Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HBB2 causes lipidation of LC3B and accumulation of p62, and enhances autophagic flux. ( a ) Immunofluorescence analysis for p62 and LC3B in U2OS cells that had been exposed to HBB2 (3 µM) (bottom) or treated with vehicle (top) for 16 h. The images were obtained by confocal microscopy. Scale bar = 20 µm. ( b–d ) Western blot analysis of U2OS whole-cell lysates was conducted to detect the changes in the levels of LC3B-II ( b ), Hsp70 ( c ) and p62 (d) in response to a 4-, 8-, 16-, or 24 h treatments with HBB2 (3 µM) and/or bafilomycin A1 (Baf-A1; 10 nM), as indicated above the lanes. DMSO (0.1%, v/v) was used as a vehicle control (indicated as −/− above the blots), whereas β-actin levels served as a loading control.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Immunofluorescence, Confocal Microscopy, Western Blot, Control

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: Knockdown of NRF2 inhibits autophagy. ( a ) Immunoblotting analysis of NRF2, p62 and LC3B in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or NRF2 siRNA (siNRF2) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with 10 nM bafilomycin A1 (Baf-A1) or vehicle (0.1% DMSO) for the last 2 h. The levels of β-actin served as a loading control. For detection of LC3B, proteins from cell lysates were resolved using 13% SDS-PAGE and transferred onto 0.45 µm PVDF membranes, whereas for detection of NRF2 and actin, proteins were blotted onto 0.45 µm NC membranes. ( b–d ) Real-time PCR analysis of NRF2 ( b ), HSF1 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment. The mRNA levels of 18 S were used for normalization. The data are represented by mean + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siNRF2 for each treatment pair.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Knockdown, Western Blot, Transfection, Control, SDS Page, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: NRF2 is required for basal and HBB2-induced autophagic flux. Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868). Wide-field microscope (Deltavision Elite) was used to collect the images where in each field 25 images were taken with an optical section of 0.2 μm. The deconvolved images represent the summed intensity projection using SoftWorX (Version 5.5). Scale bar = 20 µm.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Immunofluorescence, Transfection, Control, Microscopy

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HSF1 inhibits autophagic flux. ( a ) Immunoblotting analysis of HSF1, Hsp70, p62, mTOR, phosphorylated mTOR (pS2448), p70 S6K, phosphorylated p70 S6K (pT389) in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or HSF1 siRNA (siHSF1s or siHSF1sp) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with bafilomycin A1 (Baf-A1; 10 nM) or vehicle (0.1% DMSO) for a the last two hours of HBB2 treatment. The levels of β -actin served as a loading control. ( b–d ) Real-time PCR analysis of HSF1 ( b ), NRF2 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment, in U2OS cells transfected with siCTL (10 nM) or siHSF1sp (10 nM). The mRNA levels of 18 S were used for normalization. The data are represented by means + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siHSF1sp for each treatment pair. ( e ) Immunoblotting analysis of LC3B in lysates from U2OS cells, which had been transfected with either siCTL (10 nM) or siHSF1sp siRNA (10 nM) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, supplemented with bafilomycin A1 (Baf-A1; 10 nM) for the last 2 h. The levels of β-actin served as a loading control.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HBB2 causes lipidation of LC3B and accumulation of p62, and enhances autophagic flux. ( a ) Immunofluorescence analysis for p62 and LC3B in U2OS cells that had been exposed to HBB2 (3 µM) (bottom) or treated with vehicle (top) for 16 h. The images were obtained by confocal microscopy. Scale bar = 20 µm. ( b–d ) Western blot analysis of U2OS whole-cell lysates was conducted to detect the changes in the levels of LC3B-II ( b ), Hsp70 ( c ) and p62 (d) in response to a 4-, 8-, 16-, or 24 h treatments with HBB2 (3 µM) and/or bafilomycin A1 (Baf-A1; 10 nM), as indicated above the lanes. DMSO (0.1%, v/v) was used as a vehicle control (indicated as −/− above the blots), whereas β-actin levels served as a loading control.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Immunofluorescence, Confocal Microscopy, Western Blot, Control

