Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria
- University of Miami, United States
- Cornell University, United States
Figures
Figure 1 with 4 supplements
Perforin-2 deficiency or siRNA knockdown abrogates intracellular killing of pathogenic bacteria.
(A-C) Perforin-2 knockout, heterozygous, and wild-type macrophages and neutrophils were infected with Mycobacterium species (A) PEM infected with Mycobacterium smegmatis, (B) Neutrophils infected with Mycobacterium avium, and (C) BMDM infected with Mycobacterium tuberculosis. (D-H) Perforin-2 knockdown can be complemented in BV2 microglia cells infected with (D) M. avium, (E) M. smegmatis, (F) Salmonella typhimurium, and (G) MRSA. (H) Western blot demonstrating protein levels after complementation: BV2 transfected with (Lane 1) Perforin-2-RFP and Perforin-2 siRNA, (Lane 2) RFP and Perforin-2 siRNA, (Lane 3) RFP and Perforin-2 scramble siRNA, and (Lane 4) Perforin-2 siRNA alone. In western blots, Perforin-2-RFP is detected as a 105 kD band compared to the 72 kD band seen for endogenous Perforin-2 (lane 1 and 3 respectively). (I) Human MDM infection with MRSA. 
= MPEG1 (Perforin-2) wild-type cells (+/+), 
= MPEG1 (Perforin-2) heterozygous cells (+/−), • MPEG1 (Perforin-2) knockout cells (−/−). ■= RFP + Perforin-2 siRNA transfected cells, □= RFP + scramble siRNA transfected cells. ▼= Perforin-2-RFP + Perforin-2 siRNA transfected cells. One-way ANOVA with Tukey's multiple comparisons post-hoc test was used for A–G. (A–C) *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type cells; *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous cells. (D–G) *p < 0.05 between RFP + Perforin-2 siRNA:RFP + scramble siRNA and RFP + Perforin-2 siRNA:Perforin-2-RFP + Perforin-2 siRNA. (I) *p < 0.05 multiple t-tests with post-hoc correction for multiple comparisons using the Holm-Sidak method.
Figure 1—figure supplement 1
Perforin-2 genotype reflects total amount of Perforin-2 protein produced.
Murine peritoneal macrophages (A, B) or neutrophils (C, D) were isolated from Perforin-2 wild-type (wt), Perforin-2 heterozygous (het), or Perforin-2 homozygous knockout (ko) mice. Western blot after probing with mouse Perforin-2 and densitometry analysis demonstrates that Perforin-2 wild-type have the greatest amount of Perforin-2, heterozygous mice have a moderate level of Perforin-2, and the knockout animals have no Perforin-2 protein detected. Densitometry analysis includes a minimum of three Western Blots. Statistical analysis was conducted utilizing one-way ANOVA with Tukey's multiple comparisons post-hoc test in B and D *p < 0.05.
Figure 1—figure supplement 2
Perforin-2 dependent growth inhibition of Mtb in BMDM requires activation by LPS and IFN-γ.
BMDM were collected from Perforin-2 wild-type animals (
), Perforin-2 heterozygous animals (
), or Perforin-2 knockout animals (•). After differentiation to BMDM, macrophages were infected with Mtb. This experiment is representative of four different experiments. Statistical analysis was performed with one-way ANOVA with Tukey Post-hoc Multiple Comparisons. No significant difference was observed at any time point.
Figure 1—figure supplement 3
Perforin-2 is present in human MDM and PMN and can be knocked down in RA treated HL60.
(A) Western blot analysis of human Perforin-2 expression from two donors for primary monocyte derived macrophages and neutrophils (PMN). In addition, the efficiency of human Perforin-2 knockdown is demonstrated from two separate RA treated HL-60 experiments. (B) Densitometry analysis of both HL-60 human Perforin-2 knockdown experiments from (A). Statistical analysis was performed with Students T-test *p < 0.05.
Figure 1—figure supplement 4
Human HL-60-differentiated neutrophils require Perforin-2 to eliminate pathogens.
Human HL-60-neutrophils were generated by differentiating HL-60 with RA. One day prior to the experiment, HL-60/PMN cells were transfected with either a pool of scramble (□) or human Perforin-2 specific (■) siRNA. Cells were infected with (A) S. typhimurium, (B) MRSA, or (C) M. smegmatis 24 hr after transfection, which converged with maximal RA neutrophil differentiation. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05.
