Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , The amino-acid sequence of human MEK1 aligned to human ULK1 and human ULK2. Black letters, amino acids; blue letters, the four amino acids mutated in C u- b inding m utant (CBM) of MEK1 to decrease Cu binding and those conserved between ULK1 and ULK2. b , c , Immunoblot detection of recombinant GST-ULK1 or GST-ULK2 bound to a resin charged with or without Cu, Fe, or Zn compared to input. d , e , f , g , Immunoblot detection of recombinant phosphorylated (P)-ATG13, total (T)-ATG13, and GST-ULK1 or GST-ULK2 from ULK1 or ULK2 in vitro kinase assays treated with or without increasing concentrations of CuCl 2 from 0 to 10 μM ( d , e ), with or without increasing concentrations of TTM from 0 to 50 μM, or 10 μM MRT68921 ( f , g ). Quantification: ΔP-ATG13/T-ATG13 normalized to GST-ULK1 and GST-ATG13 alone. h , Immunoblot detection of P- and/or T-mTOR, p70S6K, ULK1, ATG13, CCS, or β-ACTIN from Ctr1 -/- MEFs stably expressing CTR1 WT (WT) or empty vector (VO) treated with vehicle (VEH) or amino acid deprivation (-AA). Quantification: ΔP-ATG13/T-ATG13 normalized to WT, VEH control. i , j , Immunoblot detection of recombinant P-ATG13, T-ATG13, or immunoprecipitated (IP)-ULK1 from immunocomplex ULK1 kinase assays from Ctr1 -/- MEFs stably expressing WT or VO treated with VEH or -AA or MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ). Quantification: ΔP-ATG13/T-ATG13 normalized to WT, VEH control or Rosa ( - ) control. k , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Ctr1 -/- MEFs stably expressing WT or VO treated with VEH, -AA, or rapamycin (RAP), with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to WT, VEH control. l , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 fluorescent intensity (FI)/mean EGFP-LC3 FI ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and mCherry-EGFP-LC3B treated with VEH, -AA, or RAP, with or without BAF normalized to WT, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Four asterisks, P<0.0001. n≥7. m , Representative fluorescence images of EGFP, mCherry, or the merge from Ctr1 -/- MEFs stably expressing WT or VO and mCherry-EGFP-LC3B treated with VEH, BAF, - AA, or RAP. n , Scatter dot plot of quantified mCherry positive ( + )-LC3 puncta per cell or mCherry + EGFP + -LC3 puncta per cell ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and mCherry-EGFP-LC3B treated with VEH, BAF, -AA, or RAP. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001; two asterisks, P<0.0001. n≥24. o , Immunoblot detection of LC3-I, LC3-II, P-ERK1/2, T-ERK1/2, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ) treated with VEH or BAF. Quantification: ΔLC3-II/T-β-ACTIN and ΔP-ERK1/2/T-ERK1/2 normalized to Rosa ( - ), VEH control. p , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 FI/mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and mCherry-EGFP-LC3B treated with VEH or BAF normalized to Rosa ( - ), VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=9. q , Representative fluorescence images of EGFP, mCherry, or the merge from MEFs stably expressing sgRNA against Rosa (-) or Atp7a ( #1 or #2 ) and mCherry-EGFP-LC3B treated with VEH or BAF. r , Scatter dot plot of quantified mCherry + -LC3 puncta per cell or mCherry + EGFP + -LC3 puncta per cell ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and mCherry-EGFP-LC3B treated with VEH or BAF. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Two asterisks, P<0.01; Four asterisks, P<0.0001. n≥26. Western blots images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Sequencing, Binding Assay, Western Blot, Recombinant, In Vitro, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Representative live cell imaging of the Cu probe CF4 every ten minutes for 60 minutes from MEFs treated with vehicle (VEH) or amino acid deprivation (-AA). b , Quantification of mean CF4 fluorescence intensity (FI) ± s.e.m. versus time (minutes, min) from MEFs treated with VEH (black circles) or -AA (blue squares) normalized to t=0, five minutes. