1. Supplemental Figure S1
Figure S1. Examples of images showing mRFP-LC3B puncta in U87 and A549 cells.
U87
72 h, 30 mM NH4Cl, hypoxia
0 h
A549
20 μm

2. Supplemental Figure S2 A B Hypoxia (h) 24 siATG5 siBECN1 siCon NH4Cl -24
siATG5
siBECN1
siCon
NH4Cl - +
A549 cells
ACTB
LC3B-I
LC3B-II
ACTB
LC3B-I
LC3B-II
NH4Cl - +
siATG5
siBECN1
siCon
U87 cells, 4 h in hypoxia
Figure S2. Knockdown of autophagy genes ATG5 or BECN1 inhibited autophagy. (A) Inhibition of autophagy by knockdown of ATG5 or BECN1 was determined by western blotting for LC3B-II in the absence and presence of NH4Cl (30 mM) in U87 cells. (B) Inhibition of autophagy by knockdown of ATG5 or BECN1 in A549 cells was determined by western blotting of LC3B-II in the absence and presence of NH4Cl. These results were representative of 3 independent experiments (n=3). ACTB was used as loading control.

3. Supplemental Figure S3 * *** (n=3) EGFR mRNA level Hypoxia (h)
Figure S3. Hypoxia treatment led to increase in EGFR mRNA level. U87 cells were treated at different time points in hypoxia. Cell pellets were collected and RNA was isolated for the measurement of EGFR mRNA level by real-time PCR. Total RNA was isolated using the Qiagen RNeasy Plus mini kit (Qiagen, 74134) according to the manufacturer’s protocol. To perform qPCR, 100 ng RNA template was used in combination with iTaq Universal SYBR Green Supermix (Bio-Rad, ).The cycling and data collection were performed on a Bio Rad CFX Real-Time Detection System (Bio-Rad, ) using the supplied software. The primers used for EGFR were the Bio-Rad PrimePCR SYBR Green Assay predesigned primer pair (Bio-Rad, qHsaCED ). Three different housekeeping genes were used to standardize the results: ACTB (qHsaCED ), RPL13A (qHsaCED ) and RPS18 (qHsaCED ), all PrimePCR predesigned primer pairs (Bio-Rad). The qPCR reaction was run as follows: 95C for 2 min and then 40 cycles of 95C for 5 sec and 60C for 30 sec. Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

4. Supplemental Figure S4
Figure S4. Interaction of EGFR with BECN1 at 4 h and 72 h in hypoxia was determined in A549 cells by immunofluorescence (IF) microscopy as described in the Materials and Methods section. Colocalization puncta are indicative in the interaction between EGFR and BECN1. These results are representative of 3 independent experiments (n=3).
Merge
BECN1
EGFR
4 h
72 h
50 μm
0 h
A549 cells, Hypoxia

5. Supplemental Figure S5 *** siCon siATG5 siEGFR siEGFR+siATG5
Cell death (%)
Normoxia
Hypoxia
***
U87 cells, 24 h
Figure S5. Effects of knockdown EGFR and ATG5 on cell death in U87 cells. EGFR and ATG5 genes were knocked down as shown in Fig. 7A and then placed in hypoxia for 24 h and the amount of cell death was determined by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

6. Supplemental Figure S6 A B *** ** * LC3B-I LC3B-II ACTB NH4Cl - + 3-MA
Spautin-1 24
Hypoxia (h)
siRNA
siCon
siEGFR
LC3B-II/ACTB
0.4
0.2
0.3
1.0
0.1
1.6
U87 cells
3-MA
Control
Spautin-1 N,
siCon
siEGFR H,
Cell death (%) * **
***
U87 cells, 24 h B
Figure S6. Autophagy inhibitors 3-MA and spautin-1 and knockdown of EGFR on autophagy and cell death in U87 cells. (A) EGFR knockdown cells as shown in Fig. 7A were treated with autophagy inhibitors 3-MA and spautin-1 and the amount of autophagy determined by western blotting for LC3B-II in the absence and presence of NH4Cl (30 mM). ACTB was used as a loading control. (B) EGFR knockdown cells were again treated with autophagy inhibitors 3-MA and spautin-1 and amount of cell death was determined at 24 h. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

7. A B C D E Supplemental Figure S7 ** *** * NH4Cl - + Hypoxia (h) 2424
siCon
siATG5
siEGFR
siEGFR + siATG5
LC3B-I
LC3B-II
ACTB
LC3B-II/ACTB
1.0
0.37
1.24
0.97
0.13
4.05
0.56
siBECN1
siEGFR+siBECN1 D E
siEGFR+siATG5
Cell death (%)
Normoxia, 4 h
Hypoxia, 4 h **
***
Normoxia, 24 h
Hypoxia, 24 h *
ATG12-ATG5
EGFR
P-EGFR (Y1068)
BECN1
siEGFR + siBECN1

