Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Matloob Husain

doi:10.3791/64189

JoVE Journal > Immunology and Infection

면역학 및 감염병학

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Published: July 21, 2022

doi:

10.3791/64189

Matloob Husain¹

¹Department of Microbiology and Immunology,University of Otago

Summary

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Abstract

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Introduction

Influenza A virus (IAV) is the prototypic member of the Orthomyxoviridae family and is known to cause global epidemics and unpredictable pandemics. IAV causes human respiratory disease, influenza, commonly known as "flu". The flu is an acute disease that results in the induction of host pro- and anti-inflammatory innate immune responses and the death of epithelial cells in the human respiratory tract. Both processes are governed by a phenomenon called programmed cell death¹. The signaling for programmed cell death is induced as soon as various pathogen recognition receptors sense the incoming virus particles in host cells. This leads to the programming of the death of infected cells and signaling to the neighboring healthy cells by three interconnected pathways called pyroptosis, apoptosis, and necroptosis-recently coined as one process, PANoptosis¹.

PANoptosis involves the proteolytic processing of many host and viral proteins from induction to execution. Such processing of proteins is primarily spearheaded by a family of cysteine proteases called caspases¹^,². Up to 18 caspases (from caspase 1 to caspase 18) are known³. Most caspases are expressed as pro-caspases and activated by undergoing their own proteolytic processing either by autocatalysis or other caspases⁴ in response to a stimulus like a virus infection. The PANoptosis of IAV-infected cells was thought to be a host defense mechanism, but IAV has evolved ways to evade and exploit it to facilitate its replication¹^,²^,⁵^,⁶. One of them is to antagonize the host factors via caspase-mediated cleavage or degradation that are either inherently antiviral or interfere with one of the steps of the IAV life cycle. To this end, host factors, cortactin, HDAC4, and HDAC6 have been discovered to undergo caspase-mediated cleavage or degradation in IAV-infected epithelial cells⁷^,⁸^,⁹. The HDAC4 and HDAC6 are anti-IAV factors⁸^,¹⁰, and cortactin interferes with IAV replication at a later stage of infection, potentially during viral assembly and budding¹¹.

In addition, various caspases are also activated, which, in turn, cleave multiple proteins to activate the host inflammatory response during IAV infection¹^,². Furthermore, nucleoprotein (NP), ion-channel M2 protein of IAV¹²^,¹³^,¹⁴, and various proteins of other viruses³^,¹⁵^,¹⁶ also undergo caspase-mediated cleavage during infection, which influences viral pathogenesis. Therefore, there is a continuous need to study caspase-mediated cleavage or degradation of host and viral proteins during IAV and other virus infections to understand the molecular basis of viral pathogenesis. Herein, the methods are presented to (1) assess the cleavage or degradation of such proteins by caspases, (2) identify those caspases, and (3) locate the cleavage sites.

Protocol

Regulatory approvals were obtained from the University of Otago Institutional Biological Safety Committee to work with the IAV and mammalian cells. Madin-Darby Canine Kidney (MDCK) or human lung alveolar epithelial A549 cells and IAV H1N1 subtypes were used for the present study. IAV was grown in chicken eggs, as described elsewhere¹⁷. Sterile and aseptic conditions were used to work with mammalian cells, and a Biosafety Level 2 (or Physical Containment 2) facility and Class II biosafety cabinet were used to work with IAV subtypes.