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: Knockdown of NRF2 inhibits autophagy. ( a ) Immunoblotting analysis of NRF2, p62 and LC3B in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or NRF2 siRNA (siNRF2) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with 10 nM bafilomycin A1 (Baf-A1) or vehicle (0.1% DMSO) for the last 2 h. The levels of β-actin served as a loading control. For detection of LC3B, proteins from cell lysates were resolved using 13% SDS-PAGE and transferred onto 0.45 µm PVDF membranes, whereas for detection of NRF2 and actin, proteins were blotted onto 0.45 µm NC membranes. ( b–d ) Real-time PCR analysis of NRF2 ( b ), HSF1 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment. The mRNA levels of 18 S were used for normalization. The data are represented by mean + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siNRF2 for each treatment pair.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Knockdown, Western Blot, Transfection, Control, SDS Page, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: NRF2 is required for basal and HBB2-induced autophagic flux. Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868). Wide-field microscope (Deltavision Elite) was used to collect the images where in each field 25 images were taken with an optical section of 0.2 μm. The deconvolved images represent the summed intensity projection using SoftWorX (Version 5.5). Scale bar = 20 µm.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Immunofluorescence, Transfection, Control, Microscopy

Journal: Scientific Reports

Article Title: Transcription factors NRF2 and HSF1 have opposing functions in autophagy

doi: 10.1038/s41598-017-11262-5

Figure Lengend Snippet: HSF1 inhibits autophagic flux. ( a ) Immunoblotting analysis of HSF1, Hsp70, p62, mTOR, phosphorylated mTOR (pS2448), p70 S6K, phosphorylated p70 S6K (pT389) in lysates from U2OS cells, which had been transfected with either control siRNA (siCTL) or HSF1 siRNA (siHSF1s or siHSF1sp) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, and supplemented with bafilomycin A1 (Baf-A1; 10 nM) or vehicle (0.1% DMSO) for a the last two hours of HBB2 treatment. The levels of β -actin served as a loading control. ( b–d ) Real-time PCR analysis of HSF1 ( b ), NRF2 ( c ), and p62 ( d ) from a parallel experiment described in ( a ) except without Baf-A1 treatment, in U2OS cells transfected with siCTL (10 nM) or siHSF1sp (10 nM). The mRNA levels of 18 S were used for normalization. The data are represented by means + SD from three independent transfections. Student’s t-test was used to test for statistical significance, where *represents p < 0.05 when comparing siCTL and siHSF1sp for each treatment pair. ( e ) Immunoblotting analysis of LC3B in lysates from U2OS cells, which had been transfected with either siCTL (10 nM) or siHSF1sp siRNA (10 nM) for 48 h, and subsequently treated with vehicle (0.1% DMSO) or HBB2 (3 µM) for 18 h, supplemented with bafilomycin A1 (Baf-A1; 10 nM) for the last 2 h. The levels of β-actin served as a loading control.

Article Snippet: Immunofluorescence images ( a ) and quantitative analysis ( b ) of the colocalization of p62 and LC3B in U2OS cells, which had been transfected with either control siRNA (top two panels) or NRF2 siRNA (bottom two panels) for 48 h, and subsequently treated with bafilomycin A1 (Baf-A1; 10 nM) for 18 h. Note that in the case of detection of LC3B, an antibody exhibiting a stronger reactivity to the lipidated form (LC3B-II) was used (LC3B D11, CST #3868).

Techniques: Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: mBio

Article Title: Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

doi: 10.1128/mBio.01134-19

Figure Lengend Snippet: Oxidative stress response in parasites with the WT and mutant PfK13 genes. (A) Ubiquitination of P. falciparum proteins following ART treatment. Representative Western blots of parasites show protein ubiquitination after ART treatment. Uninfected RBCs or trophozoite-stage parasites of the 3D7, WT, F446I, C469Y, F495L, C580Y, and N458Y strains were treated with 6 μM ART at 3% hematocrit for 90 min at 37°C. Cell extracts were separated by SDS-PAGE and probed with anti-ubiquitin IgG or anti-aldolase antibodies. (B) Quantitation of ubiquitination in parasites after ART treatment. The graph shows the results of a densitometry analysis of the anti-ubiquitin signal normalized to the anti-aldolase signal from three independent experiments (***, P <0.005, nonparametric Wilcoxon t test; the raw data are presented in Table S2B in the supplemental material). (C) Quantification of relative expression of genes upregulated in ART-resistant field isolates at the early ring stage. RT-quantitative PCR analysis was performed at the early ring stage of WT and isogenic transfected 3D7 parasites carrying the F446I, C469Y, F495L, N458Y, and C580Y mutations for the transcripts of six genes which are upregulated in the ART-resistant parasites (PF3D7_0827900, ERp72; PF3D7_0917900, BiP; PF3D7_1010700, dolichyl-phosphate-mannose protein mannosyl transferase; PF3D7_1372000, plasmodium exported protein [PHISTa]; PF3D7_1108600, endoplasmic reticulum-resident calcium binding protein; and PF3D7_1115600, peptidyl-prolyl cis-trans isomerase). Expression levels were normalized to the level for the housekeeping gene seryl-tRNA synthetase, and relative expression (fold change) was calculated using the expression levels in the WT parasites. Values of more than 1 (dashed line) indicate upregulation.

