Cell culture, transfection and lentiviral transduction
HEK293T (ATCC, CRL-3216, human embryonic kidney) and A549 (ATCC, CCL-185 human lung adenocarcinoma) cells were cultured in DMEM high glucose (Invitrogen, Darmstadt, Germany), BxPC3 (DSMZ, ACC-760, human pancreatic adenocarcinoma) cells in RPMI 1640 (Invitrogen) and HCT116 (ATCC, CCL-247, colorectal carcinoma) cells in McCoy’s 5A (Pan-Biotech, Aidenbach, Germany) supplemented each with 10% tetracycline-free FBS and antibiotics. Transient transfections were performed using Viafect (Promega, Mannheim, Germany). Production of lentiviral particles were done in HEK293T cells as described previously (Kranz et al., 2017). For transduction, 2 × 105 cells were incubated with 2 × 106 lentiviral particles in the presence of 8 µg/ml polybrene for 24 h followed by selection with 2 µg/ml puromycin for 5 days. All experiments were performed with mycoplasma-free cells.
Plasmids, shRNA sequences and knockdown induction
Plasmids used for luciferase assays: pFLAG-XBP1u-FLuc (Addgene #31239), p5xATF6-GL3 (#11976, Addgene), pNL NlucP/ATF4-RE/Hygro (Promega), pTK-GLuc- (#N8084S, NEB, Frankfurt/Main, Germany), pGL4.74 (#E6921, Promega). pCMMP-dnPERK-IRES-eGFP (Addgene #36954) was used to create V5-tagged and Cys-null mutant (C187S, C192S, C307S, C423S) of PERK luminal domain by site directed mutagenesis45. The following shRNA sequences were cloned into Tet-pLKO vector: 5′-GTGTGGTCACTGCAAACAGTT-3′ (corresponding to 1,385–1,405 bp of human PDI mRNA, GenBank acc. no. NM_000918), #2 5′-TGCTGTTCTTGCCCAAGAGTG-3′ (corresponding to 837–857 bp of human PDI mRNA, GenBank acc. no. NM_000918), 5′-GGAACGACCTGAAGCTATAAA-3′ (corresponding to 3,383–3,403 bp of human PERK mRNA, GenBank acc. no. NM_001313915.1). Knockdown of PDI and PERK was induced by addition of 250 ng/ml doxycycline to the cell culture media.
RNA extraction, RT-primer and PCR
RNA extraction was performed with the NEB monarch RNA extraction kit (NEB) following the manufacturer’s instructions. cDNA was generated using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For RT-PCR SampleIN Direct PCR Kit (highQu, Kraichtal, Germany) was used according to the manufacturer’s protocol. Expression of PERK was tested by using PERK primers 5′-ATCCCCCATGGAACGACCTG-3′ (forward), 5′-ACCCGCCAGGGACAAAAATG-3′ (reverse). β-Actin expression was assessed using Actin primers 5′-GCCGCCAGCTCACCAT-3′ (forward) and 5′-TCGATGGGGTACTTCAGGGT-3′ (reverse). Conditions for amplifications: 95 °C for 1 min followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. Samples were loaded on a 2% agarose gel containing SYBR Safe DNA Gel stain (Thermo fisher scientific, Schwerte, Germany). Bands were visualized using the FX7 chemoluminescence documentation system (Peqlab, Erlangen, Germany) and quantified using ImageJ software.
Antibodies and reagents
Tunicamycin, Thapsigargin and MG132 were obtained in p.a. quality from Sigma-Aldrich (Schnelldorf, Germany) and Santa Cruz biotechnology (Santa Cruz, California, US). Antibodies against GAPDH and Actin were from Sigma-Aldrich, against PDI from RnD-Systems (Minneapolis, MN, USA), against BiP, eIF2α, P-eIF2α, PERK, PDI, CHOP, V5, Flag and ATF4 from Cell-Signaling technologies (Frankfurt/Main, Germany). Secondary HRP coupled antibodies against rabbit and mouse were from DAKO (Hamburg, Germany).