1. Assessing the cleavage or degradation of proteins in IAV-infected cells by caspases

Seed 3 x 10⁵ MDCK or A549 cells per well in a 12-well cell culture plate.
NOTE: If using MDCK cells, ensure that the antibody against the protein of interest recognizes its canine species.
Seed the wells in pairs, i.e., a control pair-one well for the uninfected-mock sample and one for the infected-mock sample-and a test pair-one well for uninfected-inhibitor A sample and one for the infected-inhibitor A sample. Increase the number of pairs with each additional inhibitor (see references⁷^,⁸).
Incubate the cells at 37 °C under a 5% CO₂ atmosphere overnight.
The next day, infect the cells with IAV (an H1N1 subtype or another subtype).
1. For this, prepare the virus inoculum by diluting virus stock in 400 µL of serum-free minimum essential medium (MEM) (see Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell⁷^,¹¹ (a factor of the doubling of cell number when calculating the MOI).
  NOTE: For infecting MDCK cells, supplement the virus inoculum with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin at a final concentration of 1 µg/mL.
Remove the old culture medium from the cells (step 1.3) and wash the cells with 1 mL/well of serum-free MEM 2x.
Add 400 µL of virus inoculum (step 1.4.1) to the cells and incubate them for 1 h at 35 °C under a 5% CO₂ atmosphere.
In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., NH₄Cl), or a proteasome inhibitor (e.g., MG132) (see Table of Materials) at a final concentration of 40 µM, 20 mM, or 10 µM, respectively, in serum-free MEM⁷ (see Table of Materials). The latter two inhibitors serve as a control. Dilute an equal volume of the solvent (if other than water) used to reconstitute one of the inhibitors in serum-free medium as a mock.
Remove the virus inoculum and wash the cells as in step 1.5 (1x).
Add the serum-free MEM, 1 mL/well, mock or supplemented with an inhibitor, to the cells and incubate the cells as in step 1.6. This time point is considered as 0 h infection.
After 24 h, harvest the cells by scraping them with a 1 mL syringe plunger (rubber side) and transfer them into a 1.5 mL polypropylene tube.
Centrifuge the tube at 12,000 x g for 1-2 min at room temperature. Collect the supernatant using a pipette.
NOTE: This supernatant could be discarded or used for a plaque assay⁸ to measure the titer of the released virus progeny.
Wash the cell pellet with 250 µL of phosphate-buffered saline (PBS) by re-centrifugation as in step 1.11.
Remove the supernatant and lyse the cells by adding 80-100 µL of cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see Table of Materials) and vortexing.
Heat the tube at 98 °C for 10 min to completely lyse and prepare the total cell lysate. Store the lysates at 4 °C for performing steps 1.15-1.17 the next day, but, for best results, finish these steps on the same day.
Estimate the protein amount in each sample using a BCA assay kit (see Table of Materials).
Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE)¹¹ along with molecular weight markers¹¹.
Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see Table of Materials).
NOTE: PVDF membrane may give a high background in some western blot imagers; check for compatibility.
Perform western blotting to detect the protein of interest using the method described elsewhere¹¹.
Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.
NOTE: If the protein level has recovered in the caspase inhibitor-treated infected sample lane, then the protein is cleaved or degraded by caspases. Otherwise, it is degraded by either lysosome or proteasome.
Quantify the protein recovery by measuring its band intensity in each lane from at least three replicates of the same experiment and normalizing it with the corresponding loading control band.
Use any contemporary imager and associated software (see Table of Materials) to image the western blots and quantify the protein recovery.

2. Confirmation of caspase-mediated cleavage or degradation of proteins in IAV-infected cells by RNA interference

Design or obtain a pre-design small-interfering RNA (siRNA) targeting either canine or human caspase 3, caspase 6, and caspase 7 (executioner caspases¹) and a non-targeting control siRNA (see Table of Materials).
Deliver each siRNA to MDCK or A549 cells by reverse transfection⁸^,¹¹.
For this, dilute in separate tubes 100 nM of control siRNA or caspase siRNA or a recommended volume of transfection reagent (see Table of Materials) in 100 µL of appropriate medium (like OptiMEM, see Table of Materials), and incubate for 5 min at room temperature.
Prepare each dilution in duplicate.
NOTE: This duplicate includes one replicate for analyzing the knockdown of caspase expression by RT-qPCR or western blotting (if antibodies to caspases are available) and another for assessing the recovery of the protein of interest by western blotting.
Mix 100 µL of each siRNA solution with 100 µL of transfection reagent solution (step 2.3) and incubate for 20-45 min at room temperature to form the siRNA-transfection reagent complex.
In the meantime, split and add 1 x 10⁵ MDCK or A549 cells in 800 µL of the complete growth medium, mix with 200 µL of siRNA-transfection reagent complex (step 2.5), and add 1 mL of the suspension to a well of a 12-well culture plate. This dilution brings the final concentration of control siRNA or caspase siRNA to 10 nM.
Incubate the cells at 37 °C under a 5% CO₂ atmosphere for 72 h.
Harvest the cells from one replicate as in step 1.10, and process them to validate or confirm the knockdown of the expression of caspase 3, caspase 6, and caspase 7 either by RT-qPCR or western blotting, respectively, using standard methods and protocols⁸^,¹¹.
Infect the cells in the other replicate with IAV as described in steps 1.4-1.9 (without inhibitors).
After 24 h, harvest, process, and analyze the cells by western blotting and measure and quantify the protein recovery as described in steps 1.10-1.20.

