Pbabepuro gfp lc3

The plasmids pBABE-puro mCherry-EGFP-LC3B and pBABEpuro GFP-LC3 (N#22418 and 22405), engineered by Jayanta Debnath [21] , were obtained from Addgene. Cell transduction and selection were performed as previously described [20] (link), [22] (link). Genetic inhibition of autophagy was achieved using ATG7 shRNA-mediated knock down (Santa Cruz). Non-specific shRNA (control) as well as copGFP were applied according to manufacturer's instructions (Santa Cruz). Transduction was performed using lentiviral particles with up to five distinct expression constructs as described elsewhere [20] (link). Transduction efficiency was quantified by the number of GFP-positive cells and in general exceeded 94% at the end of puromycin selection.

The lentiviral shRNA plasmid pLKO.1 targeting BRCA1 (clone ID TRCN0000039833, TRCN0000039837, and TRCN0000010305), DRP1 (TRCN0000318424), ATM (TRCN0000010299), LKB1 (TRCN0000000408), CaMMKβ (TRCN0000002299), and shRNA control plasmid were purchased from Sigma. MBP‐BRCA1 expressing vector was constructed in previous study.[qv: 50] Mfn1 and Mfn2 luciferase reporters were requested from Dr. Martin Lidell. Six BRCA1 fragments expressing vector were requested from Dr. Rong Li. pCHAC‐mt‐mKeima and pBMN‐mCherry‐Parkin were gifts from Richard Youle (Addgene plasmid # 72342 and # 23956); pRK5‐HA‐Parkin was a gift from Ted Dawson (Addgene plasmid # 17613); pBABEpuro GFP‐LC3 was a gift from Jayanta Debnath (Addgene plasmid # 22405); pLV‐mitoDsRed was a gift from Pantelis Tsoulfas (Addgene plasmid # 44386); LAMP1‐mRFP‐FLAG was a gift from David Sabatini (Addgene plasmid # 34611); pLenti.CMV/TO_PRKAA1 and pLenti.CMV/TO_PRKAA2 were gifts from Reuben Shaw (Addgene plasmid # 74446/74447).

The following plasmids were obtained from Addgene: pBABEpuro GFP‐LC3 from Jayanta Debnath (Addgene plasmid #22405)^[16] and FUW mCherry‐GFP‐LC3 from Anne Brunet (Addgene plasmid #110060).^[17] Cx43‐mCherry was constructed on pLPCX‐Cx43‐IRES‐GFP from Trond Aasen (Addgene plasmid #65433).^[18] Transfection of HeLa, PC12, and N2A cells with indicated plasmids was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Transduced cells were grown in the medium as described above. Selection of transduced cells was performed with 1 mg mL⁻¹ Geneticin (G418) (Thermo Fisher Scientific) for about 15 days. High‐expressing cell clones were identified using fluorescence microscopy. Following selection, stably transfected HeLa and PC12 cells were cultured and supplemented with 500 µg mL⁻¹ G418, and N2A cells were cultured with 800 µg mL⁻¹ G418.

The following plasmids were from Addgene:
pMXs-puroGFP-DFCP1,pCDNA6-myc ULK1 wt, pCDNA3 Flag ULK1, pCDNA3 myc AMPK
Alpha 2, pBABE-puro-mCherry-GFP-LC3,and pBABE-puro-GFP-LC3. pCMV-AMPK alpha
2 Flag was from Sinobiologicals. Our lab generated pCMV-IPMK-GST and
different fragments tagged with GST, pCMV-myc-IPMK, pCMV-HA-IPMK, pMX-myc,
pMX-myc IPMK WT, and pMX-myc KSA. Constitutively active AMPK was a kind gift
from Dr. Anne Burnet (Stanford University School of Medicine, Stanford, CA).
We purchased recombinant hIPMK from Origene. Recombinant hULK1 and AMPK were
purchased from Signalchem.

To detect the LC3B signal, 3–4 × 10⁵ cells were seeded in 6-well plates. The next day, the cells were washed twice with pre-warmed phosphate buffered saline (PBS) before treatment with 100 μM chloroquine (CQ; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. After protein extraction, LC3B (Sigma-Aldrich) and β-actin (Santa Cruz) were detected using immunoblotting. The chemiluminescent signals were detected and visualized using a LAS-3000 luminescent image analyzer (FUJIFILM, Tokyo, Japan). The autophagy flux unit (A.F.U. = [LC3B-II/LC3B-I]_CO(+)/[LC3B-II/LC3B-I]_CO(-)) was calculated by analyzing the band intensities of LC3B-I and LC3B-II from three independent experiments using the ImageJ software.
PK59 and HPAF-II cells stably expressing GFP-LC3 were transfected with retrovirus (pBABEpuroGFP-LC3; Addgene, Watertown, MA, USA). After treatment with CQ (100 μM) for 5 h, GFP signals from LC3 were analyzed using a fluorescence microscope (Inverted Microscope Eclipse Ti-S; Nikon, Tokyo, Japan). Five images were selected to manually count the number of total and GFP-LC3 positive cells.

Recombinant plasmid for retroviral expression of GFP‐tagged LC3 (pBABEpuro GFP‐LC3; Addgene plasmid number 22405) was created by Dr Jayanta Debnath.20 Another retroviral vector expressing GFP‐LC3‐RFP‐LC3ΔG fluorescence probe (pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG; Addgene plasmid number 84572) was constructed by Dr Noboru Mizushima.21 Both plasmids were obtained from Addgene. Generation of retrovirus and transduction of experimental cell lines were performed following standard protocol.
Chemically synthesized shRNA sequences targeting Atg7,20, 22 Bak23 and scrambled control (http://www.hollingscancercenter.org/research/shared-resources/shRNA/Vectors.pdf) were procured from Integrated DNA Technologies, Inc. The sequences were annealed and sub‐cloned into the AgeI and EcoRI sites of the pLKO.1 puro vector. Standard protocol was followed for generation of lentivirus and transduction of cells.
Cells, transduced with retro/lentiviruses encoding desired genes/shRNAs, were cultured in growth media as mentioned above. Puromycin was added into the media after 48 hours of transduction. After maintaining the cells for ~10 days in puromycin‐containing media, viable stable clones were propagated and used for experiments.