8. Figure S7. Effects of knockdown of ATG5, BECN1 and EGFR on autophagy in A549 cells. (A) Cells were knocked down for EGFR and ATG5 or BECN1 and western blotted for tyrosine phosphorylated EGFR (P-EGFR), total EGFR, ATG5 and BECN1. EGFR and (B) ATG5 or (C) BECN1 were knocked down and the cells were then western blotted for LC3B-II in the absence and presence of NH4Cl (30 mM) to determine autophagy levels. (D, E) A549 cells with knockdown of EGFR and ATG5 or BECN1 were placed in hypoxia for 4 h and 24 h and the amount of cell death determined by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). ACTB was used as the loading control. Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

9. Supplemental Figure S8 ** * *** N, siCon N, siEGFR H, siCon H, siEGFR
Cell death (%)
A549 cells, Time (h)
*** * **
Figure S8. A549 cells with knockdown of EGFR were placed in hypoxia for 4, 24, 72 and 144 h and amount of cell death determined by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

10. Supplemental Figure S9 A B C *** **
Figure S9. Effects of the EGFR tyrosine kinase inhibitor erlotinib on autophagy and cell death in U87 cells. (A) Cells were treated with erlotinib (10 µM) and western blotted for tyrosine phosphorylated EGFR and EGFR. (B) Cells were treated with erlotinib and 3-MA (4 mM) and amount of autophagy determined by western blotting for LC3B-II in the absence and presence of NH4Cl (30 mM). (C) The amount of cell death was determined after erlotinib and 3-MA treatment after 24 h in hypoxia as described above. These results were representative of 3 independent experiments (n=3). ACTB was used as the loading control. Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.
LC3B-I
LC3B-II
ACTB
NH4Cl - +
Erlotinib
3-MA
Hypoxia (h) 24
LC3B-II/ACTB
1.0
2.9
U87 cells
Control
Cell death (%)
Control, N H
Erlotinib,
*** **
U87 cells, 24 h
EGFR
P-EGFR (Y1068) 4
P-EGFR/EGFR
0.1

11. Supplemental Figure S10 A B
Figure S10. Effect of EGFR tyrosine kinase inhibitor gefitinib (Gef) on autophagy in A549 cells. (A) Cells were treated with Gef (40 µM) and western blotted for tyrosine phosphorylated EGFR (P-EGFR Y1068) and EGFR. (B) Cells treated with Gef and autophagy inhibitors 3-MA (4 mM) or spautin-1 (3 μM) were lysed and western blotted for LC3B-II in the absence and presence of NH4Cl (30 mM) after 4 h in hypoxia. These results were representative of 3 independent experiments (n=3). ACTB was used as the loading control.
Hypoxia (h)
LC3B-I
LC3B-II
ACTB
NH4Cl - +
Gef
3-MA
Spautin-1 4
LC3B-II/ACTB
1.0
5.3
A549 cells
EGFR
P-EGFR (Y1068)
P-EGFR/EGFR
0.9
0.1

12. Supplemental Figure S11 A B C D * *** ** *** ** * ACTB CAV1 EGFR siCon
siCAV1
CAV1/ACTB
P-EGFR/EGFR
1.0
0.47
0.44
U87 cells, 4 h, hypoxia B
LC3B-I
LC3B-II
ACTB
NH4Cl - +
siCon
siCAV1
LC3B-II/ACTB
0.7
1.0
1.31
1.83
U87 cells, 24 h, hypoxia
P-EGFR (Y1068) C D
siCon, - 3-MA *
***
Normoxia
Hypoxia
siCon,+3-MA
siCAV1, - 3-MA
siCAV1,+3-MA
Cell death (%) **
U87 cells, 4 h
siCon, - 3-MA
siCon,+3-MA
siCAV1, - 3-MA
siCAV1,+3-MA
Normoxia
Hypoxia
*** ** *
Cell death (%)
U87 cells, 24 h
Figure S11. Effects of CAV1 knockdown on autophagy and cell death in U87 cell. (A) CAV1 was knocked down and then the cells were placed in hypoxia for 4 h and then lysed and western blotted for P-EGFR (Y1068), EGFR and CAV1. (B) CAV1 was knocked down and then the cells were placed in hypoxia for 24 h. Cells were lysed and western blotted for LC3B-II protein levels. NH4Cl (30 mM) was added to determine autophagic flux. (C, D) CAV1 was knocked down and then the cells were treated with autophagy inhibitor 3-MA for 4 h and 24 h in normoxia and hypoxia. The amount of cell death was then determined by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). ACTB was used as the loading control. Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

