*4.2. Cell Culture*

Frozen (liquid-vapor) cell lines were cultured in complete growth media [A549 and H2347 cells in Roswell Park Memorial Institute 1640 (RPMI-1640) with 10% Fetal Bovine Serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% Penicillin/Streptomycin (Caisson Laboratories, Smithfield, VA, USA); MRC-5 in Minimum Essential Medium (MEM HyClone, Chicago, IL, USA), with 10% FBS, 1% Penicillin/Streptomycin, and 1% Nonessential Amino acids (10 mM 100×, Solarbio Biotechnology, Beijing, China) (EMEM)] [26]. For freezing A549, H2347, and MRC-5 cell line, 10% Dimethyl sulfoxide (DMSO, MP Biomedicals, Shanghai, China) in FBS, RPMI-1640, and MEM complete growth medium, respectively, was used. Adherent cells were detached using 1–3 mL of 0.25% Trypsin EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 2–4 min at room temperature (RT).

#### *4.3. Cell Viability Assay*

Subconfluent cells (<90%) were aseptically trypsinized and diluted to 5 × <sup>10</sup><sup>4</sup> cell/mL using complete growth media RPMI-1640 (for A549 and H2347) or EMEM (for MRC-5). Cells were then seeded aseptically in 96 welled plates at 1 × 104 cells/well (200 <sup>μ</sup>L/well) and incubated for 12 h at 37 ◦C in a 5% CO2 incubator before dosing. After 12 h, we exposed the cells to different concentrations of Lactucin for 24 h. All Lactucin concentrations were prepared using complete growth media and contained equal vehicle volume DMSO

(*v*/*v*). Cell viability was determined using a MTT cell proliferation kit (M1020, Solarbio Biotechnology, Beijing, China) following the manufacturer's instructions. UV absorbance was scanned at 490 nm using a Spark microtitration plate reader (Tecan, Männedorf, Switzerland). Each experiment was repeated three times, the viability of the control group was set to 100%, and cell viability and IC50 values were calculated.

#### *4.4. DNA Content/Cell Cycle Assay*

Subconfluent A549 and H2347 cells (<90%) were cultured at 1 × 105 cell/mL concentration using complete growth media RPMI-1640 for 12 h at 37 ◦C in 5% CO2. Cells were then treated with respective IC50 Lactucin solution or equivalent volume DMSO (*v*/*v*) solution and incubated for 24 h. After treatment, cells were trypsinized, washed, and fixed for 24 h. Fixed cells were washed and stained using a Propidium Iodide (PI) Flow Cytometry kit (Abcam, Cambridge, UK) following manufacturer instructions. The DNA contents were scanned using a flow cytometer (CytoFLEX, Beckman Coulter Inc., Miami, FL, USA).

### *4.5. Apoptosis Assay*

Subconfluent A549 and H2347 cells (<90%) were cultured at 1 × 105 cell/mL concentration using complete growth media RPMI-1640 for 12 h at 37 ◦C in 5% CO2. Cells were then treated with respective IC50 Lactucin solution or equivalent volume DMSO (*v*/*v*) solution and incubated for 24 h. After treatment, cells were trypsinized and washed. Following manufacturer instructions, cells were double-stained using the FITC Annexin V Apoptotic kit (BD Pharmingen, San Diego, CA, USA). Samples were filtered (70 μM Nylon cell strainer, Falcon, Corning, Durham, NC, USA) and scanned using a flow cytometer.

#### *4.6. Protein Extraction*

<sup>5</sup> × <sup>10</sup><sup>6</sup> cell/mL untreated [A549 for Co-IP] and treated (Lactucin and DMSO in A549 and H2347 for WB) cells were washed (1× PBS chilled), scraped (cell scraper, Corning, Durham, NC, USA), collected in 1.5 mL centrifuge tubes, and then washed again twice (1× PBS chilled). Between each washing, cells were centrifuged at 600× *g* for 5 min at 4 ◦C. After the final centrifuge, 300–500 μL of chilled modified RIPA buffer (1% PMSF + 1% SDS in RIPA) was added to each tube, briefly vortexed and then homogenized using probe sonicator at 30% power of 3 × 5 s with a 10 s gap in between. Homogenized cells were then left to chill on ice for 1 h. After 1 h, cells were centrifuged at 14000× *g* at 4 ◦C for 10 min, and intact cells and nuclear materials were separated by pipetting the supernatant into new centrifuge tubes and stored at −20 ◦C. The protein concentrations were determined using the Bicinchoninic Acid (BCA) protein assay kit (Solarbio Biotechnology, Beijing, China) following manufacturer instructions. UV absorbance was scanned at 562 nm using a microtitration plate reader.

