Guo_Fig. S1. Atg7 +/+ Atg7 -/- FBP_M+6 DHAP_M+3. Glucose Uptake Rate G6P_M+6. nmol/ul cell/hr

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1 Guo_Fig. S1 A C D nmol/ul cell/hr nmol/ul cell/hr Glucose Uptake Rate *. Lactate Secretion Rate Atg7-/- B Glucose G3P Lac Glucose G6P FBP 3-PG Pyr DHAP cycle Carbon from labeled glucose Carbon from labeled glucose unlabeled carbon Glucose Acetyl-CoA CoA [U6-13 C] Glc labeling (%) [U6-13 C] Glc labeling (%) [U6-13 C] Glc labeling (%) G6P_M+6 3PG_ _ [U6-13 C] Glc labeling (%) FBP_M α-ketogutarate_m _ DHAP_ _ _ Acetyl-CoA CoA Glutamine Carbon from unlabeled glutamine Carbon from labeled glucose Acetyl-CoA CoA [U6-13 C] Glc labeling (%) 1 1 α-ketoglutarate M+ M+5 [U6-13 C] Glc labeling (%) M+ M+5 M M+ M+

2 A B nmol/ ul cell/ hr Glutamine Uptake Rate Acetyl-CoA CoA Glutamine Carbon from labeled glutamine unlabeled carbon Glutamine [U5-13 C] Gln labeling (%) 8 α-ketoglutarate (M+5) 8 () 8 () [U5-13 C] Gln labeling (%) () Guo_Fig. S2 Glutamine C Glutamine Acetyl-CoA CoA Glutamine Carbon from labeled glutamine unlabeled carbon [U5-13 C] Gln labeling (%) [U5-13 C] Gln labeling (%) α-ketoglutarate M+ M+5 M+ M+5 M M+ M M+

3 Guo_Fig. S3 A Asp Asp Asp Asp Ser Leu Ser Leu Ser Leu Ser Leu Asn Phe+Ala +Tyr Trp+Cys +Thr+Gly Asn Phe+Tyr Trp+Cys +Thr+Gly Asn Phe+Ala +Tyr Phe+Tyr Trp+Cys +Thr+Gly Trp+Cys +Thr+Gly Tyr+ Ile+Lys Pro+ Arg+His Tyr+ Ile+Lys Pro+ Arg+His Tyr+ Ile+Lys Pro+ Arg+His Tyr+ Ile+Lys Pro+ Arg+His Ile+ Val+Met Met Ile+ Val+Met Met Ile+ Val+Met Met Ile+ Val+Met Met Atg7 -/ Pro + Asn 2 days treatment + 2 days recovery B Arg His 1 day treatment + 1 day recovery

4 Guo_Fig. S4 Relative OCR (%) Relative OCR (%) vs (min) RPMI +Q 71-9 vs (min) RPMI Q RPMI +Q RPMI Q vs (min) RPMI +Q 71-3 vs (min) RPMI Q RPMI +Q RPMI Q vs (min) RPMI +Q 71-2 vs (min) RPMI Q RPMI +Q RPMI Q Relative OCR (%) vs (min) RPMI Q RPMI Q vs (min) RPMI Q RPMI Q RPMI vs (min) Q RPMI Q 71-1 vs (min) Relative OCR (%) RPMI Q RPMI Q RPMI 71-1 vs (min) Q RPMI Q

5 Guo_Fig. S5 um um um UTP *** ** UDP RPMI RPMI RPMI ** * 1 8 *** RPMI RPMI RPMI CTP 15 CDP 2 CMP *** 1 15 *** ** RPMI RPMI RPMI UMP GTP GDP GMP ***

6 Guo_Fig S6 A Ion Count RPMI + U+A+G RPMI + U+A+G RPMI + U+A+G RPMI + U+A+G B OCR (%) vs 2 vs RPMI +UAG RPMI +UAG

7 Tissues Genotype Sample ID R1 read Tumor Normal lung Supplemental Table 1 Average Coverage across chrm mm1 genome Total number of Reads (R1+R2) Properly Paired Alignment Average coverage* Alignement percentage (%) Coverage p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7+/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53-/-;krasg12d/+;atg7-/ p53f/f;krasg12d/+;atg7f/f p53f/f;krasg12d/+;atg7f/f Note: *samtools view Coverage using q1 and Q2, read length = 2X15 bp, Coverage = (Number of reads * Read Length)/Target size.

8 Supplemmental Table 2 MtDNA mutations in Kras-driven tumors Total Tumors with mtdna Total mtdna Syn Non-Syn mt-rnr1 Genotype tumors mutation mutations mutataion mutation mutation ATG7+/ ATG7-/ P=.2676 (Bernard s test, one-tail)

