amaxatm basic nucleofectortm kit for primary mammalian epithelial cells  (Lonza)

Journal: bioRxiv

Article Title: Antisense oligonucleotide allele-specific targeting of EFEMP1 in a patient-derived model of Doyne honeycomb retinal dystrophy

doi: 10.1101/2025.07.16.664883

Figure Lengend Snippet: (A-D) iRPE were transfected with ASO1.1, 1.2, 1.3, 1.4 or control ASO (CTRL) (200 nM) at day 72 of differentiation and analysed 48 hours later. (A) Fluorescence and bright field images of R345W iRPE showing efficient delivery of 6-FAM conjugated ASO. (B) Quantitation of EFEMP1 transcript levels by qPCR. All ASO mediate a significant decrease in EFEMP1 levels. N = 3 independent experiments, n = 2 technical replicates of each condition per experiment. Statistical significance was determined by one-way ANOVA followed by post-hoc Tukey’s (HSD) test where *, ** and *** denotes a p value <0.05, 0.01 and 0.005 respectively. ns = not significant. Bars represent mean ± SEM. (C) Sanger sequencing chromatograms of untreated (NT) R345W iRPE and R345W iRPE treated with CTRL ASO, ASO1.1, ASO1.2, ASO1.3 or ASO1.4 (200 nM). The heterozygous ‘C/T’ peak in the NT and CTRL treated R345W iRPE is resolved to a homozygous ‘C’ peak in all ASO treated samples. (D) Quantitation of ASO-mediated allele-specific EFEMP1 targeting by NGS. WT (%C) and R345W (%T) EFEMP1 transcript levels were quantified in untreated R345W iRPE (NT) or R345W iRPE treated with CTRL or therapeutic ASO1.1, 1.2, 1.3 or 1.4. NGS results for gDNA and cDNA from corresponding patient renal epithelial cells are also shown. N = 3 independent experiments, n = 2 technical replicates of each condition per experiment. Bars represent mean ± SD. (E) Integrated analysis of the qPCR and NGS data showing a significant reduction of the R345W transcript with all ASO. Statistical significance was determined by one-way ANOVA followed by post-hoc Tukey’s (HSD) test where *, ** and *** denotes a p value <0.05, 0.01 and 0.005 respectively. ns = not significant. Bars represent mean ± SD.

Article Snippet: A. Thomson, Regenerative Biology, Morgridge Institute for Research, Madison, Wisconsin, USA; Howden et al., 2015) using the AmaxaTM Basic NucleofectorTM Kit for primary mammalian epithelial cells, program T-020 (Lonza).

Techniques: Transfection, Control, Fluorescence, Quantitation Assay, Sequencing

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A ) A schematic of the GFP-tagged PKCδ plasmids used, with red circles indicating mutations. ( B ) ParC5 and ( C ) HEK293 cells were transiently transfected with the indicated plasmids for 72 hrs, harvested and assessed for DNA damage using a neutral comet assay. ( D ) ParC5 shNT, shδ110, and shδ680 cells, and ( E ) HEK293 shSCR, shδ193, and shδ203 cells were exposed to 5 Gy IR, harvested at the indicated times and assessed for DNA damage using a neutral comet assay. Data is the mean ± SEM from a representative experiment that was repeated three or more times. ( F ) Representative images of RPE cells stably expressing pGFP or pPKCδGFP. The white arrows indicate micronuclei. Scale bar = 20 µm. ( G ) The percent micronuclei was determined by normalizing the number of micronuclei ( N = 20 fields/cell line) to the total number of nuclei identified by DAPI staining. Statistics represent one-way ANOVA in (B) and (C) followed by Dunnett’s multiple comparisons to the pGFP controls, two-way ANOVA in (D) and (E) followed by Dunnett’s multiple comparisons within each time point to the corresponding shNT or shSCR control and unpaired t-test in (G). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Transfection, Neutral Comet Assay, Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A to C ) DNA repair was analyzed in (A) ParC5 shNT, shδ110, and shδ680 cells containing stably integrated NHEJ or HR reporters, (B) HEK293 shSCR, shδ193, and shδ800 cells containing NHEJ reporters, or (C) HEK293 shSCR, shδ193, and shδ203 cells containing HR reporters. Cells were co-transfected with plasmids expressing pI-SceI to induce DSBs and pDsRed to normalize transfection efficiency. Reporter cells were allowed to grow for 72 hrs post-electroporation and repair was quantified by flow cytometry. ( D ) ParC5 NHEJ or HR reporter cells were transfected with 1 µg pEV (black bars) or increasing amount of pPKCδFLAG, and DNA repair was assayed. ( E ) ParC5 cells transfected with 0.1 ug of PKCδFLAG for 72 hrs were collected, lysed, and assayed for caspase-3 activities. ( F ) ParC5 shNT and shδ110 reporter cells were co-transfected with plasmids expressing pI-SceI, pDsRed, and pEV or pPKCδFLAG and DNA repair was analyzed. Each data point represents an average from three independent biological replicates. Data is shown as mean ± SEM. Statistics represent two-way ANOVA or one-way ANOVA for (B) and (C), followed by Dunnett’s multiple comparisons within NHEJ or HR reporter cell lines to their corresponding shNT, shSCR, or pEV control, and unpaired t-test for (E). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Stable Transfection, Transfection, Expressing, Electroporation, Flow Cytometry