Figure 2 with 2 supplements
Antimicrobial compounds (ROS and NO) enhance Perforin-2 mediated killing of S. typhimurium by PEM but have limited activity in the absence of Perforin-2.
(A–D) Wild-type S. typhimurium infection of PEMs isolated from either MPEG1 (Perforin-2) +/+ (A, C), or MPEG1 (Perforin-2) −/− mice (B, D). Non-filled symbols indicated MPEG1 (Perforin-2) +/+ PEMs; whereas filled symbols are MPEG1 (Perforin-2) −/− PEMs. Cells were incubated with NAC (blue line), NAME (green line), or mock (black line). To assess bacterial resistance mechanisms against these effectors, (E) SodC1 or (F) HmpA knockout S. typhimurium were used to infect MPEG1 (Perforin-2) −/− or +/+ PEMs. The above experiments were conducted with six biologic replicates and are representative of four independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05.
Figure 2—figure supplement 1
Nitrite and reactive oxygen production in PEMs following addition of inhibitors.
(A) PEM nitrite production following stimulation with LPS and IFN-γ and incubation with ROS (NAC) or NO (NAME) inhibitors as indicated. (B) Reactive oxygen production of PEMs after LPS or PMA stimulation with addition of indicated inhibitors.
Figure 2—figure supplement 2
Antimicrobial compounds (ROS and NO) enhance Perforin-2 mediated killing of M. smegmatis by PEM.
PEMs were isolated from either (A, C) wild-type, or (B, D) Perforin-2 knockout mice. □= MPEG1 (Perforin-2) +/+ PEMs, ■= MPEG1 (Perforin-2) −/− PEMs. Cells were incubated with NAC (blue line), NAME (green line), or mock (black line). The above graphs were conducted with eight biologic replicates and are representative of three experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05.
Figure 3 with 2 supplements
Perforin-2 significantly contributes to intracellular killing in non-hematopoietically derived cells.
One day prior to the infection, cells were transfected with either a pool of scramble (□) or Perforin-2 specific (■) siRNA and 14 hr prior to the infection induced with IFN-γ. (A) HUVEC cells infected with M. smegmatis, (B) MIA-PaCa-2 cells infected with S. typhimurium, (C) UM-UC-9 infected with MRSA, (D) Perforin-2 MEF infected with MRSA, (E) Human Kc infected with MRSA induced with IFN-γ, (F) Human Kc infected with MRSA with no IFN-γ induction. 
= MPEG1 (Perforin-2) +/+, 
= MPEG1 (Perforin-2) +/−, •= MPEG1 (Perforin-2) −/−. (A–C, E, F) The above graphs contain 5–9 biologic replicates, and are representative of 3–7 independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05. (D) One-way ANOVA with Tukey post-hoc multiple comparisons. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type mice *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous mice. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type, Perforin-2 knockout:Perforin-2 heterozygous, and Perforin-2 heterozygous:Perforin-2 wild-type.
Figure 3—figure supplement 1
Perforin-2 knockdown is complementable and is able to replicate endogenous Perforin-2 bactericidal function.
Murine cells were transfected with either a pool of murine Perforin-2 specific siRNA and a RFP vector control plasmid (■); a pool of scramble siRNA and a RFP vector control plasmid (□); or a pool of murine Perforin-2 specific siRNA and a siRNA resistant Perforin-2-RFP vector (▼) and stimulated for 14 hr with IFN-γ. (A) C2C12 infected with S. typhimurium, (B) CMT93 infected with MRSA, and (C) PEM infected with M. smegmatis. The above graphs were conducted with biologic triplicates and are representative of four experiments. Statistical analysis was performed with one-way ANOVA with Tukey Post-hoc Multiple Comparisons. *p < 0.05.
Figure 3—figure supplement 2
Perforin-2 protein expression in human primary keratinocytes after knockdown compared to human monocyte derived macrophages (MDM).
