Journal: Nucleic Acids Research

Article Title: Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS

doi: 10.1093/nar/gkac037

Figure Lengend Snippet: Oligonucleotides with chimeric PN-containing backbones have improved potency and durability in CNS. ( A ) Schematic representation of dosing regimen with black arrow indicating administration of intracerebroventricular dose (day 0, D0) and blue arrow indicating day of analysis (day 7, D7). Relative Malat1 expression (normalized to Hprt1) in spinal cord (left) and cortex (right) one-week post treatment with PBS (beige), stereopure PS oligonucleotide (Malat1-200, light blue) or chimeric PN containing oligonucleotide (Malat1-244, navy blue) at the indicated dose. Data are presented as box and whisker plots with box from min to max with data from individual mice shown, n = 8 * P < 0.05, ** P < 0.01, *** P < 0.001 mixed-effects model with multiple comparisons. ( B ) Schematic representation of dosing regimen with black arrow indicating administration of ICV dose (day 0, D0) and blue arrow indicating day of analysis (day 28, D28). Relative Malat1 expression (normalized to Hprt1) in spinal cord (top left) and cortex (top right) 4 weeks post treatment with PBS (beige), stereopure PS oligonucleotide (Malat1-200, light blue) or chimeric PN containing oligonucleotide (Malat1-244, navy blue). Concentration of oligonucleotide detected in spinal cord (bottom left) and cortex (bottom right) 4-weeks post treatment with PBS (beige), stereopure PS oligonucleotide (Malat1-200, light blue) or chimeric PN containing oligonucleotide (Malat1-244, navy blue). Data are presented as in panel A, n = 4, **** P < 0.0001 one-way ANOVA with multiple comparisons. ( C ) Schematic representation of dosing regimen with black arrow indicating administration of intracerebroventricular dose (day 0, D0) and blue arrow indicating day of analysis (day 70, D70). Relative Malat1 expression (normalized to Hprt1) in the indicated tissue in CNS 10-weeks post treatment with PBS (beige), stereopure PS oligonucleotide (Malat1-200, light blue) or chimeric PN containing oligonucleotide (Malat1-244, navy blue). Data are presented as in panel A, n = 3 one-way ANOVA with multiple comparisons. Red asterisks show comparison of MALAT1-244 to PBS; blue asterisks MALAT1-244 to MALAT1-200.

Article Snippet: Mouse Hprt1 transcripts were quantified with the following primers: forward primer: CAA ACT TTG CTT TCC CTG GTT; reverse primer: TGG CCT GTA TCC AAC ACT TC; probe sequence:/5HEX/AC CAG CAA G/Zen/C TTG CAA CCT TAA CC/3IABkFQ/ (Integrated DNA Technologies).

Techniques: Expressing, Whisker Assay, Concentration Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

doi: 10.1186/1477-7827-7-49

Figure Lengend Snippet: Expression of uterine TRPV6 mRNA during pregnancy in rats . Uteri of gestating rats (n = 2) were collected daily from P0.5 to P21.5, and expression of TRPV6 mRNA was assayed by RT-PCR (Top panel, agarose gel image) and Real-Time PCR (Bottom panel, line graph). The unattached uteri containing uterine epithelial cells were used as a RNA preparation from P1.3.5. The line graph shows the analysis of Real-Time PCR data expressed as a percentage of TRPV6/HPRT1 (mean ± SEM of duplicates).

Article Snippet: Real-time PCR was performed in 20 μl reactions containing 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA), and 1 μl of 20×Assays-on-Demand TM Gene Expression Assay Mix (Applied Biosystems; rat TRPV6 , Rn00586673_A1 [Assay ID in the Applied Biosystmes]; mouse TRPV6 , Mm00499069_m1; rat HPRT1 (hypoxanthine guanine phosphoribosyl transferase 1, an internal control), Rn01527840_A1; mouse HPRT1 , Mm00446968_m1) and 2 μl cDNA.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

doi: 10.1186/1477-7827-7-49

Figure Lengend Snippet: Placental TRPV6 mRNA expression during pregnancy in rats . Placentas were collected daily from P13.5 to P21.5. Placenta TRPV6 mRNA was examined by RT-PCR (Top panel, agarose gel image) and Real-Time PCR (Bottom panel, line graph). The line graph represents the analysis of Real-Time PCR data expressed as a percentage of TRPV6/HPRT1 (mean ± SEM of duplicates).

Article Snippet: Real-time PCR was performed in 20 μl reactions containing 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA), and 1 μl of 20×Assays-on-Demand TM Gene Expression Assay Mix (Applied Biosystems; rat TRPV6 , Rn00586673_A1 [Assay ID in the Applied Biosystmes]; mouse TRPV6 , Mm00499069_m1; rat HPRT1 (hypoxanthine guanine phosphoribosyl transferase 1, an internal control), Rn01527840_A1; mouse HPRT1 , Mm00446968_m1) and 2 μl cDNA.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

doi: 10.1186/1477-7827-7-49

Figure Lengend Snippet: Effects of steroid receptor antagonists on uterine and placental TRPV6 mRNA expressions . Panel A (uteri at P5.5) and B (placenta at P20.5) presented the rat TRPV6 mRNA levels. Four groups of pregnant rats (n = 4 per group) were treated with ethanol as a negative control (VE), progesterone receptor antagonist (RU, 2.5 mg per rat), or estrogen receptor antagonist (ICI, 0.5 mg per mouse). Panel C (uteri at P10.5) and D (placenta at P10.5) showed the mouse TRPV6 mRNA expressions. Four groups of pregnant mice (n = 4 per group) were treated with ethanol as a negative control (VE), progesterone receptor antagonist (RU, 25 μg per mouse) or estrogen receptor antagonist (ICI, 2 μg per mouse). Murine TRPV6 mRNA levels were examined by real-time PCR. The bar graph represents the analysis of real-time PCR data expressed as a percentage of TRPV6/HPRT1 (mean ± SEM of duplicates). a, statistically significant compared to a vehicle ( P < 0.05).

