multiple unpaired t tests with welch’s correction and no correction for multiple comparisons  (GraphPad Software Inc)

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A) Western blot of proteins extracted from rHA- Tg ApiCox13 parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days, separated by SDS-PAGE and detected using anti-HA antibodies (to detect the HA -Tg ApiCox13 protein) and anti- Tg Tom40 antibodies (loading control). (B) Plaque assays measuring proliferation of WT, rHA- Tg ApiCox13 and complemented rHA- Tg ApiCox13/cFLAG- Tg ApiCox13 WT parasites cultured in the absence (top) or presence (bottom) of ATc for 7 days. Assays are from a single experiment and are representative of three independent experiments. Scale bar represents 1 cm. (C) Basal mitochondrial O 2 consumption rate (mOCR) of WT (blue), rHA- Tg ApiCox13 (green) and rHA- Tg ApiCox13/cFLAG- Tg ApiCox13 WT (purple) parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. A linear mixed-effects model was fitted to the data and values depict the estimated marginal mean ± 95% CI of three independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed with relevant p values shown. (D) Basal mOCR versus basal extracellular acidification rate (ECAR) of WT (blue), rHA- Tg ApiCox13 (green) and rHA- Tg ApiCox13/cFLAG- Tg ApiCox13 WT (purple) parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. Data depict the estimated marginal mean mOCR and ECAR values ± 95% CI of three independent experiments.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Western Blot, Cell Culture, SDS Page, Control

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A) Schematic of the assay probing ETC function, illustrating where substrates and inhibitors act on the ETC. Malate donates electrons (e - ) to coenzyme Q (CoQ), increasing OCR when ETC is functional; atovaquone (ATV) inhibits Complex III (III), decreasing OCR; TMPD donates e - to cytochrome c (CytC), increasing OCR when Complex IV (IV) is functional; sodium azide (NaN 3 ) inhibits Complex IV, decreasing OCR. (B-D) WT (blue), rHA- Tg ApiCox13 (green) and cFLAG- Tg ApiCox13/rHA- Tg ApiCox13 (purple) parasites were cultured in the absence of ATc or in the presence of ATc for 1–3 days and the assay was performed. (B) Representative trace depicting OCR over time. Data represent the mean ± SD of 3 technical replicates and are representative of 3 independent experiments. (C-D) Quantification of (C) malate-elicited mOCR (mOCR Malate ) and (D) TMPD-elicited mOCR (mOCR TMPD ). A linear mixed-effects model was fitted to the data and values depict the estimated marginal mean ± 95% CI of 3 independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed with relevant p values shown. (E) In-gel Complex IV activity assay performed on samples extracted from an equal number of rHA- Tg ApiCox13 parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. The intensity of the band at ~720 kDa corresponds to the extent of Complex IV activity.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Functional Assay, Cell Culture, Activity Assay

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A-C) Western blots of proteins extracted from (A) rHA- Tg ApiCox13/ Tg ApiCox25-FLAG, (B) rHA- Tg ApiCox13/ Tg Cox2a-FLAG, and (C) rHA- Tg ApiCox13/ Tg MPPα-FLAG parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. Samples were prepared in 1% (v/v) TX-100, separated by BN-PAGE, and detected with anti-FLAG and anti- Tg Tom40 antibodies. (D-F) Western blots of proteins extracted from (D) rHA- Tg ApiCox13/ Tg ApiCox25-FLAG, (E) rHA- Tg ApiCox13/ Tg Cox2a-FLAG, and (F) rHA- Tg ApiCox13/ Tg MPPα-FLAG parasites cultured as described for (A-C). Samples were separated by SDS-PAGE, and probed with anti-HA, anti-FLAG and anti- Tg Tom40 antibodies. Western blots are representative of three independent experiments, with matched BN-PAGE and SDS-PAGE samples harvested from the same experiment. The abundance of FLAG-tagged proteins was quantified, normalised relative to the Tg Tom40 loading control and expressed as a percentage of the abundance on 0 days ATc. These data are shown as graphs below each blot. Bars represent the mean ± SD of three independent experiments, with each replicate shown as a dot. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p -values shown. Colours match those in with Complex IV subunits in green and the Complex III subunit in orange.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Western Blot, Cell Culture, SDS Page, Control