Human primary keratinocytes were transfected with a pool of scramble siRNA or human Perforin-2 specific siRNA 24 hr prior to cell collection for western blot. (A) Western blot analysis demonstrating Perforin-2 protein levels in (Lane 1) human MDM, (Lane 2) human keratinocytes with Perforin-2 siRNA ablation, (Lane 3) human keratinocytes transfected with scramble siRNA. (B) Densitometry analysis from A analyzing five different experiments. Statistical analysis was performed with one-way ANOVA with Tukey post-hoc Multiple comparisons. *p < 0.05, ns = not significant.
Figure 4 with 3 supplements
Endogenous Perforin-2 is located in intracellular sites allowing for rapid translocation to bacteria.
(A) Schematic demonstrating proposed orientation of Perforin-2 in vesicles. (B) Fractionation results of endogenous Perforin-2 from human macrophages. (Lane L) is a post-nuclear lysate control, (Lane 1–8) are individual fractions corresponding with specific indicated organelles. (C) Overexpression of murine Perforin-2-GFP in murine BV2 microglial cells. (D–F) Confocal images taken 5 min after S. typhimurium infection in Perforin-2-GFP + Perforin-2 siRNA transfected BV2 cells. White arrows denote extracellular S. typhimurium, red arrows highlight a DNA cloud corresponding with S. typhimurium (D) DAPI only, (E) Perforin-2-GFP only, (F) Merge of DAPI and Perforin-2-GFP. (G–I) Confocal images taken 5 min after Escherichia coli-GFP infection in Perforin-2-RFP + Perforin-2 siRNA transfected BV2 cells. Arrows point to extracellular E. coli-GFP that has made contact but is still extracellular with normal bacilli morphology maintained. (G) E. coli-GFP only, (H) Perforin-2-RFP only, and (I) merge E. coli-GFP and Perforin-2-RFP. Fractions in B were probed as follows: Cytoplasm—MEK1/2; Early Endosome—EEA1; Lysosome—Lamp1; ER—calreticulin; Golgi—Golgin-97; Mitochondria—Prohibitin; Peroxisome—Catalase; Plasma Membrane—Cadherin.
Figure 4—figure supplement 1
Perforin-2-RFP colocalizes with ER, Golgi, and early endosomes.
(A-L) We used RAW264.7 macrophages which constitutively express Perforin-2 and studied the subcellular localization of fluorescent Perforin-2. RAW264.7 macrophages were transfected with Perforin-2-RFP and stimulated with IFN-γ and LPS for 14 hr to induce a shift towards M1 macrophages. Cells were fixed and stained as indicated. These images are representative of 3 separate experiments.
Figure 4—figure supplement 2
S. typhimurium infection of macrophages with Perforin-2 and DAPI localization through the cell (multiple Z-sections).
(A-F) BV2 cell line overexpressing Perforin-2-GFP infected with S. typhimurium. Images are collected 5 min post-infection. Arrows indicate extracellular S. typhimurium that has maintained the normal shape of S. typhimurium-likely attributed to not being surrounded by the bactericidal Perforin-2-GFP.
Figure 4—figure supplement 3
Perforin-2-RFP colocalizes with E. coli-GFP within minutes of infection.
BV2 microglia were transfected with Perforin-2-RFP and stimulated overnight with IFN-γ. Transfected cells were infected with E. coli-GFP for several minutes upon which the cells were fixed and imaged. Arrows point to extracellular E. coli-GFP. (A) Perforin-2-RFP only, (B) E. coli-GFP only, (C) Merge of Perforin-2-RFP and E. Coli-GFP. Yellow in (C) corresponds with colocalization of Perforin-2-RFP with E. coli-GFP.
Figure 5 with 1 supplement
Perforin-2 forms pores in bacterial surfaces after infection and in Perforin-2 overexpressed eukaryotic membranes that are visible by negative stain transmission electron microscopy (TEM).
(A, B) Electron micrograph of polymerized Perforin-2 membrane lesions from Perforin-2-GFP transfected HEK-293 cells, with Perforin-2 activated to form pores by trypsin digestion to the enriched membrane fraction. Panel A Demonstrates the quantity of pores on the Perforin-2 overexpressed membranes after trypsin activation. Panel B denotes a higher magnification to illustrate the uniform pore structure. (C-G) Perforin-2 wild-type MEFs were treated with IFN-γ for 14 hr, and infected with (C–E) MRSA or (F, G) M. smegmatis. After 5 hr the infected bacteria were isolated and imaged utilizing negative stain TEM. Arrows point to black, stain-filled pores on the bacterial cell wall surrounded by white, stain excluding borders created by polymerized Perforin-2. Round pores measure 8.5–10 nm inner diameter, the size typical for polymerized Perforin-2-pores. Panels E and G are close-up images of the boxed region in C and F. Blinded quantification of pore amount with different conditions is demonstrated in Figure 6—figure supplement 1.