#### *4.7. Western Blot Assay*

Western blot was performed using the method adopted from Lu. et al. [26]. A549 and H2347 cells protein (10 μg/lane) along with a pre-stained page ruler (5 μg/lane) were resolved using 8, 10, and 12% SDS-PAGE, along with pre-stained protein ladder (#26617; Thermo Fisher Scientific, Carlsbad, CA, USA) and transferred to polyvinylidene fluoride (PVDF) transfer membrane (Immobilon®-P, Billerica, MA, USA) by wet transfer at 350 mA for 70–110 min (depending on targeted proteins molecular weight). PVDF membranes were washed thrice using Tris-buffered saline with 0.1% Tween® 20 Detergent (TBST) (10 min each) and blocked using 5% Bovine Serum Albumin (BSA) in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% tween 20) at room temperature (RT) for 1 h. The PVDF was then washed thrice using TBST (10 min each), cut, and probed using primary antibodies at 4 ◦C overnight. The primary antibodies include anti-Akt (AA326), anti-Bax (AB026), anticleaved Caspase-3 (AC033), anti-CDK-4 (AC251-1), anti-Cyclin D1 (AC853-1), anti-p53 (AF0255), anti-ERK1/2 (AF1051), anti-MEK1/2 (AF1057), anti-cleaved-PARP-1 (AF1567), anti-mTOR (AF1648), anti-Ki67 (AF1738), anti-p-ERK (AF1891), anti-p-Akt1/2/3(Thr 308) (AF5734), anti-p21 (AP021-1), anti-PTEN (AP686) from Beyotime Biotechnology (Shanghai, China); anti-LC3B (NB100-2220) from Novus Biologicals (Littleton, CO, USA); anti-β-actin (SC47778), anti-Cdk-2 (SC6248), anti-Cyclin B1 (SC245), anti- p-MEK1/2 (SC81503), anti-Bcl-2 (SC7882) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Next, the PVDF was washed thrice using TBST (10 min each) and probed with appropriate goat anti-mouse (ab150113) or anti-rabbit (ab97051) secondary antibodies (Abcam, Cambridge, UK) for 1 h at RT. Finally, the PVDF was washed (thrice for 10 min each), exposed using luminol reagents (Millipore, Billerica, MA, USA), and photographed using the CLiNX Chemiluminescence imaging system (Shanghai, China).

#### *4.8. Synthesis of Lactucin Probe*

To synthesize the biotinylated Lactucin probe, Lactucin was conjugated with an alkynyl group through Michael's addition reaction, Figure 6A. To a solution of lactucin (5.0 mg, 0.018 mM) in dry pyridine (0.3 mL), Propargylamine (1.5 mg, 0.027 mM) (innochem, Beijing, China) was added and stirred at 0 ◦C for 36 h under N2 air. The reaction was monitored by thin-layer chromatography (TLC). The solvent was concentrated and purified through column chromatography (Dichloromethane-CH3OH, 15:1) and dried. For structural confirmation, the probe was analyzed using 1H-NMR at 600 MHz (Oxford NMR AS600, Abingdon, UK) in deuterated DMSO (d-DMSO, Merck KGaA, Darmstadt, Germany), Figure 5A. The yield of the probe was estimated using HPLC.

### *4.9. Cytotoxicity Test and Protein Labelling by Lactucin Probe*

Cytotoxicity of the Lactucin probe and Propargylamine was examined by incubating A549 cells with incriminating (0 to 150 μM) concentrations and incubation periods (12, 24, and 48 h) of each. The total A549 protein lysate was prepared using the method described in Section 4.6. The Lactucin binding proteins were labeled and pulled out using a biotinylated Lactucin probe following the ABPP method adopted from the method described by Speers and Cravatt [35]. Two 400 μL of protein lysate (2 mg/mL in PBS) were aliquoted into a 1.5mL microcentrifuge tube, and Lactucin-probe or Propargylamine was added (final concentration 100 μM). Both tubes were incubated at RT overnight in a shaker. It was followed by the addition of biotin-azide (Institute of Medicinal Plant Development, Beijing, China) (final concentration 100 μM), then Tris (2-carboxyethyl) phosphine (TCEP, T1656) (final concentration 1 mM), and Tris [(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]-amine (TBTA, T2993) (final concentration 100 μM) (Tokyo Chemical Industry, Japan), and Copper sulfate pentahydrate (CuSO4·5H2O) (final concentration 1 mM) (Merck KGaA, Darmstadt, Germany) to each tube with vortex after each addition. Both tubes were incubated at room temperature for 1 h with a vortex every 30 min. Then the tubes were centrifuged at 12,000× *g* for 10 min at 4 ◦C, and the supernatant was removed. Protein precipitate was dissolved by adding 750 μL of pre-cooled methanol and sonicating for 3–4 s at 4 ◦C using a probe sonicator (~30% power level). It was followed by methanol wash thrice with centrifugation (12,000× *g*, 4 ◦C for 10 min) in between.