3. Site-directed mutagenesis for locating the caspase cleavage site(s) in the polypeptide

Clone the gene-encoding protein of interest in a mammalian expression plasmid¹¹ or obtain it from a research lab or commercial source (see Table of Materials).
NOTE: It is preferred if the gene is cloned with an epitope tag or as GFP fusion to distinguish the ectopically expressed polypeptide from the endogenously expressed one during western blotting.
Using caspase substrate databases⁴ or protein alignment tools, locate the consensus caspase cleavage motifs, XXXD and DXXD, on the polypeptide. Almost all caspase cleavage motifs possess the aspartic acid at the cleavage site (P1)³^,⁴.
Mutate the putative P1 aspartic acid to glutamic acid or a non-polar amino acid (alanine, valine) by site-directed mutagenesis followed by DNA sequencing¹¹ using standard methods and protocols.
Deliver the wild-type (WT) and mutant plasmid DNA to MDCK or A549 cells by reverse transfection¹¹.
1. For this, dilute 2 µg of plasmid DNA or a recommended volume of the transfection reagent in 100 µL of an appropriate medium (see step 2.3 and Table of Materials) in separate tubes, and incubate for 5 min at room temperature.
2. Mix 100 µL of each plasmid DNA solution with 100 µL of the transfection reagent solution and incubate for 20-45 min at room temperature to form the DNA-transfection reagent complex.
In the meantime, split and add 3 x 10⁵ MDCK or A549 cells to 800 µL of the complete growth medium, mix with 200 µL of DNA-transfection reagent complex, and add the 1 mL suspension to a well of a 12-well culture plate.
Incubate the cells at 37 °C under a 5% CO₂ atmosphere.
After 48 h, infect the cells with IAV as described in steps 1.4-1.9 (without adding the inhibitors).
After 24 h, harvest, process, and analyze the cells by western blotting (if applicable, using the antibody to an epitope tag or GFP). Then, measure and quantify the protein recovery as described in steps 1.10-1.20.

Representative Results

Treatment with caspase 3 inhibitor
It has been discovered that host cortactin, HDAC4, and HDAC6 polypeptides undergo degradation in response to IAV infection in both canine (MDCK) and human (A549, NHBE) cells⁷^,⁸^,⁹. By using the above approaches, it was uncovered that IAV-induced host caspases, particularly caspase 3, cause their degradation⁷^,⁸^,⁹. The cortactin was degraded in an infection dose- and time-dependent manner and a host cell- and IAV strain-independent manner⁷. Similar results were obtained for HDAC4⁸ and HDAC6⁹. Interestingly, the degradation of all three host proteins coincided with the cleavage of viral NP⁷^,⁸^,⁹, which is known to undergo caspase-mediated cleavage¹⁴. Thus, it was hypothesized that cortactin, HDAC4, and HDAC6 also undergo caspase-mediated cleavage and subsequent degradation. To test this, first, the IAV-infected cells were treated with a caspase 3 inhibitor, and the rescue of cortactin, HDAC4, and HDAC6 polypeptides was analyzed by western blotting. All three polypeptides (as well as viral NP) recovered from the IAV infection-induced degradation after caspase 3 inhibitor treatment⁷^,⁸^,⁹. The representative data⁷ for cortactin are shown in Figure 1A. This recovery was quantified by measuring the intensity of protein bands and normalizing the value of the cortactin band by the value of the corresponding actin (loading control) band. Subsequently, the normalized value of cortactin in the corresponding uninfected samples was considered 100% to determine its value in the infected samples. This quantitation method accounted the recovery of cortactin polypeptide in caspase 3 inhibitor-treated infected cells as 78.8% from 20.7% in untreated infected cells⁷ (Figure 1B).

