Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: Rat NRVMs and BMDMs on coverslips were fixed in 4% PFA for 30 min and permeabilized with 1% Triton X-100 for 10 min. After blocking with 1% bovine serum albumin (BSA) for 1 h, NRVMs were incubated with rabbit anti-Ki67 (1:200; Cell Signaling Technology, 12075S), anti-pH3 (1:200; Cell Signaling Technology, 53348S) and mouse anti-α-actinin (1:200; Cell Signaling Technology, 69758S) at 37°C for 2 h. After washing with 1% PBS-Tween 20, Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 555-conjugated anti-mouse secondary antibodies (Abcam, ab150077, ab150118) were added.

Techniques: Western Blot, Staining

Journal: bioRxiv

Article Title: HIVtat Alters Epithelial Differentiation State and Increases HPV16 Infectivity in Oral Keratinocytes

doi: 10.1101/2023.03.08.531567

Figure Lengend Snippet: A. The level of luciferase gene expression driven by the HIV LTR was measured by RT-qPCR using Luciferase-specific primers to ensure that HIVtat was being expressed in these cells. Fold increase was determined after normalization to GAPDH levels. B. Western blot analysis was performed on total cell extracts obtained after introduction of HPV16 episomes into OKF6tert1 cells in the presence or absence of HIVtat followed by treatment with 0.6 mM NaB. The blot was probed with anti-p53 antibody to determine p53 levels. Total histone H3 was used as loading controls. Relative p53 levels are indicated after normalization to the total H3 signal. C. HIVtat did not significantly affect transactivation of the HPV16 LCR in OKF6tert1 cells. OKF6tert1 cells were cotransfected with an HPV16 LCR/luciferase reporter plasmid and either empty vector or a tat-expression vector (pCMV-tat). No increase in luciferase expression from the HPV16LCR construct was detected when HIVtat was included (left panel). HIVtat was capable of significantly increasing HIVLTR transactivation after cotransfection of OKF6tert1 cells with HIVLTR/luciferase plasmids and the HIVtat expression vector (right panel).

Article Snippet: The following primary antibodies were used to detect proteins on western blots: anti-involucrin (sc-28557; Santa Cruz), anti-loricrin (PRB-145P; Biolegend), anti-GAPDH (sc-25778; Santa Cruz), anti-γ-H2A.X Ser 139 (sc-101696; Santa Cruz), anti-p-Chk2 Thr 68 (sc-16297-R; Santa Cruz), anti-actin (sc-1616-R; Santa Cruz), anti-p38 MAPK (9212S; Cell Signaling), anti-phospho p38MAPK (9215S; Cell Signaling), anti-p53 (sc-6243; Santa Cruz), anti-histone H3 (D1H2; Cell Signaling), The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Antiserum to HIV-1 Tat from Dr. Bryan Cullen (catalog #705) ( ).

Techniques: Luciferase, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Construct, Cotransfection

Journal: bioRxiv

Article Title: HIVtat Alters Epithelial Differentiation State and Increases HPV16 Infectivity in Oral Keratinocytes