Clonogenic survival assay
400 A549 cells were plated in six-well plates and KD was induced immediately afterwards. Cells were irradiated with different doses after 40 h using an X-ray machine (X-rad 320, PXI) operated at 320 kV, 12.5 mA with a 1.65 mm aluminum filter (dose rate: 3.6 Gy/min). After 11 to 13 days of incubation, colonies were fixed and stained with 0.25% PFA, 70% EtOH and Coomassie brilliant blue (0.1 Coomassie blue, 5% acetic acid, 45% methanol). Colonies were counted automatically by using the colony area plugin for ImageJ as described previously46. Plating efficiency (PE) and survival fraction (SF) were calculated with the formulas “PE = colonies formed/number of cells seeded” and “SF = colonies formed/number of cells seeded × PE”.
Tumor spheroid growth experiments
Tumor spheroid growth was performed in 96-well round bottom plates coated with 1.5% agarose. 5 × 103 A549 shPDI cells were seeded, KD was induced immediately and plates were centrifuged with 800 rpm for 30 min. After 2 days, spheroids were measured and irradiated with 5 Gy. Spheroid area was measured using ImageJ plugin as described previously 47 and volume was calculated using the sphere formula (4/3 * π * r3). Controls had to be terminated before treated samples due to exponential growth and rapidly exceeding the size of the well.
Cell lysis, SDS-PAGE and western blotting
Cells were seeded in 6-Well or P60-dishes at densities between 7 × 104 and 2 × 105 cells per well and KD was induced directly afterwards. After 72 h cells were treated with ER-stress inducers for the indicated time and lysed in RIPA buffer (50 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.5% sodium deoxycholate and protease/phosphatase inhibitor cocktail (Cell Signaling)). Protein concentration was measured with a BCA assay kit (Thermo fisher scientific) and 10–30 µg protein were dissolved in SDS sample buffer (62.5 mM Tris (pH 7.4)) 2% SDS, 3% β-mercaptoethanol, 10% glycerol, 0.25 mg/ml bromophenol blue, 25 mM DTT). Samples were loaded on 7.5–10% polyacrylamide gels and afterwards transferred to PVDF membranes using the Trans-Blot Turbo system (Bio Rad, Munich, Germany). Membranes were blocked in 5% skim milk in TBS-T (50 mM Tris/HCL, 150 mM, NaCl, 0.1% Tween-20, pH 7.5). Primary antibodies were diluted as recommended by the manufacturer and incubated with the membrane overnight. Secondary antibodies were diluted 1:2000 in TBS-T and incubated for 1 h. ECL kit (Thermo Fisher Scientific, Oberhausen, Germany) and FX7 chemiluminescence documentation system (Peqlab) were used for protein detection. To show results of the western blots thoroughly through the whole manuscript, we avoided presenting multiple gels in one subfigure. Therefore, it was not possible to present all targets of interest in every subfigure.
8 × 104 HEK293T cells were plated in four wells of a 24-well plate and transfected with 500 ng V5-PERK and 500 ng flag-PDI per well. After 72 h of incubation four wells were pooled together to achieve adequate protein concentration for immunoprecipitation. Cells were lysed in caspase lysis buffer (Tris (pH 7.3) 50 mM, NaCl 150 mM, NP-40 1% (v/v)) and incubated for 4 h at 4 °C with V5 antibody. Afterwards the lysate was incubated for 1 h with protein S and G magnetic beads (Cell-Signaling) for pull down. Lysates were transferred to SDS-sample buffer containing DTT and heated at 95 °C for 10 min before loaded on a 7.5% polyacrylamide gel.
7 × 104 HEK293T cells were seeded in 24-well plates, KD was induced immediately and cells were transfected with 500 ng of firefly luciferase reporter gene plasmid and 100 ng of renilla luciferase plasmid. After 72 h cells were harvested and prepared for measurement using the Dual Glo luciferase assay kit (Promega) as recommended by the manufacturer. Firefly and renilla luciferase were measured using the GloMax detection system (Promega) and normalized to renilla values to exclude variations in transfection efficiency. For testing the secretion efficiency, cells were transfected with 500 ng gaussia luciferase and 100 ng of renilla luciferase plasmid. Luciferase activity was measured in cell culture media and normalized to intracellular renilla luciferase activity.
For bright field microscopy images a Zeiss Axiovert 200M (Carl Zeiss, Oberkochen, Germany) was used with a 4 × magnification objective and 2.5 NA. Quantification of cell confluence was performed using ImageJ.
All results were obtained in at least two independent experiments. In bar graphs mean plus standard deviation is shown for one representative experiment run in triplicates. For comparison between groups, two-way ANOVA with Bonferroni or Sidak post-hoc test was used. Significance is presented as *P < 0.05.