RNA interference-mediated knockdown of caspase 3 expression
Next, a genetic tool, RNA interference (RNAi), was employed, followed by western blotting to confirm the role of caspases in IAV infection-induced degradation of cortactin¹¹ and HDAC4⁸. In caspase 3-depleted cells (Figure 2C)¹¹, both cortactin (Figure 2A)¹¹ and HDAC4⁸ polypeptides recovered from the IAV infection-induced degradation. However, in caspase 6- or caspase 7-depleted cells (Figure 2C)¹¹, no significant recovery in cortactin polypeptide levels was observed (Figure 2A)¹¹. Specifically, when quantified and compared to their levels in corresponding uninfected cells as mentioned above, the cortactin polypeptide level in infected cells with depleted caspase 3 expression recovered to 83% from 28.6% in infected cells with normal caspase 3 expression (Figure 2B)¹¹.

Caspase cleavage motifs in cortactin and HDAC6 polypeptides
Finally, the amino acid sequence in cortactin and HDAC6 polypeptides were screened and, in putative caspase cleavage motifs, the aspartic acid at the P1 position was mutated to glutamic acid⁹^,¹¹. This approach identified the aspartic acid 116 in cortactin polypeptide¹¹ and the aspartic acid 1088 in HDAC6 polypeptide⁹ as a caspase cleavage site. The mutation of both aspartic acid residues to glutamic acid rendered the cortactin (Figure 3A)¹¹ and HDAC6⁹ polypeptides resistant to IAV infection-induced degradation. Specifically, when quantified and compared to their levels in corresponding uninfected cells as mentioned above, the level of plasmid-expressed mutant cortactin polypeptide in infected cells was 77.9%, whereas the level of plasmid-expressed wild-type cortactin polypeptide in infected cells was 23.6% (Figure 3B)¹¹.

Figure 1: Cortactin polypeptide undergoes caspase 3-mediated degradation in IAV-infected cells. (A) MDCK cells were infected with influenza virus A/New Caledonia/20/1999/H1N1 strain at an MOI of 0.5 and subsequently treated as mock or with caspase 3 inhibitor (Cas3-I, 40 µM). After 24 h, the cortactin, viral NP, and actin polypeptides were detected in total cell lysates by western blotting. UNI, uninfected; INF, infected. Arrows point to the full-length and cleaved NP. (B) The intensity of cortactin and actin bands was quantified. Then, the value of cortactin was normalized with the value of actin. The normalized amount of cortactin in UNI samples was considered 100% to determine its amount in the corresponding INF samples. Data presented are means ± SE of three biological replicates. ***P = 0.0006, calculated using two-way ANOVA. ns, not significant. This figure has been modified from Chen et al.⁷. Please click here to view a larger version of this figure.

Figure 2: Knockdown of caspase 3 expression rescued cortactin polypeptide degradation in IAV-infected cells. (A) A549 cells were transfected in duplicates with 10 nM of control (Ctrl), caspase 3 (Cas3), caspase 6 (Cas6), or caspase 7 (Cas7) siRNA. After 72 h, the cells in one replicate were harvested and processed to extract total RNA. The cells in the other replicate were infected with influenza virus A/WSN/1933/H1N1 subtype at an MOI of 3.0. After 24 h, the cortactin, viral NP, and actin polypeptides were detected by western blotting. UNI, uninfected; INF, infected. (B) The intensity of cortactin and actin bands was quantified, and the value of cortactin was normalized with the value of actin. Then, the normalized amount of cortactin in the UNI samples was considered 100% to determine its amount in the corresponding INF samples. Data presented are means ± SE of three biological replicates. ****P < 0.0001, ***P = 0.0007, ***P = 0.0002, calculated using two-way ANOVA. ns, not significant. (C) Total RNA extracted above was converted to cDNA, which was then used as a template to detect caspase 3, caspase 6, caspase 7, and actin mRNA levels by RT-qPCR. The actin mRNA level was used as a reference, and the mRNA level of each caspase in control siRNA (Ctrl) and respective caspase siRNA (Cas) transfected cells was calculated using the standard 2^−ΔΔCT method. ****P < 0.0001, calculated using two-way ANOVA. This figure has been modified from Chen et al.¹¹. Please click here to view a larger version of this figure.