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A) Schematic of the assay illustrating where substrates and inhibitors act on the ETC. (B-E) OCR measurements of WT (blue) and r Tg NFS1/FLAG- Tg ApiCox13 (purple) parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. (B) Representative trace depicting OCR over time. Data depict the mean ± SD of 3 technical replicates and are representative of 3 independent experiments. (C-E) Quantification of (C) malate-elicited mOCR (mOCR Malate ), (D) TMPD-elicited mOCR (mOCR TMPD ) and (E) fold stimulation of mOCR elicited by TMPD relative to malate (mOCR TMPD/Malate ). A linear mixed-effects model was fitted to the data and values depict the estimated marginal mean ± 95% CI of 3 independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed with relevant p values shown. (F-G) Western blots of proteins extracted from r Tg NFS1/FLAG- Tg ApiCox13 parasites cultured in the absence of ATc or in the presence of ATc for 1–3 days. Samples were separated by (F) BN-PAGE or (G) SDS-PAGE and detected with anti-FLAG and anti- Tg Tom40 antibodies. Western blots are representative of three independent experiments, with matched BN-PAGE and SDS-PAGE samples harvested from the same experiment. FLAG- Tg ApiCox13 abundance was quantified, normalised relative to Tg Tom40 and expressed as a percentage of the abundance on 0 days ATc. These data are shown as graphs below each blot. Bars represent the mean ± SD of three independent experiments, with each replicate shown as a dot. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p -values shown.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Cell Culture, Western Blot, SDS Page

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A-C) Western blots and immunofluorescence assays of rHA- Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing (A) cFLAG- Tg ApiCox13 H49Q , (cH49Q) (B) cFLAG- Tg ApiCox13 H85Q (cH85Q), or (C) cFLAG- Tg ApiCox13 H49/85Q (cH49/85Q). Proteins were extracted from the parasites, separated by SDS-PAGE, and probed by western blotting with anti-FLAG antibodies (left). Immunofluorescence assays (right) depict two to eight parasites in a vacuole, with FLAG- Tg ApiCox13 proteins detected with anti-FLAG (green) and the mitochondrion with anti- Tg Tom40 (magenta) antibodies. DIC, differential interference contrast; scale bars represent 2 μm. (D) Plaque assays measuring the proliferation of Tg ApiCox13/ Tg ApiCox25-GFP parasites (r Tg ApiCox13), or Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing the cH49Q, cH85Q, or cH49/85Q proteins, and cultured in the absence (top) or presence (bottom) of ATc for 8 days. Assays are from a single experiment and are representative of 3 independent experiments; scale bar represents 1 cm. (E) Basal mitochondrial oxygen consumption rate (mOCR) of Tg ApiCox13/ Tg ApiCox25-GFP parasites (r Tg ApiCox13), or Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing the cWT, cH49Q, cH85Q or cH49/85Q proteins, and cultured in the absence (white) or presence (red) of ATc for 3 days. A linear mixed-effects model was fitted to the data and values depict the estimated marginal mean ± 95% CI of three independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed with relevant p values shown.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Western Blot, Immunofluorescence, Expressing, SDS Page, Cell Culture

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A and C) Western blot of proteins extracted from rHA- Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing cFLAG- Tg ApiCox13 WT (cWT), cFLAG- Tg ApiCox13 H49Q (cH49Q), cFLAG- Tg ApiCox13 H85Q (cH85Q) or cFLAG- Tg ApiCox13 H49/85Q (cH49/85Q) proteins, cultured in the absence (-, white) or presence (+, red) of ATc for 3 days, separated by (A) SDS-PAGE or (C) BN-PAGE, and probed using anti-FLAG (top), anti-GFP (upper middle), anti-HA (lower middle) or anti- Tg Tom40 (bottom) antibodies. Western blots are representative of 3 independent experiments, with matched BN-PAGE and SDS-PAGE samples harvested from the same experiment. Asterisks in the anti-GFP western blots mark the presence of likely degradation products of the Tg ApiCox25-GFP protein. (B and D) The abundance of FLAG- (top) and GFP- (bottom) tagged protein (B) monomers or (D) complexes was quantified, normalised relative to the Tg Tom40 loading control and expressed as a percentage of the cWT abundance in the absence of ATc. These quantifications are shown as column graphs with bars representing the mean ± SD of three independent experiments. ANOVA followed by Tukey’s multiple comparisons test was performed with relevant p -values shown.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Western Blot, Expressing, Cell Culture, SDS Page, Control