doi: 10.1101/2023.03.08.531567

Figure Lengend Snippet: A. DNA damage occurs in OKF6tert1 cells expressing HIVtat. Cells were transfected with control vector (pCMV-HA) or HIVtat expression vector (pCMV-tat). 48 hours post transfection cells were assayed for DNA damage by TUNEL assay. B. The levels of phospho-chk2 (p-chk2) were analyzed in either control cells or HPV16+ cells in the presence or absence of HIVtat. OKF6tert1 cells were cotransfected with pSVGFP and either control vector (pCMV-HA) or the HIVtat expression vector, pCMV-tat. Another set of cells were cotransfected with pHPV16loxp and either control vector (pCMV-HA) or the HIVtat expression vector, pCMV-tat. 48 hours post transfection cell lysates were obtained and analyzed by western blot analysis for the DNA damage marker, p-chk2. β-actin was detected on the same blot to show equal loading of the lysates (left panel). OKF6tert1 cells expressing HPV16 E7 and HIVtat have an increased DNA damage response. OKF6tert1 cells were cotransfected with either HPV16 E6 or E7 expression vectors and either control vector (pCMV-HA) or the HIV-tat expression vector, pCMV-tat. 48 hours post transfection cell lysates were obtained and analyzed by western blotting for the DNA damage marker, γ-H2AX. HIVtat was detected using a rabbit anti-tat antibody. Cell lysate from OKF6tert1 cells treated with the DNA damage-inducing agent, etoposide was used as a positive control. Numbers below each lane indicate the fold increase in γ-H2AX over control transfected cells (pcDNA3 + pCMV-HA) as determined by densitometry after normalization to β-actin (right panel). C. OKF6tert1 cells were transfected with either control vector (pCMV-HA) or the tat expression vector, pCMV-tat. 48 hours post transfection total RNA was isolated and used to perform RT-qPCR for the detection of transcripts encoding the oxidative stress markers, glutathione peroxidase (GPX) and superoxide dismutase (SOD). While HIVtat expression caused a slight decrease in GPX transcripts it was not statistically significant (p=0.18). However, HIVtat expression did result in a statistically significant decrease in SOD transcripts (p=.0019). D. Cell lysates from OKF6tert1 cells treated with vehicle or 0.6mM NaB for 72 hours were subjected to SDS_PAGE and western blot analysis. Monomeric involucrin was detected in all extracts and is indicated by the arrow. Cells were transfected with SVGFP or HPV16 and were co transfected with HIVtat. In cells transfected with SVGFP, a 130 kD band representing crosslinked involucrin was detected when cells are grown in the presence of NaB as indicated by the arrowhead. In cells transfected with HPV16 episomes, lower levels of the 130 kD band representing crosslinked involucrin were detected when cells are grown in the presence and absence of NaB as indicated by the arrowhead. Fold increase in crosslinked involucrin relative to monomeric involucrin is indicated in the graph. E. Cells were cotransfected with SVGFP or HPV16 and pCMVtat. Cell lysates from cotransfected OKF6tert1 cells treated with vehicle or 0. 0.25, 0.5 or 1 mM NaB for 72 hours were subjected to SDS_PAGE and western blot analysis. Levels of cytokeratin K10, loricrin, and acetylated H3 and HIVtat were detected in the extracts. Total H3 served as a loading control.

Article Snippet: The following primary antibodies were used to detect proteins on western blots: anti-involucrin (sc-28557; Santa Cruz), anti-loricrin (PRB-145P; Biolegend), anti-GAPDH (sc-25778; Santa Cruz), anti-γ-H2A.X Ser 139 (sc-101696; Santa Cruz), anti-p-Chk2 Thr 68 (sc-16297-R; Santa Cruz), anti-actin (sc-1616-R; Santa Cruz), anti-p38 MAPK (9212S; Cell Signaling), anti-phospho p38MAPK (9215S; Cell Signaling), anti-p53 (sc-6243; Santa Cruz), anti-histone H3 (D1H2; Cell Signaling), The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Antiserum to HIV-1 Tat from Dr. Bryan Cullen (catalog #705) ( ).

Techniques: Expressing, Transfection, Plasmid Preparation, TUNEL Assay, Western Blot, Marker, Positive Control, Isolation, Quantitative RT-PCR, SDS Page

Journal: bioRxiv

Article Title: ACRC/GCNA is an essential protease for the repair of DNA-protein crosslinks during vertebrate development