Figure 3: Mutation of aspartic acid 116 to glutamic acid rendered the cortactin polypeptide resistant to IAV infection-induced degradation. (A) MDCK cells were transfected with a plasmid expressing the wild-type (WT) or mutated (D116E) GFP-cortactin fusion. After 48 h, cells were infected with influenza virus A/WSN/1933/H1N1 subtype at an MOI of 3.0. After 24 h, the GFP-cortactin, viral NP, and actin polypeptides were detected by western blotting. UNI, uninfected; INF, infected. (B) The intensity of cortactin and actin bands was quantified, and the value of cortactin was normalized with the value of actin. Then, the normalized amount of cortactin in UNI samples was considered 100% to determine its amount in the corresponding INF samples. Data presented are means ± SE of three biological replicates. **P = 0.001, calculated using two-way ANOVA. ns, not significant. This figure has been modified from Chen et al.¹¹. Please click here to view a larger version of this figure.

Discussion

It is established that viruses tailor the host factors and pathways to their benefit. In turn, the host cells resist that by employing various strategies. One of those strategies is PANoptosis, which host cells use as an antiviral strategy against virus infections. However, viruses like IAV have evolved their own strategies to counter PANoptosis and exploit it to their advantage¹^,³^,⁶. This interplay involves the cleavage of various host and viral proteins by caspases. Identifying those caspases and their cleavage sites in substrate proteins are important to elucidate the mechanisms and significance of such interplay.

To this end, standard biochemical and molecular methods were used to identify the caspases and their cleavage sites in host cortactin, HDAC4, and HDAC6⁷^,⁸^,⁹^,¹¹. The protocols described above can be used for such studies involving influenza viruses, other mammalian viruses, other microbes, and external stimuli like toxins and chemicals. Furthermore, these methods can be replicated in a standard molecular biology laboratory with pertinent skills and do not require specialized equipment and software training. The 12-well cell culture plate format was successfully adapted for these and similar studies to generate sufficient sample amounts for downstream analyses by western blotting and RT-qPCR. Nevertheless, as needed, this format can be downscaled or upscaled accordingly. When doing an experiment involving a caspase inhibitor for the first time, using a pan-caspase inhibitor (Z-VAD-FMK) alongside a lysosome and proteasome inhibitor is suggested to assess whether the protein of interest undergoes caspase-mediated degradation. Further, in addition to NH₄Cl and MG132, other lysosome and proteasome inhibitors like Bafilomycin A and Epoxomicin, respectively, can be used. After positive results with a pan-caspase inhibitor, either specific inhibitors of individual caspases can be used, or RNA interference can be employed. For the former, it is important to optimize the effective inhibitory concentrations to avoid toxicity to the target cell type and non-specific results.

A genetic tool like RNA interference or CRISPR must be employed next to confirm the involvement of caspases because inhibitors do cross-react. Alternatively, an RNA interference or CRISPR screen individually targeting all known caspases can be carried out. The siRNA, gRNA, and RT-qPCR primer pairs to all known caspases could be procured as pre-designed or designed using online tools. In addition, two or more caspases can be depleted simultaneously to assess the sequential or cooperative cleavage of polypeptides (this strategy was recently used in a yet-to-be-published study). RNA interference is believed to be an ideal tool for virus-host cell interaction research. The latter requires a controlled manipulation of host gene expression so that the host cells remain relatively viable for virus infection and completion of the life cycle to display the phenotype of that manipulation. However, optimizing an effective siRNA concentration and siRNA-transfection reagent combination is needed for each cell type for an optimal knockdown and viability ratio for subsequent infection.