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=30. c , d , e , f , Representative fluorescence images of EGFP-ATG13 ( c ), EGFP-FIP200 ( d ), EGFP-WIP1 ( e ), or EGFP-DFCP1 ( f ) from Ctr1 -/- MEFs stably expressing CTR1 WT (WT) or empty vector (VO) and EGFP-ATG13 , EGFP-FIP200 , EGFP-WIP1 , or EGFP-DFCP1 treated with VEH or -AA with or without bafilomycin (BAF). g , h , i , j , Scatter dot plot of quantified EGFP + puncta per cell ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and EGFP-ATG13 ( g ), EGFP-FIP200 ( h ), EGFP-WIP1 ( i ), or EGFP-DFCP1 ( j ) treated with VEH or -AA with or without bafilomycin (BAF). Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. EGFP-ATG13, ≥26; EGFP-FIP200, n≥21; EGFP-WIP1, n≥18; EGFP-DFCP1, n≥24. k , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from Ctr1 -/- MEFs stably expressing WT (black circles) or VO (red squares) and EGFP-LC3B treated with VEH, -AA, or RAP with or without BAF normalized to WT, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Four asterisks, P<0.0001. n=12. l , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-, black circles) or Atp7a ( #1 or #2 , blue squares, blue triangles) and EGFP-LC3B treated with VEH or BAF normalized to Rosa ( - ), VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Immunoblot detection of recombinant HA-ULK1 WT or C u b inding m utant HA-ULK1 CBM bound to a resin charged with or without Cu compared to input. b , Immunoblot detection of recombinant phosphorylated (P)-ATG13, total (T)-ATG13, and HA-ULK1 WT or HA-ULK1 CBM from ULK1 in vitro kinase assays. c , Immunoblot detection of recombinant P-ATG13, T-ATG13, or immunoprecipitated (IP)-ULK1 from Ulk1/2 -/- MEFs stably expressing HA-ULK1 WT (WT) or HA-ULK1 CBM (CBM) treated with amino acid deprivation (-AA). d , Immunoblot detection of LC3-I, LC3-II, P-ULK1, T-ULK1, P-ATG13, T-ATG13, or β-ACTIN from MEFs stably expressing sgRNA against Rosa (-) reconstituted with empty vector (VO) or sgRNA against Ulk1 and Ulk2 reconstituted with empty vector (VO), HA-ULK1 WT (WT), or HA-ULK1 CBM (CBM) treated with vehicle (VEH) or -AA with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN and ΔP-ATG13/T-ATG13 normalized to Rosa (-), VO, VEH control. e , Scatter dot plot of flow cytometry analysis of autophagic flux quantified by the ratio of mean mCherry-LC3 fluorescent intensity (FI)/mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and mCherry-EGFP-LC3B treated with VEH or -AA with or without BAF normalized to Rosa ( - ), VO, VEH control. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05; Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9. f , g , Representative fluorescence images of EGFP-FIP200 ( f ) or EGFP-WIP1 ( g ) from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM and EGFP-FIP200 or EGFP-WIP1 treated with VEH or - AA with or without BAF. h , i , Scatter dot plot of quantified EGFP + puncta per cell ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and EGFP-FIP200 ( h ) or EGFP-WIP1 ( i ) treated with VEH or -AA with or without BAF. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. EGFP-FIP200, n≥26; EGFP-WIP1, n≥27. j , Scatter dot plot of flow cytometry analysis of the number of LC3-II positive autophagosomes quantified by the mean EGFP-LC3 FI ± s.e.m. from MEFs stably expressing sgRNA against Rosa (-) reconstituted with VO (dark grey circles) or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangles), or CBM (red inverted triangles) and EGFP-LC3B treated with VEH or -AA with or without BAF normalized to Rosa ( - ), VO, VEH control. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. One asterisk, P<0.05; Three asterisks, P<0.001; Four asterisks, P<0.0001. n=9. k , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Ctr1 flox / flox ( fl/fl ) or Ctr1 -/- ( -/- ) MEFs stably expressing sgRNA against Rosa (-) or sgRNA against Ulk1 and Ulk2 ( + ) treated with VEH or -AA with or without BAF. Quantification: ΔLC3-II/β-ACTIN normalized to Ctr1 flox/flox , Rosa (-), VEH control. Western blot images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Western Blot, Recombinant, In Vitro, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma

doi: 10.1101/816587

Figure Lengend Snippet: a , Immunoblot detection of LC3-I, LC3-II, or β-ACTIN from Kras G12D ;p53 flox/flox (KP) lung adenocarcinoma cell line #2474 (KP #2474) stably expressing sgRNA against Rosa (-) reconstituted with empty vector (VO) or sgRNA against Ulk1 and Ulk2 reconstituted with empty vector (VO), HA-ULK1 WT (WT), or HA-ULK1 CBM (CBM) treated with vehicle (VEH) or amino acid deprivation (-AA) with or without bafilomycin (BAF). Quantification: ΔLC3-II/β-ACTIN normalized to Rosa (-), VO, VEH control. b , Immunoblot detection of phosphorylated (P)-ULK1, total (T)-ULK1, P-ATG13, T-ATG13, or β-ACTIN from Kras G12D ;p53 flox/flox (KP) lung adenocarcinoma cell line #2474 cells stably expressing sgRNA against Rosa (-) reconstituted with VO or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM. Quantification: ΔP-ATG13/T-ATG13 normalized to Rosa (-), VO, VEH control. c , Mean tumor volume (mm 3 ) ± s.e.m. versus time (days) in mice injected with KP #2474 cells stably expressing sgRNA against Rosa reconstituted with VO (dark grey line, dark grey circles) , Atg5 (pink line, pink hexagons), or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey line, grey squares), WT (black line, black triangle), or CBM (red line, red inverted triangle). Results were compared using a paired, two-tailed Student’s t-test. One asterisk, P<0.05. n=4. d , Representative dissected tumors from mice injected with KP #2474 cells stably expressing sgRNA against Rosa (-) reconstituted with VO, sgRNA against Atg5 , or sgRNA against Ulk1 and Ulk2 reconstituted with VO, WT, or CBM. e , Scatter dot plot of mean tumor weight (g) ± s.e.m. of tumors at endpoint from KP #2474 cells stably expressing sgRNA against Rosa reconstituted with VO (dark grey circles), Atg5 (pink hexagons), or sgRNA against Ulk1 and Ulk2 reconstituted with VO (grey squares), WT (black triangle), or CBM (red inverted triangle). Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Three asterisks, P<0.001; Four asterisks, P<0.0001. n=4. f , Representative crystal violet images of KP #2474 cells stably expressing sgRNA against Rosa , Ctr1 ( #2 ), Ulk1 and Ulk2 , or Ulk1 , Ulk2 , and Ctr1 (#2 ) from days 3, 4, and 5 of recovery. g , Scatter dot plot of mean absorbance of extracted crystal violet at 590nM ± s.e.m. of KP #2474 cells stably expressing sgRNA against Rosa (black circles) , Ctr1 (#2, pink squares), Ulk1 and Ulk2 (grey triangles), or Ulk1 , Ulk2 , and Ctr1 (#2 , red inverted triangles) from days 3, 4, and 5 of recovery normalized to Rosa , day 3 control. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. Two asterisks, P<0.01; Three asterisks, P<0.001; Four asterisks, P<0.0001. n≥9. Western blot images are representative of at least three biological replicates.

Article Snippet: pWZLblasti-CTR1 WT (Addgene plasmid #53157) , pBABEpuro-mCherry-EGFP-LC3B (Addgene plasmid #22418) , pBABEpuro-EGFP-LC3B (Addgene plasmid #22405) , pLenti-CRISPRV2blasti (Addgene plasmid #83480), pLenti-CRISPRV2puro (Addgene plasmid #52961) , pWZLblasti-MEK1 WT (Addgene plasmid #53161) , pWZL-MEK1 CBM (CBM:M187A/H188A/M230A/H239A) , pBABEpuro-HA-ERK2 GOF (GOF: R67S/D321N; Addgene plasmid #53203) , pMXs-IP-EGFP-ATG13 (Addgene plasmid #38191) , pMXs-IP-EGFP-FIP200 (Addgene plasmid #38192) , pMXspuro-EGFP-DFCP1 (Addgene plasmid #38269) , pMXs-IP-EGFP-WIPI1 (Addgene plasmid #38272) , and pLentiCRISPRv2Cre (Addgene plasmid #82415) were previously described.

Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Injection, Two Tailed Test

Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: PCGF isoforms colocalize with cytoplasmic E. chaffeensis vacuoles during the late stage (48 h) of infection. (A) Immunofluorescence analysis of E. chaffeensis-infected THP-1 cells. Immunofluorescence micrographs demonstrate distribution of PCGF4 isoforms in uninfected and E. chaffeensis-infected cells at 12, 24, and 48 hpi. PCGF4 was mainly localized in the nucleus of an uninfected cell or during early infection (12 and 24 hpi) but was distributed to the ehrlichial vacuole (rectangle) at a late stage (48 hpi). Scale bar, 10 μm. (B) Bar graph depicting percentages of PCGF-TRP colocalized cells for individual PCGF isoforms (n = 3; Student's two-tailed t test; *, P ≤ 0.05; **, P ≤ 0.005). (C) Immunofluorescence micrographs demonstrating colocalization (infected, rectangles) of E. chaffeensis expressing TRP120 (green) with PCGF2, -3, -4, -5, and -6 isoforms (red) at the ehrlichial vacuole (arrow) at 48 hpi compared to uninfected cells. The signal intensities of red, green, and blue fluorescence channels are shown on each image using a table (a.u., arbitrary units) to cross-reference index numbers to output values. The color map is used to look up the actual colors corresponding to each index number. The Pearson's correlation coefficients shown next to the regions of interest (rectangles) indicate a strong/very strong colocalization for PCGF2, -3, -4, -5, and -6 isoforms with TRP120 in the ehrlichial vacuole. Scale bar, 10 μm. (D) Coimmunoprecipitation using PCGF2, -3, -4, -5, and -6 antibodies and chemiluminescence detection of TRP120 using TRP120-specific antibody from E. chaffeensis-infected and uninfected THP-1 cell lysate. (E) Western blot analysis of E. chaffeensis-infected and uninfected input lysates using TRP120, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6 antibodies; GAPDH was used as a loading control. (F) Western blot analysis of immunoprecipitation eluates using PCGF2, -3, -4, -5, and -6 isoform-specific antibodies.

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Immunofluorescence, Two Tailed Test, Expressing, Fluorescence, Western Blot, Control, Immunoprecipitation

Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: Decrease in PCGF isoforms occurs via proteosomal degradation in E. chaffeensis-infected cells. (A) Graph representing fold changes in PRC1 core component gene expression in E. chaffeensis-infected cells at 4, 10, 24, and 48 hpi; n = 3. Biologically significant (approximately ≤2-fold increase or decrease) and statistically significant (Student's two-tailed t test; *, P ≤ 0.05) changes in gene transcription were observed for CBX7, PCGF2, PCGF5, and RING1 genes. (B) Heat map showing relative expression levels of PRC1 core component genes at 4, 10, 24, and 48 hpi. Each position in the heat map represents an individual gene (listed next to the heat map). The colors indicate differential expression (red indicates induction, green indicates repression, and white represents no significant change) from the average gene expression level in uninfected cells. The intensity of the color represents the amplitude of induction/repression. (C) Western blot analysis of bortezomib (BORTZ)- or DMSO-treated THP-1 (E. chaffeensis-infected and uninfected) whole-cell lysates using isoform-specific anti-PCGF antibodies. E. chaffeensis-infected or uninfected THP-1 cells were treated with 100 nM bortezomib 38 h postinfection for 10 h. The whole-cell lysates were then subjected to SDS-PAGE separation, and the relative abundance of PCGF isoforms was determined using chemiluminescence. (D) Graph representing GAPDH-normalized relative abundance of PCGF isoforms in bortezomib- or DMSO-treated E. chaffeensis-infected or uninfected THP-1 cell lysates. Error bars indicate standard deviations between experiments (n = 3; Student's two-tailed t test; *, P ≤ 0.05).

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Gene Expression, Two Tailed Test, Expressing, Quantitative Proteomics, Western Blot, SDS Page

Journal: Infection and Immunity

Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

doi: 10.1128/IAI.00845-17

Figure Lengend Snippet: siRNA-mediated silencing of PCGF isoforms increases E. chaffeensis infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with E. chaffeensis at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial dsb to the host cell gapdh in individual PCGF knockdown using real-time qPCR at 48 hpi (n = 3; *, P ≤ 0.05).

Article Snippet: E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described ( 54 ). siRNAs and antibodies. siRNAs used in this study were PCGF2 (sc-38191), PCGF3 (sc-89157), PCGF4 (sc-29814), PCGF6 (sc-90663), and nontargeting siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA). siRNA for PCGF5 (M-007089-01) was obtained from GE Dharmacon (Lafayette, CO).

Techniques: Infection, Transfection, Western Blot, Control, Knockdown