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A to C ) ParC5 shNT, shδ110, and shδ680 cells were plated on coverslips and exposed to 1 Gy IR. Cells were harvested at indicated times post IR by permeabilization, fixation, and stained for foci, (A) γH2AX, (B) DNA-PK pSer 2056 , and (C) Rad51 foci (legend in C is for A to C). An average of three independent experiments is shown. Error bars show ± SEM. ( D ) Western blots of ParC5 shNT, shδ110, and shδ680 cells immunoblotted with the indicated antibodies. ( E ) Densitometry of Western blots shown in D; expression of each protein of interest (POI) was normalized to the actin control. ( F ) ParC5 cells were transiently transfected with empty vector (pEV) or pPKCδFLAG for 48 hrs. Cells were then collected and immunoblotted with the indicated antibodies (First and second blots were probed with antibodies against PKCδ and FLAG-tag respectively). Note that panels D and F show two different experiments separated by lines, each normalized to their own β-actin loading control. ( G ) Densitometry of Western blots shown in F; expression of each POI was normalized to the actin control. Data are from three independent biological replicates and shown as mean ± SEM. Statistics represent two-way ANOVA with Dunnett’s multiple comparisons and Sidak’s multiple comparisons for (G) to the corresponding control (black bars). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Staining, Western Blot, Expressing, Transfection, Plasmid Preparation, FLAG-tag

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A and B ) MNase assay was performed on (A) ParC5 shNT, shδ110, and shδ680 cells, and on (B) HEK293 shSCR, shδ193, and shδ800 cells. ( C and D ) ParC5 shNT cells were transfected with pGFP only, while shδ110 cells were transfected with (C) pGFP, pPKCδCF, or pPKCδCFKN, or (D) pGFP, pPKCδNLS, or pPKCδNLM for 24 hrs prior to harvesting. Percentage of DNA in each [NaCl] fraction was assayed as described in Materials and Methods. Shown is data (mean ± SEM) from at least three independent biological replicates. Statistics represent two-way ANOVA followed by Dunnett’s multiple comparisons within each fraction to their corresponding shNT or shSCR control for (A) and (B), or two-way ANOVA followed by Tukey’s multiple comparisons within each fraction for (C) and (D). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Transfection

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A and B ) DNA repair was analyzed in ParC5 shNT-NHEJ and shδ110-NHEJ reporter cells pretreated with (A) 100 nM trichostatin A (TSA) or 5 mM nicotinamide (NAM), or (B) 5 mM succinate (SUC) for 24 hrs and then co-transfected with plasmids expressing pI-SceI to induce DSBs and pDsRed to normalize transfection efficiency. NHEJ and HR reporter cells were allowed to grow for 72 hrs post-transfection and repair was quantified. ( C and D ) MNase assay was performed on ParC5 shNT and shδ110 cells pretreated with (C) 5 mM NAM or (D) 5 mM SUC for 48 hrs before harvesting. Percentage of DNA in each [NaCl] fraction was assayed. Shown is data (mean ± SEM) from at least three independent biological replicates. ( E ) Histone modifications of ParC5 shNT, shδ110, and shδ680 were analyzed by mass spectrometry (Northwestern University). Heat map shows the percent change of PTMs on H3 in each modification in shδ110 and shδ680 cells relative to shNT (top and bottom 10 shown). ( F ) Profile of H3K36 methylation in shNT, shδ110, and shδ680 cells. ( G ) Densitometry of H3K36me2 normalized to total H3. Shown is data (mean ± SEM) from three independent biological replicates. Statistics represent two-way ANOVA followed by Sidak’s for (A), Tukey’s for (B to D), and Dunnett’s multiple comparisons for (F), and one-way ANOVA followed by Dunnett’s multiple comparisons for (G). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Transfection, Expressing, Mass Spectrometry, Modification, Methylation