13. Supplemental Figure S12 A B * *** ** LC3B-I LC3B-II ACTB NH4Cl - +
Vector alone
CAV1
LC3B-II/ACTB
0.51
1.0
0.54
0.43
HEK293 cells, 24h, hypoxia A
Vector, Control
Vector, 3-MA
CAV1, Control
CAV1, 3-MA
Normoxia
Hypoxia
Cell death (%) *
*** **
HEK293 cells, 48 h B
Figure S12. Effects of overexpression of CAV1 on autophagy and cell death in HEK293 cells. (A) CAV1 over-expressing HEK293 cells (as shown in Fig. 9D) were place in hypoxia for 24 h and the amount of autophagy determined by western blotting for LC3B-II protein levels. NH4Cl (30 mM) was added to determine the autophagic flux. ACTB was used as the loading control. (B) CAV1 over-expressing HEK293 cells were treated with 3-MA in hypoxia for 48 h. The amount of cell death was determined. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

14. Supplemental Figure S13 A N
0.5 μm
Phase 1/2 M
E-PNS
Phase 2
Phase 1

15. Figure S13. Extent examples of A549 cells showing morphological features of autosis. A549 cells were treated in hypoxia for 72 h and were analyzed by electron microscopy as described in Materials and Methods section. Different stages of autosis are described in the text and in Figure 10. White arrows show dilated and fragmented ER. Black arrows show the swollen perinuclear space that contains cytoplasmic materials. Triangles show an empty ballooning space with membrane starting to merge with the outer nuclear membrane. A, autophagic body (autophagosome or autolysosome); E-PNS, empty focal ballooning perinuclear space; M, mitochondria; N, nucleus.

16. Supplemental Figure S14 * Cell death (%) Digo-0.5 Normoxia Hypoxia
A549 cells, 4 h
Digo-1
Digo-2
Digo-5
Di-0.5
Di-1
Di-2
Di-5
Nec1-10
Control *
Figure S14. Effect of autosis inhibitors on cell death. A549 cells were treated with and without the autosis inhibitor Digo and Di at different concentrations (Digo/Di-#, Digo/Di-# μM), and the necroptosis inhibitor necrostatin-1 (Nec-1, 10 μM), in normoxia and hypoxia for 4 h, and cell death was determined by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

17. Supplemental Figure S15 A B C *** ** * Control Di-0.5 Di-1 Di-2 Di-5
Gef
Gef+Di-0.5
Gef+Di-1
Gef+Di-2
Gef+Di-5
Normoxia
Hypoxia
*** **
Cell deat h (%)
A549 cells, 24 h *
Nec1-5
Nec1-10
Nec1-40
Gef+Nec1-5
Gef+Nec1-10
Gef+Nec1-40
zVAD
Gef+zVAD

18. Figure S15. Effects of inhibitors of different cell death pathways on gefitinib-induced cell death in A549 cells. (A) Cells were treated with and without gefitinib (Gef, 40 μM) and the autosis inhibitor digitoxigenin (Di) at different concentrations (Di-#, Di-# μM), in normoxia and hypoxia for 24 h and cell death was determined as by trypan blue exclusion assay. (B) Effects of necroptosis inhibitor necrostatin-1 (Nec-1) (Nec-1-#, Nec-1-# μM) on Gef-induced cell death. (C) Effect of apoptosis inhibitor zVAD-fmk (zVAD, 10 μM) on Gef-induced cell death. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

19. Supplemental Figure S16 * *** Cell death (%) Control Digo-0.5 Digo-5
Gef
Gef+Digo-0.5
Gef+Digo-5
Gef+Di-0.5
Gef+Di-1
Gef+Di-5
Normoxia
Hypoxia
U87 cells, 24 h
*** *
Figure S16. Effects of autosis inhibitors on gefitinib-induced cell death in U87 cells. Cells were treated with and without gefitinib (Gef, 40 μM) and the autosis inhibitors digoxin (Digo) and digitoxigenin (Di) at different concentrations (Digo/Di-#, Digo/Di-# μM), in normoxia and hypoxia for 24 h and cell death was determined as by trypan blue exclusion assay. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.

20. Relative colony number (%)
Supplemental Figure S17
Cell death (%)
Digo-0.5
Control
Di-0.5
- zVAD
+ zVAD
***
Normoxia
Hypoxia
A549 cells, 144 h A B
Digo-0.2
Di-0.2
A549 cells, 72 h
Relative colony number (%) *

21. Figure S17. Combinational effects of inhibitors of autosis and apoptosis on cell death and cell survival in A549 cells. (A) Cells were treated with and without the autosis inhibitors digoxin (Digo-0.5, 0.5 μM) and digitoxigenin (Di-0.5, 0.5 μM) and the apoptosis inhibitor zVAD-fmk (zVAD, 10 μM), in normoxia and hypoxia for 144 h, respectively. Then, cell death was determined by trypan blue exclusion assay. (B) Cells were treated with and without the autosis inhibitors digoxin (Digo-0.2, 0.2 μM) and digitoxigenin (Di-0.2, 0.2 μM) and the apoptosis inhibitor zVAD-fmk (zVAD, 10 μM), in normoxia and hypoxia for 72 h, respectively. cell survival was measured by clonogenic assay. These results were representative of 3 independent experiments (n=3). Error bars represent standard deviation and statistical significance calculated as *, P<0.05; **, P<0.01; ***, P<0.001.