Article Snippet: For colocalization experiments, 3D7 or GFP::PfK13 parasites were fixed and probed with anti-BiP mouse antisera as the endoplasmic reticulum (ER) marker, anti-HAP mouse sera as the food vacuole marker (catalog number MRA-811A; BEI Resources, VA), or anti-PI3P mouse antibodies (Echelon Biosciences Inc., UT) at a 1:200 dilution ( ).

Techniques: Mutagenesis, Ubiquitin Proteomics, Western Blot, SDS Page, Quantitation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Binding Assay

Journal: mBio

Article Title: Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

doi: 10.1128/mBio.01134-19

Figure Lengend Snippet: Expression and localization of PfK13 during asexual erythrocytic growth. (A) Western blots of PfK13 expression at the ring (R), trophozoite (T), and schizont (S) stages of 3D7 parasites with anti-PfK13 antibodies. Aldolase was used as the control for equal protein loading from different stages. (B) Localization of PfK13 in the ring (R), trophozoite (T), early schizont (ES), mature schizont (MS), and merozoite (M) stages by IFA with anti-PfK13 antibodies. (C) Colocalization analysis of PfK13 with BiP (an ER marker) and HAP (a food vacuole marker) in 3D7 using anti-PfK13 antibodies. (D) Colocalization analysis of PfK13 with BiP and HAP in GFP::PfK13 parasites using anti-GFP antibodies. DIC, differential interference contrast.

Article Snippet: For colocalization experiments, 3D7 or GFP::PfK13 parasites were fixed and probed with anti-BiP mouse antisera as the endoplasmic reticulum (ER) marker, anti-HAP mouse sera as the food vacuole marker (catalog number MRA-811A; BEI Resources, VA), or anti-PI3P mouse antibodies (Echelon Biosciences Inc., UT) at a 1:200 dilution ( ).

Techniques: Expressing, Western Blot, Marker

Journal: mBio

Article Title: Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

doi: 10.1128/mBio.01134-19

Figure Lengend Snippet: Oxidative stress response in parasites with the WT and mutant PfK13 genes. (A) Ubiquitination of P. falciparum proteins following ART treatment. Representative Western blots of parasites show protein ubiquitination after ART treatment. Uninfected RBCs or trophozoite-stage parasites of the 3D7, WT, F446I, C469Y, F495L, C580Y, and N458Y strains were treated with 6 μM ART at 3% hematocrit for 90 min at 37°C. Cell extracts were separated by SDS-PAGE and probed with anti-ubiquitin IgG or anti-aldolase antibodies. (B) Quantitation of ubiquitination in parasites after ART treatment. The graph shows the results of a densitometry analysis of the anti-ubiquitin signal normalized to the anti-aldolase signal from three independent experiments (***, P <0.005, nonparametric Wilcoxon t test; the raw data are presented in Table S2B in the supplemental material). (C) Quantification of relative expression of genes upregulated in ART-resistant field isolates at the early ring stage. RT-quantitative PCR analysis was performed at the early ring stage of WT and isogenic transfected 3D7 parasites carrying the F446I, C469Y, F495L, N458Y, and C580Y mutations for the transcripts of six genes which are upregulated in the ART-resistant parasites (PF3D7_0827900, ERp72; PF3D7_0917900, BiP; PF3D7_1010700, dolichyl-phosphate-mannose protein mannosyl transferase; PF3D7_1372000, plasmodium exported protein [PHISTa]; PF3D7_1108600, endoplasmic reticulum-resident calcium binding protein; and PF3D7_1115600, peptidyl-prolyl cis-trans isomerase). Expression levels were normalized to the level for the housekeeping gene seryl-tRNA synthetase, and relative expression (fold change) was calculated using the expression levels in the WT parasites. Values of more than 1 (dashed line) indicate upregulation.

Article Snippet: For colocalization experiments, 3D7 or GFP::PfK13 parasites were fixed and probed with anti-BiP mouse antisera as the endoplasmic reticulum (ER) marker, anti-HAP mouse sera as the food vacuole marker (catalog number MRA-811A; BEI Resources, VA), or anti-PI3P mouse antibodies (Echelon Biosciences Inc., UT) at a 1:200 dilution ( ).

Techniques: Mutagenesis, Western Blot, SDS Page, Quantitation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Binding Assay