Journal: PLOS Pathogens

Article Title: An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites

doi: 10.1371/journal.ppat.1011430

Figure Lengend Snippet: (A) Western blot and immunofluorescence assay of rHA- Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing cFLAG- Tg ApiCox13 H49/85C (cH49/85C). Proteins were extracted from the parasites, separated by SDS-PAGE, and probed by western blotting with anti-FLAG antibodies (left). Immunofluorescence assay (right) depicts eight parasites in a vacuole, with FLAG- cH49/85C detected with anti-FLAG (green) and the mitochondrion with anti- Tg Tom40 (magenta) antibodies. DIC, differential interference contrast; scale bar represents 2 μm. (B) Plaque assays measuring the proliferation of Tg ApiCox13/ Tg ApiCox25-GFP parasites (r Tg ApiCox13), or Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing WT Tg ApiCox13 (cWT) or cH49/85C proteins, and cultured in the absence (top) or presence (bottom) of ATc for 8 days. Assays are from a single experiment and are representative of 3 independent experiments; scale bar represents 1 cm. (C) Basal mitochondrial oxygen consumption rate (mOCR) of Tg ApiCox13/ Tg ApiCox25-GFP parasites (r Tg ApiCox13), or Tg ApiCox13/ Tg ApiCox25-GFP parasites constitutively expressing the cWT or cH49/85Q proteins, and cultured in the absence (white) or presence (red) of ATc for 3 days. A linear mixed-effects model was fitted to the data and values depict the estimated marginal mean ± 95% CI of three independent experiments. ANOVA followed by Tukey’s multiple pairwise comparisons test was performed with relevant p values shown.

Article Snippet: Statistical differences were tested using ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test with Welch’s correction in GraphPad Prism.

Techniques: Western Blot, Immunofluorescence, Expressing, SDS Page, Cell Culture

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: The SIV-induced IL-1β production requires receptor-interacting protein kinase 1 (RIPK1) kinase activity. ( A ) PAMs were transfected with a negative control siRNA (siControl) or RIPK1 -targeting siRNA (siRIPK1) for 24 h and infected with Sk02 at an MOI of 1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β was analyzed by Western blotting. RIPK1 knockdown efficiency was determined by real-time PCR. ( B ) PAMs were infected with Sk02 at an MOI of 1 for 20 h in the presence of a vehicle (DMSO) or increasing concentrations (80 and 160 μM) of RIPK1 kinase inhibitor, Nec-1. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β and viral NS1 was analyzed by Western blotting. ( C ) Amino acid sequences of the N-terminal ends of the kinase domains in porcine, murine, and human RIPK1 (based on the GenBank accession numbers XM_005665536, NM_009068, and NM_003804, respectively) were aligned using Clustal Omega. Amino acid positions are based on the human RIPK1 and asterisks indicate conserved residues among the three species. K41/K42 in porcine RIPK1, K45/K46 in murine RIPK1, and K45 in human RIPK1 are underscored. ( D ) For the NLRP3 inflammasome reconstitution assay, HEK293T cells were co-transfected with plasmids expressing porcine NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β along with Myc-vector or Myc-tagged WT/mutant human RIPK1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Protein expression was analyzed by Western blotting. ( E ) HEK293T cells were transfected as in ( D ) along with the Myc-vector or Myc-tagged porcine RIPK1 WT/mutant for 16 h. ELISA and Western blotting were done as in ( D ). Statistical analysis was done with one-way ANOVA except for the knockdown efficiency data done with an unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. Results are representative of three independent experiments.

Article Snippet: One-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests or unpaired t -test with Welch’s correction was performed using GraphPad Prism 7.

Techniques: Activity Assay, Transfection, Negative Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Knockdown, Real-time Polymerase Chain Reaction, Reconstitution Assay, Plasmid Preparation, Mutagenesis