Figure 5—figure supplement 1
Quantification of pores from negative stain transmission electron microscopy.
10 blinded fields were counted from the above eight conditions. Pores are defined as regions containing stain (corresponding to a ‘hole” in the surface) that is approximately 10 nm in diameter. Around the “hole” ultrastructure needs to be visible to ensure that the presumed “hole” is actually a pore. Statistical analysis was performed by with one-way ANOVA with Tukey Post-hoc Multiple Comparisons.*p < 0.05.
Figure 6 with 1 supplement
Cleaved Perforin-2 recovered after cellular infection with bacteria.
Perforin-2 −/− MEFs were transfected with human Perforin-2-GFP or GFP induced with murine IFN-γ and infected with the extracellular bacteria MRSA or EPEC. Following infection, bacteria were isolated, goat IgG was added to assess for nonspecific protein loss, and a portion filtered to distinguish bacteria from debris/soluble proteins. A noninfected control (first lane), demonstrates the selectivity of the differential centrifugations to remove mammalian cells. Figure 6—figure supplement 1 illustrates the recognition domain for human each Perforin-2 specific peptide generated antibody as well as verification. Fractions were probed against peptide-generated antibodies against the Perforin-2 domain (C93), and peptide generated antibodies against the MACPF domain (C186, C252). Commercial antibodies against ADA of EPEC and PBP of MRSA were utilized as bacterial markers. An additional band was observed following PBP immunoblot with a slightly higher molecular weight. This band was unspecific because it occurred in all samples including those derived from the experiments using EPEC. No signal was detected with previously validated peptide derived antibodies targeting the cytoplasmic domain of human Perforin-2 (C174), or peptide derived antibodies targeting a N-terminal portion of the Perforin-2 domain (C267) (Data not shown). In addition, commercial anti-human Perforin-2 antibody (detecting the cytoplasmic domain), clathrin, actin, and GFP also did not generate any signal (data not shown).
Figure 6—figure supplement 1
Human Perforin-2 peptide antibody validation.
(A) Six peptide antibodies were generated against different regions of human Perforin-2. Two antibodies detect in the MACPF domain of Perforin-2: C252 detects in the N-terminal portion; C186 detects in the C terminal portion of the domain. Two antibodies detect in the Perforin-2 domain of Perforin-2: C93 detects in the N terminal portion; C267 detects in the C terminal portion of the domain. Two antibodies detect in the same portion of the cytoplasmic tail of Perfroin-2: C246 and C174 (B) Antibodies were screened against either Perforin-2 constitutively expressing human macrophages from PMA differentiated Thp1s or murine macrophages (BV2 microglia cell line). All bands are located at ∼72kD, the predicted weight of Perforin-2. (C, D) Example antibodies C186 and C174 with increasing concentrations of Thp1 or BV2 protein loaded to assess detection sensitivity of the peptide antibody.
Figure 7 with 3 supplements
Perforin-2 is required for in vivo survival after MRSA epicutaneous challenge.
(A) Aggregated survival curves of 60 C57BL/6 × 129 × 1/SJV mice challenged epicutaneously with 109 MRSA. (B) Aggregated survival curves of 75,129X1/SVJ mice challenged epicutaneously with 109 MRSA. (C–F) Organ load twelve days after MRSA epicutaneous infection in (C) blood, (D) spleen, (E) kidney, and (F) skin. (G) Perforin-2 ex vivo infection of murine neutrophils with MRSA. 
= MPEG1 (Perforin-2) wild-type animals (+/+), 
= MPEG1 (Perforin-2) heterozygous animals (+/−), •= MPEG1 (Perforin-2) knockout animals (−/−). Log-rank (Mantel–Cox) test was performed for A and B with statistical significance p < 0.0001. One-way ANOVA with Tukey post-hoc multiple comparisons was performed in C-G. *p < 0.05 as indicated. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous neutrophils. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type, Perforin-2 knockout:Perforin-2 heterozygous, and Perforin-2 heterozygous:Perforin-2 wild-type neutrophils.