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A ) Top: Whole cell lysates of ParC5 shNT, shδ110, and shδ680 cells were immunoblotted with the indicated antibodies. Bottom: Densitometry of SIRT6 expression normalized to β-actin. ( B ) Relative expression of SIRT6 mRNA in ParC5 shNT, shδ110, and shδ680 cells was assayed by qRT-PCR. ( C ) Top: ParC5 cells transfected with pEV or pPKCδFLAG were analyzed for SIRT6 expression by immunoblot analysis. Bottom: Densitometry of SIRT6 expression normalized to β-actin. ( D ) Relative expression of SIRT6 mRNA in ParC5 overexpressing either pEV or pPKCδFLAG cells was assayed by qRT-PCR. Shown is data from at least three independent biological replicates. Data is shown as mean ± SEM. ( E ) Top: ParC5 shNT, shδ110, and shδ680 cells were fractionated into cytoplasm, nuclear and chromatin fractions, and each fraction was immunoblotted with the indicated antibodies. Bottom: Percentage of KDM2A in nuclear and chromatin were calculated. ( F ) Top: Immunoprecipitation of KDM2A from ParC5 shNT, shδ110, and shδ680 nuclear fractions using KDM2A antibody or normal IgG control, and immunoblotted using anti-mono-ADP-ribose (MAR) and KDM2A antibodies. Bottom: Densitometry of MAR-KDM2A normalized to KDM2A. Statistics represent one-way ANOVA followed by Dunnett’s multiple comparisons for (A), (B) and (F), unpaired t-test for (C) and (D), and two-way ANOVA followed by Dunnett’s multiple comparisons for (E). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: PKCδ regulates chromatin remodeling and DNA repair through SIRT6

doi: 10.1101/2023.05.24.541991

Figure Lengend Snippet: ( A ) ParC5 shNT and shδ110 NHEJ reporter cells were generated that stably express shSCR, shSIRT6- 1, shSIRT6-2, or shSIRT6-3. Lysates were immunoblotted for the indicated proteins. ( B ) Top: Histone fractions from ParC5 shNT/shδ110-shSCR/shSIRT6-2/shSIRT6-3 cells were acid-extracted and immunoblotted for H3K36me2 and total H3. Bottom: Densitometry of H3K36me2 normalized to total H3. ( C ) MNase assay was performed on ParC5 shNT/shδ110-shSCR/shSIRT6- 2/shSIRT6-3 cells. Percentage of DNA in each [NaCl] fraction was assayed as described in Materials and Methods. ( D ) DNA repair was analyzed in ParC5 shNT/shδ110-shSCR/shSIRT6- 2/shSIRT6-3 NHEJ reporter cells and quantified as described in Materials and Methods. ( E to H ) Lysates from ParC5 shNT/shδ110-shSCR/shSIRT6-2/shSIRT6-3 cells were immunoblotted with the indicated antibodies. Densitometry of DNA Lig4 (F), XRCC4 (G), and XLF (H) normalized to their corresponding β-actin control from the Western blots shown in (E). ( I ) ParC5 shNT/shδ110-shSCR/shSIRT6-2/shSIRT6-3 cells were IR at 10 Gy and allowed to recover for 48 hrs. Cells were harvested and caspase-3 activity was assayed. Shown is the fold induction of caspase-3 activities in each cell line over their own corresponding no IR controls. Shown is data from at least three independent biological replicates. Data is shown as mean ± SEM. Statistics represent two-way ANOVA followed by Tukey’s multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 µg) were transfected into parental ParC5 cells, ParC5 shδ cell lines (shNT, shδ110, and sh680), or HEK293 shδ cell lines (shSCR, shδ193, shδ203, and sh800) cells using Amaxa Nucleofector 2B (Basic Nucleofector Kit Primary Mammalian Epithelial Cells, Cat # VPI-1005, Program T-020 for ParC5 cells, and Cell Line Nucleofector Kit V, Cat # VCA-1003, Program Q-001 for HEK293 cells, Lonza, Walkersville, MD).

Techniques: Generated, Stable Transfection, Western Blot, Activity Assay

Journal: iScience

Article Title: BRPF1 bridges H3K4me3 and H3K23ac in human embryonic stem cells and is essential to pluripotency

doi: 10.1016/j.isci.2023.105939

Figure Lengend Snippet:

Article Snippet: AMAXA Basic Nucleofector Kit for Prim. Mammalian Epithelial cells , Lonza , Cat#VPI-1005.

Techniques: Recombinant, DNA Extraction, Cloning, Plasmid Preparation, DNA Ligation, Flow Cytometry, Software