To locate the putative caspase cleavage motifs, many databases such as CASBAH¹⁸, CaspDB¹⁹, CutDB²⁰, DegraBase²¹, MEROPS²², and TopFIND²³^,²⁴ have been developed. A visual screening was performed to locate these motifs on HDAC6 polypetide⁹, but for cortactin¹¹, the CaspDB was used (though the CaspDB website has been inaccessible lately). Those motifs were successfully confirmed as caspase cleavage sites by mutating the P1 aspartic acid to glutamic acid. However, mutating the aspartic acid to a non-polar amino acid-like alanine or valine first is suggested because some caspases can cleave after P1 glutamic acid⁴. This exercise may promptly result in the identification of caspase cleavage site(s) but could require generating tens of mutants if the target polypeptide is rich in aspartic acids and the putative target motifs are non-canonical. For the delivery of plasmids (and siRNA) to cells, forward, instead of reverse, transfection can be carried out. Further, the infection dose and time kinetics should be performed using a 4%-20% gradient SDS-PAGE for western blotting to assess the degradation kinetics of the polypeptides and identify any smaller-sized cleavage products. Finally, to confirm the protein of interest as a direct caspase substrate, in vitro biochemical assays containing purified caspases and target proteins (WT or mutant) and any cofactors could be developed.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The author acknowledges Jennifer Tipper, Bilan Li, Jesse vanWestrienen, Kevin Harrod, Da-Yuan Chen, Farjana Ahmed, Sonya Mros, Kenneth Yamada, Richard Webby, the BEI Resources (NIAID), the Health Research Council of New Zealand, the Maurice and Phyllis Paykel Trust (New Zealand), the H.S. and J.C. Anderson Trust (Dunedin), and the Department of Microbiology and Immunology and School of Biomedical Sciences (University of Otago).

Materials

A549 cells	ATCC	CRM-CCL-185	Human, epithelial, lung
Ammonium chloride	Sigma-Aldrich	A9434
Caspase 3 Inhibitor	Sigma-Aldrich	264156-M	Also known as 'InSolution Caspase-3 Inhibitor II – Calbiochem'
cOmplete, Mini Protease Inhibitor Cocktail	Roche	11836153001
Goat anti-NP antibody			Gift from Richard Webby (St Jude Children’s Research Hospital, Memphis, USA) to MH
Lipofectamine 2000 Transfection Reagent	ThermoFisher Scientific	31985062
Lipofectamine RNAiMAX Transfection Reagent	ThermoFisher Scientific	13778150
MDCK cells	ATCC	CCL-34	Dog, epithelial, kidney
MG132	Sigma-Aldrich	M7449
Minimum Essential Medium (MEM)	ThermoFisher Scientific	11095080	Add L-glutamine, antibiotics or other supplements as required
MISSION siRNA Universal Negative Control #1	Sigma-Aldrich	SIC001
Odyssey Fc imager with Image Studio Lite software 5.2	LI-COR		Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software.
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23225
Plasmid expressing human cortactin-GFP fusion	Addgene	50728	Gift from Kenneth Yamada to Addgene
Pre-designed small interferring RNA (siRNA) to caspase 3	Sigma-Aldrich	NM_004346	siRNA ID: SASI_Hs01_00139105
Pre-designed small interferring RNA to caspase 6	Sigma-Aldrich	NM_001226	siRNA ID: SASI_Hs01_00019062
Pre-designed small interferring RNA to caspase 7	Sigma-Aldrich	NM_001227	siRNA ID: SASI_Hs01_00128361
Pre-designed SYBR Green RT-qPCR Primer pairs	Sigma-Aldrich	KSPQ12012	Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1
Protran Premium nitrocellulose membrane	Cytiva (Fomerly GE Healthcare)	10600003
Rabbit anti-actin antibody	Abcam	ab8227
Rabbit anti-cortactin antibody	Cell Signaling	3502
Rabbit anti-GFP antibody	Takara	632592
SeeBlue Pre-stained Protein Standard	ThermoFisher Scientific	LC5625
Transfection medium, Opti-MEM	ThermoFisher Scientific	11668019
Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100	Merck
Trypsin, TPCK-Treated	Sigma-Aldrich	4370285

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Husain, M. Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection. J. Vis. Exp. (185), e64189, doi:10.3791/64189 (2022).