Figure 7—figure supplement 1
Characterization of lymphocytes in Perforin-2 knockout mice.
Frequency of CD4, CD8, and NK cells in the peripheral blood of Perforin-2 knockout and wild-type littermate mice. The phenotype of peripheral blood lymphocytes was determined by 7-color flow cytometer analysis. Each bar (A–D, G) represents the Mean ± SD from 10 to 14 mice, numbers represent the percentage of cells among live lymphocyte (A, D, G) or CD3 gated cell population (B, C). Plots represent (A) CD3+ population, (B) CD4 population, (C) CD8 population, (D) NK population, and (G) B cell population. Percentage of memory markers on (E) CD3+CD4 and (F) CD3+CD8 positive cells was determined by expression of CD44 and CD62L: Naïve CD62L + CD44-; Central memory CD62L + CD44+; and Effector memory CD62L-CD44+. The data are compiled percentage averages of 10–14 mice per group analyzed in two independent experiments. No statistical differences were observed for any adaptive immune populations by Students T-test p < 0.05.
Figure 7—figure supplement 2
Weight loss curves after MRSA epicutaneous infection.
Mice were shaved, tape-stripped, weighed for preinfection baseline, and infected with 109 S. aureus. Blinded individuals weighed the animals at indicated time points. 
= Perforin-2 wild-type animals, 
= Perforin-2 heterozygous animals, • = Perforin-2 knockout animals. (A) Weight loss in 25 mice after infection throughout the first six days post-infection. (B) Overall weight loss of 25 animals infected with 109 S. aureus, after 30% weight loss animals were euthanized, causing the apparent rebound in weight of surviving animals at day 10. The above experiments portray a representative experiment of three repeats. Statistical analysis was preformed with one-way ANOVA with Tukey Post-hoc Multiple Comparisons. * indicates p < 0.05 between both knockout:wild-type and knockout:heterozygous animals.
Figure 7—figure supplement 3
Epicutaneous MRSA infection Day 6 organ load.
Mice were shaved, tape-stripped, and infected with 109 S. aureus. 
= Perforin-2 wild-type animals, 
= Perforin-2 heterozygous animals, • = Perforin-2 knockout animals. (A–D) Organs from seven mice were collected six days after MRSA infection, weighed, homogenized, serially diluted, plated, and enumerated. Samples were normalized in weight to one another. (A) Blood was collected from cardiac puncture, (B) Spleen, (C) Kidney, and (D) Skin—site of bacterial inoculation. All samples were analyzed by Kruskal–Wallis test with Dunn's post-hoc multiple comparisons test. *p < 0.05.
Figure 8 with 3 supplements
Perforin-2 is required for in vivo survival after orogastric S. typhimurium challenge.
(A) Aggregated survival curves of 70 C57BL/6 × 129 × 1/SJV mice challenged with 105 S. typhimurium. (B) Aggregated survival curves of 45,129X1/SVJ mice challenged with 105 S. typhimurium. (C–F) Organ load five days after 105 S. typhimurium infection in C57BL/6 × 129 × 1/SJV mice in (C) blood, (D) small intestine, (E) liver, and (F) spleen. 
= MPEG1 (Perforin-2) wild-type animals (+/+), 
= MPEG1 (Perforin-2) heterozygous animals (+/−), •= MPEG1 (Perforin-2) knockout animals (−/−). Log-rank (Mantel–Cox) test was performed for A and B with statistical significance p < 0.0001. One-way ANOVA with Tukey post-hoc multiple comparisons was performed in C-F. *p < 0.05 as indicated.
Figure 8—figure supplement 1
Weight loss curves after in vivo orogastric S. typhimurium challenge.
Representative weight loss curves of 15 mice in each group challenged with 105 S. typhimurium. 
= MPEG1 (Perforin-2) wild-type (+/+) animals, 
= MPEG1 (Perforin-2) heterozygous (+/−) animals, •= MPEG1 (Perforin-2) knockout (−/−) animals. One-way ANOVA with Tukey post-hoc multiple comparisons was performed with *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type mice *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type and Perforin-2 knockout:Perforin-2 heterozygous mice. *p < 0.05 between Perforin-2 knockout:Perforin-2 wild-type, Perforin-2 knockout:Perforin-2 heterozygous, and Perforin-2 heterozygous:Perforin-2 wild-type.