9 Mouse ID #chrm POS Supplemental Table3 mtdna mutations detected in ATG7+/+ and ATG7-/- lung tumors Genomic Annotation Normal Lung Tissue Lung Tumor Tissue Variant Effect Predictor REF ALT VAF ref/fwd, ref/rev, var/fwd,var/rev VAF ref/fwd, ref/rev, var/fwd,var/rev Genotype ATG7-/- 698 chrm 231 mt-rnr1 G A.9% 9779,198,4, % 11391, 11679, ATG7-/ chrm 231 mt-rnr1 G A.9% 9779,198,4, % 9654, 116, 1485, 15 ATG7-/ chrm 9461 mt-nd3 Syn C T.46% 7924,834,14, % 5582, 593, 1386, 1455 ATG7-/- 698 chrm 138 mt-nd4 Syn T C.3% 772,814,3, % 7464, 7861, 2521, 2664 ATG7-/- 61 chrm 138 mt-nd4 Syn T C.3% 772,814,3,1 19.3% 8943, 9449, 2129, 227 ATG7-/ chrm 138 mt-nd4 Syn T C.3% 772,814,3, % 8279, 867, 12, 125 ATG7-/ chrm mt-nd5 Syn T C.3% 767,6773,2,2 1.97% 8792, 7942, 162, 1 ATG7-/ chrm mt-cyth Syn T C.13% 1582,172,16, % 8663, 8694, 718, 748 ATG7+/+ 529 chrm 9461 mt-nd3 Syn C T.46% 7924,834,14, % 4195, 442, 5632,5871 Note: POS:chrM Genome Position; REF:reference; ALT:alternative; VAF: Variant Allele Frequency %, Strand read counts : ref/fwd (Reference Forward), ref/rev(reference Reverse), var/fwd(variant Forward),var/rev(Variant Reverse), Syn : synonymous variants # read count is based on base Quality filter of 2 and Unique Mapping Quality filter (1) # red color read count represents variants allele

10 Supplemental Table 4.1 Autophagy mediates substrate recycling: prelabeling metabolites by uniformly 13C- and 15N-labeled amino acids and 13C-labeled glucose R [13C][15N] labeling (%) Atg7+/+ Atg7-/ asparagine serine aspartate G6P a-ketoglutarate sn-glycerol-3-phosphate glutamate CTP ADP-D-glucose proline glutathione arginine O8P UDP-D-glucose dttp ribose-phosphate CDP cyclic-amp methionine histidine S7P Sedoheptoluse bisphosphate tyrosine valine threonine tryptophan UTP GTP AMP FBP leucine/isoleucine UMP UDP-N-acetyl-glucosamine GDP ATP N-acetyl-glucosamine-1/6-phosphate coenzyme A alanine glutathione disulfide malate UDP UDP-D-glucuronate datp Note: R, the time point after cells were cultured in uniformly C and 15N-labeled amino 95.1 acids (AAs) and 13C-labeled glucose for three days to label endogenous components.

11 Supplemental Table 4.2 Autophagy mediates substrate recycling: chasing labeled metabolites with unlabeled RPMI medium R3 [13C][15N] labeling (%) Atg7+/+ Atg7-/ asparagine serine aspartate G6P a-ketoglutarate sn-glycerol-3-phosphate glutamate CTP ADP-D-glucose proline glutathione arginine O8P UDP-D-glucose dttp ribose-phosphate CDP cyclic-amp methionine histidine S7P Sedoheptoluse bisphosphate tyrosine valine threonine tryptophan UTP GTP AMP FBP leucine/isoleucine UMP UDP-N-acetyl-glucosamine GDP ATP N-acetyl-glucosamine-1/6-phosphate coenzyme A alanine glutathione disulfide malate UDP UDP-D-glucuronate datp Note: cells were cultured in uniformly 13C-labeled glucose and glutamine for three days to label endogenous components and followed by three hours culture in unlabeled RPMI to replace labeled with unlabeled metabolites. R3, the time point after cells were cultured in unlabed RPMI medium for three hours.

12 Supplemental Table 4.3 Autophagy mediates substrate recycling:the breakdown of intracellular components in starvation H4 WT H4 vs. [13C][15N] labeling (%) Atg7+/+ Atg7-/- Null H4 Fold Change (H4/R3) T test Atg7+/+ Atg7-/- asparagine serine aspartate E G6P a-ketoglutarate sn-glycerol-3-phosphate E glutamate E CTP ADP-D-glucose proline glutathione arginine O8P UDP-D-glucose dttp ribose-phosphate E CDP cyclic-amp methionine histidine S7P Sedoheptoluse bisphosphate tyrosine E valine threonine tryptophan E UTP GTP AMP FBP leucine/isoleucine UMP UDP-N-acetyl-glucosamine GDP ATP N-acetyl-glucosamine-1/6-phosphate coenzyme A alanine glutathione disulfide malate UDP UDP-D-glucuronate E datp Note: H4, the time point after 4 hour starvation to degrade intracellular compoments for recycling. 23 of 43 examined metabolites significantly incresed labeling after 4 hour sarvation, which is measured by fold change (H4/R3) (fold change>2 was showed in Bold number). Of these 23 metabolites that were derived substantially from macromolecule degradation, 18 (labeled with red color) of these showed significant differences in labeling between Atg7 WT and deletion cells, with greater labeling in the WT cells in all cases. T-test (FDR<.5) is used to compare the substrate recycing between Atg7 WT and Atg7 deficient TDCLs in starvation. P value in bold indicates motabolites in Atg7+/+_TDCLs had significantly more [13C], [15] labeling than that in Atg7-/-_TDCLs after 4 hour starvation.