SF solution (Lonza) and mixed with 500 nM pool of siRNA (Sigma-Aldrich) targeting V-ATPase. In each reaction a scrambled sequence (SCR) was used as a control.
After transfection, 50,000 cells were plated out in 96 well Cytostar-T plates, and grown for 24 h, before treated with different concentrations of bafilomycin A1 and concanamycin A (Enzo Life Sciences), respectively, for up to 48 h. Cell proliferation, by incorporation of [14C]-thymidine, was measured as described above.

Transfections into K562 cells (either wild-type or engineered cells) were performed by combining 2E5 cells with either plasmid- or mRNA-encoding ZFNs and SF solution (Lonza) using the Amaxa 96-well Shuttle System as per the manufacturer’s protocols. Following nucleofection, cells were placed into RPMI media containing 10% fetal bovine serum. A variation of a transient cold shock protocol^{74 (link)} was employed by placing the 96-well plate of cells at 30 °C for 24 h followed by incubation at 37 °C for an additional 48 h. Cells were then pelleted and genomic DNA was isolated using QuickExtract (Lucigen, Middleton, WI) as per the manufacturer’s instructions. Target loci were then analyzed by PCR amplification followed by deep sequencing (MiSeq).

The Itga4 KO MM1.S-GFP were generated by using CRISPR-Cas9. Briefly, the α₄ knockout guide RNA sequence was cloned into the MLM3636 plasmid (Addgene). 250,000 MM.1S cells were resuspended in 20μL SF solution (Lonza), 1 μg gRNA plasmids and 250 ng Cas9-HF plasmid (Addgene # 43,945 pc3 CAS9hc) and electroporated using the Lonza 4-D Nucleofector DS-137 program (optimized for these cells, not shown). α₄ negative cells were sorted using a MoFlow sorter.
Six to ten-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) were used in all MM.1S mice experiments. Briefly, 500,000 MM.1S parent or alpha4 KO cells were injected i.v. into tail veins of mice. Tumors were allowed to grow for ~ 2–3 weeks and tumor progression was followed by bioluminescence imaging. Flow cytometry for CD138 (Biolegend) was used to assess percent MM.1S cells in multiple tissues. Flow was run on an Attune and analyzed using FlowJo (Treestar).

A Nucleoflector 4D (Lonza, Basel, Switzerland) was used for HeLa cells and HEK 293T cells. The SpCas9 plasmid (500 ng) and the sgRNA plasmid (500 ng) were electroporated into 1.0 × 10⁵ cells according to the manufacturer’s protocol. SE solution (Lonza, Switzerland) and SF solution (Lonza, Basel, Switzerland) were used for HeLa cells and HEK 293T cells, respectively. In the presence of the dsODN, the SpCas9 plasmid (500 ng), the sgRNA plasmid (500 ng) and the dsODN (100 pM, 1 μL) were electroporated into 1.0 × 10⁵ cells using the same protocol. For the large deletion experiment, the SpCas9 plasmid (750 ng) and each sgRNA plasmid (250 ng) were transfected. The genomic DNA was isolated from the whole cell population using NucleoSpin Tissue (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) 72 h after transfection.

MNT1 cells were cultured as previously described (26 (link)). For microscopy experiments, 0.3 × 10⁶ cells were resuspended in 20 μl Lonza SF solution, mixed with 0.4 μg of each plasmid, transferred to 16-well nucleocuvette strips, and nucleofected using program DS-137 in a Lonza 4D nucleofector. After 10 min, cells were transferred to 35 mm glass bottom dishes with 2 ml 37 °C KGM Gold media and grown 24 to 72 h. Nonidentified human neonatal primary epidermal melanocytes (Thermo Fisher Scientific; catalog no.: C0025C) were cultured in supplemented Medium 254 per manufacturer instructions and nucleofected as described for MNT1 cells. imMKCL cells were cultured as previously described (58 (link), 62 (link)) and nucleofected as described for MNT1 cells except program EN-138 was used for nucleofection. About 24 h after transfection, imMKCL cells were transferred to Matrigel-coated glass bottom dishes, and the media were replaced with fresh media lacking doxycycline to induce differentiation. imMKCL cells were imaged after 72 h in differentiation media.