Figure 8—figure supplement 2
Equal Colonization in feces 12 hr following 105 S. typhimurium inoculation.
Feces was collected, homogenized, serially diluted, plated, and enumerated from 30 animals of each genotype 12 hr after S. typhimurium oral-gastric inoculation. No statistical differences were observed in fecal shedding between groups at this time point. Statistical analysis was performed with one-way ANOVA with Tukey post-hoc multiple comparisons.
Figure 8—figure supplement 3
Organ load 60 hr after 105 S. typhimurium oral-gastric infection.
(A–D) Organs from 10 mice were collected 60 hr after S. typhimurium infection, weighed, homogenized, serially diluted, plated, and enumerated. Samples were normalized in weight to one another. (A) Blood was collected from cardiac puncture, (B) Small Intestine, (C) Liver, and (D) Spleen. All samples analyzed by Kruskal–Wallis test with Dunn's post-hoc multiple comparisons test. *p < 0.05.
Tables
Table 1
Murine perforin-2 expression
| Cell type: | Perforin-2 expression: |
|---|---|
| Peritoneal macrophage | Constitutive |
| Bone marrow derived macrophage (BMDM) | Constitutive |
| Bone marrow derived dendritic cell (BMDC) | Constitutive |
| BV-2 microglia cell line | Constitutive |
| Raw264.7 macrophage cell line | Constitutive |
| J774A.1 macrophage cell line | Constitutive |
| Microglia | Constitutive |
| Neutrophil (peritoneum stimulation) | Constitutive |
| Neutrophil (bone marrow) | Constitutive |
| Gamma delta (γδ) T cell (from Skin) | Constitutive |
| Gamma delta (γδ) T cell (from Gut) | Constitutive |
| Gamma delta (γδ) T cell (from Vagina) | Constitutive |
| Marginal zone B cell | Constitutive |
| Keratinocyte (Back) | Constitutive |
| Intestinal epithelial cells | Constitutive |
| Splenocytes | Constitutive |
| OT1 CD8 T cell induced with TGFβ, RA, and IL2 | Constitutive |
| OT1 CD8 T cell | Inducible |
| CD4 T cell | Inducible |
| B cell | Inducible |
| Astrocyte | Inducible |
| Neuron | Inducible |
| Cath.a neuroblastoma cell line | Inducible |
| Neuro-2A neuroblastoma cell line | Inducible |
| Adult CNS fibroblast | Inducible |
| Embryonic fibroblast | Inducible |
| NIH 3T3 fibroblast cell line | Inducible |
| Balb/c 3T3 fibroblast cell line | Inducible |
| C2C12 myoblast cell line | Inducible |
| Neonatal ventricular myocytes | Inducible |
| CMT-93 rectal carcinoma cell line | Inducible |
| CT26 colon carcinoma cell line | Inducible |
| B16-F10 melanoma cell line | Inducible |
| B16-F0 melanoma cell line | Inducible |
| MOVCAR 5009 ovarian cancer cell line | Inducible |
| MOVCAR 5447 ovarian cancer cell line | Inducible |
| LL/2 Lewis lung carcinoma cell line | Inducible |
| ED-1 lung adenocarcinoma cell line | Inducible |
-
Italics: Ex vivo primary cells utilized for analysis.