doi: 10.1101/2023.03.07.531502

Figure Lengend Snippet: A – Total DPC levels (percentage of DNA-protein crosslinks vs. free DNA) as quantified using the KCl/SDS method: increased DPC levels in acrcrbi8/9 maternal mutants relative to WT. An unpaired Student’s t-test was performed for 3 biological replicates, pools of 100 embryos each (***: p-value = 0.0254). B- C – RADAR assays on 6 hpf embryos. Unpaired Student’s t-test were performed for 4 biological replicates, pools of 50 embryos each (****: p-value < 0.0001). B – Total DPCs were isolated from n = 50 embryos each for WT and acrcrbi5/rbi5 homozygotes, resolved on an SDS acrylamide gel, and stained with silver. Dot-blots showing DNA loading controls for DPC analysis prior to benzonase treatment are shown below; a DPC equivalent of 50 ng total DNA was loaded per well. C – Quantifications of total DPC staining intensities normalized to WT (top), and of staining intensities for high, middle and low molecular weight DPCs (bottom) based on data shown here and in (4 biological replicates). D – Histone H3-DPC detection by western blot, with corresponding dot blot for dsDNA as loading control; a DPC equivalent of 50 ng total DNA was loaded per well. Total DPCs were isolated from 6-hpf old embryos, separated by SDS-PAGE and detected with histone H3-specific antibody. Quantification normalized to WT based on data shown here and in (5 biological replicates). An unpaired Student’s t-test was performed for 3 biological replicates, pools of 50 embryos each (****: p- value < 0.0001). L = ladder. Data shown are means with S.E.M.

Article Snippet: For the detection of histone H3, a DPC amount of 150 ng DPC-DNA (for 2dpf embryo samples) or 50 ng DPC-DNA (for 6 hpf embryo samples) was subjected to western blotting and immunostained with the anti-H3 antibody (Cell Signaling, #9715, 1:3000).

Techniques: Isolation, Acrylamide Gel Assay, Staining, Molecular Weight, Western Blot, Dot Blot, SDS Page

Journal: bioRxiv

Article Title: ACRC/GCNA is an essential protease for the repair of DNA-protein crosslinks during vertebrate development

doi: 10.1101/2023.03.07.531502

Figure Lengend Snippet: A – Total DPCs isolated from WT and acrc rbi5/rbi5 homozygous maternal mutants were separated on an SDS acrylamide gel and stained with silver. Dot blots beneath show equal dsDNA loading; the equivalent of 50 ng of DNA was loaded per well. Shown are 4 biological replicates whose quantification is shown in . B – Histone H3-DPC detection by western blot, with dot blot for dsDNA. Shown are 5 biological replicates whose quantification is shown in . L = ladder.

Article Snippet: For the detection of histone H3, a DPC amount of 150 ng DPC-DNA (for 2dpf embryo samples) or 50 ng DPC-DNA (for 6 hpf embryo samples) was subjected to western blotting and immunostained with the anti-H3 antibody (Cell Signaling, #9715, 1:3000).

Techniques: Isolation, Acrylamide Gel Assay, Staining, Western Blot, Dot Blot

Journal: bioRxiv

Article Title: ACRC/GCNA is an essential protease for the repair of DNA-protein crosslinks during vertebrate development

doi: 10.1101/2023.03.07.531502

Figure Lengend Snippet: A – RADAR assays followed by histone H3-DPC detection by western blot, with dot blot for dsDNA as a loading control (the equivalent of 150 ng of DNA was loaded per well). Shown are 2 biological duplicates and the quantification of H3 staining intensities normalized to WT. RADAR performed on 2 dpf WT and rbi5 heterozygous embryos treated or not with 10 mM FA for 30 minutes. A two-way ANOVA analysis was performed, followed by Tukey’s multiple comparisons test; *: p- value = 0.0277. B – Total DPC levels (percentage of DNA-protein crosslinks vs. free DNA) as quantified using the KCl/SDS method. DPC levels quantified in 3dpf embryos after exposure to 5 mM formaldehyde (FA) for 1 hour. Acrc samples consist in the progeny of incrossed heterozygous acrc rbi5/+ parents: 25% WT, 50% heterozygotes and 25% mutants; all embryos are viable due to maternal Acrc deposition in the eggs. Two-way ANOVA analysis followed by Tukey’s multiple comparisons tests were performed for 3 biological replicates, pools of 10 embryos each; *: p- value < 0.05. L = ladder. Data shown are means with S.E.M; ns = not significant.

Article Snippet: For the detection of histone H3, a DPC amount of 150 ng DPC-DNA (for 2dpf embryo samples) or 50 ng DPC-DNA (for 6 hpf embryo samples) was subjected to western blotting and immunostained with the anti-H3 antibody (Cell Signaling, #9715, 1:3000).

Techniques: Western Blot, Dot Blot, Staining