Table 2
Human peforin-2 expression
| Cell type: | Perforin-2 expression: |
|---|---|
| Monocyte derived macrophage (MDM) | Constitutive |
| Monocyte derived dendritic cell (MDC) | Constitutive |
| PBMC isolated NK cell | Constitutive |
| Polymorphonuclear granulocyte (neutrophil) | Constitutive |
| HL-60 promyelocyte cell line RA differentiated to PMN | Constitutive |
| HL-60 cell line PMA differentiated to Macrophage | Constitutive |
| Fetal keratinocyte | Constitutive |
| Adult keratinocyte | Constitutive |
| PMA differentiated Thp-1 monocyte cell line | Constitutive |
| NK-92 cell line | Constitutive |
| Normal colon biopsy | Constitutive |
| Normal skin biopsy | Constitutive |
| Umbilical endothelial cell (HUVEC) | Inducible |
| HeLa cervical carcinoma cell line | Inducible |
| A2EN endocervical epithelial cell line | Inducible |
| UM-UC-3 bladder cancer cell line | Inducible |
| UM-UC-9 bladder cancer cell line | Inducible |
| CaCo-2 colorectal carcinoma cell line | Inducible |
| HEK293 embryonal kidney cell line | Inducible |
| MIA-PaCa-2 pancreatic cancer cell line | Inducible |
| Skin fibroblast | Inducible |
| Thp-1 monocyte cell line | Inducible |
| HL-60 promyelocyte cell line | Inducible |
| OVCAR3 ovarian carcinoma cell line | Inducible |
| A549 alveolar adenocarcinoma cell line | Inducible |
| U-1752 bronchiolar epithelial cell line | Inducible |
| Jeg-3 placental choriocarcinoma cell line | Inducible |
-
Italics: Ex vivo primary cells utilized for analysis.
Additional files
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Supplementary file 1
Type I and Type II interferon increase Perforin-2 message in murine non-hematopoietic cell lines. (A–D) Select murine cell lines from Table 1 indicating qPCR delta CT (Perforin-2 normalized to GAPDH) (five experimental replicates) following Type I (Interferon-α, β stimulation), Type II (Interferon-γ stimulation), or both Type I and II (Interferon-αβγ stimulation). (A) Ovarian cancer cell line MOVCAR 5009, (B) Cath.a neuroblastoma cell line, (C) C2C12 myoblast cell line, (D) B16-F10 melanoma cell line. (E–H) Interferon stimulation corresponds with an increase in Perforin-2 protein. (E) Ovarian Cancer MOVCAR 5009 and (F) C2C12 myoblast cell line. Densitometry analysis of five experimental replicates of (G) MOVCAR 5009 or (H) C2C12. (A–D) Statistical analysis was performed with one-way ANOVA with Tukey post-hoc multiple comparisons. (G, H) Statistical analysis was performed with Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.032
-
Supplementary file 2
Type I and Type II interferon increase Perforin-2 message in human non-hematopoietic cell lines. Select human cell lines from Table 2 analyzed by qPCR demonstrating delta CT (Perforin-2 normalized to GAPDH) (five experimental replicates) after Type I (Interferon-αβ stimulation), Type II (Interferon-γ stimulation), or both Type I and II (Interferon-αβγ stimulation). (A) Primary HUVEC cells, (B) HEK293 cell line, and (C) MIA-PaCa-2 pancreatic cancer cell line. Interferon stimulation also increased human Perforin-2 protein with (D) MIA-PaCa-2 and (E) HUVEC cell lines. Densitometry analysis of five experimental replicates of (F) MIA-PaCa-2 or (G) HUVEC. (A–C) Statistical analysis was performed with one-way ANOVA with Tukey post-hoc multiple comparisons. (F, G) Statistical analysis was performed with Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.033
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Supplementary file 3
Perforin-2 significantly contributes to intracellular killing in murine non-hematopoietically derived cells. (A–C) One day prior to the experiment, cells were transfected with either a pool of scramble (□) or murine Perforin-2 specific (■) siRNA and 14 hr prior to the experiment induced with IFN-γ. (A) MOVCAR 5009 infected with S. typhimurium, (B) CT26 infected with MRSA, or (C) C2C12 infected with M. smegmatis. The above graphs contain 5 biologic replicates, and are representative of 3 independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.034
-
Supplementary file 4
Perforin-2 siRNA knockdown is efficient in both murine and human cells. (A–D) Representative blots from knockdown of selected cells from Table 1, Table 2, and Figure 3. (A, C) Human HUVEC cells representing knockdown of human Perforin-2; (B, D) Murine C2C12 myoblast cell line-demonstrating knockdown of murine Perforin-2. Cells were transfected with either a pool of human or mouse Perforin-2 specific siRNA or scramble siRNA. Cells were induced for 14 hr with IFN-γ and 24 hr post-transfection lysed for protein quantification. Statistical analysis was conducted utilizing Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.035
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Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria
eLife 4:e06508.
https://doi.org/10.7554/eLife.06508