Article Snippet: Real-time PCR was performed in 20 μl reactions containing 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA), and 1 μl of 20×Assays-on-Demand TM Gene Expression Assay Mix (Applied Biosystems; rat TRPV6 , Rn00586673_A1 [Assay ID in the Applied Biosystmes]; mouse TRPV6 , Mm00499069_m1; rat HPRT1 (hypoxanthine guanine phosphoribosyl transferase 1, an internal control), Rn01527840_A1; mouse HPRT1 , Mm00446968_m1) and 2 μl cDNA.

Techniques: Negative Control, Real-time Polymerase Chain Reaction

Journal: Communications Biology

Article Title: The role of RNF149 in the pre-emptive quality control substrate ubiquitination

doi: 10.1038/s42003-023-04763-9

Figure Lengend Snippet: A Left-293 cell lysates expressing RNF149-Flag alongside the indicated AIRAPL isoform or AIRAP (used as a specificity control), were immunopurified (IP) with AIRAP or AIRAPL antibodies and RNF149 content was revealed with a Flag immunoblot. Total content prior to IP is indicated as input. Right- 293 cell lysates expressing AIRAPL alongside the indicated RNF149-GFP or GFP (used as a specificity control), were IPed with a GFP antibody. AIRAPL and endogenous p97 content were revealed by immunoblot. Total content prior to IP is indicated as input. B 293 cell lysates expressing RNF149 alongside the indicated AIRAPL isoforms were IPed with an RNF149 antibody. AIRAPL content was revealed by immunoblot. Total content prior to IP is indicated as input. C A time course in-vitro ubiquitin ligase assay using recombinant cytosolic portion of RNF149 (AA222-400) as a E3 ligase, in the presence or absence of the E2 UbcH5b (upper panel) or a RING mutant VW271/299AA (lower panel). D Binding of endogenous RNF149 to endogenous Bag6 was evaluated by an IP of RNF149 or a non-relevant (N.R.) antibody IP from 293 cells stably expressing the pEQC substrate VCAM1-YFP. Bag6 content was revealed by immunoblot. Total content prior to IP is indicated as input. E Cells expressing VCAM-1 alongside GFP, RNF149WT-GFP or RNF149RINGmut-GFP were treated with CAM741 or velcade as indicated. The presence of the non-translocated (VCAM-1) and glycosylated translocated protein (Glc-NAC-VCAM-1) are indicated. GFP immunoblot show the expression level of the free or fused RNF149 forms and p97 IB serves as a loading control of lysates.

Article Snippet: Quantitative expression of RNF149 transcript was performed using R.T. PCR predesigned primers for mouse RNF149 and HPRT1 genes (Mm01234645 and Mm00446968) according to manufacturer conditions (Applied Biosystems).

Techniques: Expressing, Control, Western Blot, In Vitro, Ubiquitin Proteomics, Recombinant, Mutagenesis, Binding Assay, Stable Transfection

Journal: Communications Biology

Article Title: The role of RNF149 in the pre-emptive quality control substrate ubiquitination

doi: 10.1038/s42003-023-04763-9

Figure Lengend Snippet: A 293 cells expressing RNF149UBAIT and the pEQC substrate VCAM-1 were treated as indicated with velcade and CAM741. Cell lysates were adjusted to RIPA buffer and a Flag IP was applied followed by extensive RIPA buffer washes. IP content was evaluated towards RNF149 and VCAM-1 content by Flag and HA immunoblots respectively. Total content prior to IP is indicated as input. B 293 cells expressing IGF1R-GFP and RNF149UBAIT were subjected to a Flag IP under RIPA conditions. Following extensive RIPA buffer washes the IP content was split into two equal fractions and the deubiquitinating enzyme Usp2 was added to half of the purified content. Deubiquitintion was terminated by addition of laemmli buffer and RNF149 and IGF1R content was evaluated by Flag (RNF149) and GFP (IGF-1R) immunoblots. C 293 cells expressing RNF149UBAIT wt or mutant forms were purified from cell extracts and protein content identification was evaluated by LC-MS. A volcano plot showing the enrichment fold ( X -axis) and significance ( Y -axis) is shown. In addition to AIRAPL, the top ranked specific interactor with RNF149UBAIT wt is labeled (LTN1).

Article Snippet: Quantitative expression of RNF149 transcript was performed using R.T. PCR predesigned primers for mouse RNF149 and HPRT1 genes (Mm01234645 and Mm00446968) according to manufacturer conditions (Applied Biosystems).

Techniques: Expressing, Western Blot, Purification, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Labeling

Journal: Communications Biology

Article Title: The role of RNF149 in the pre-emptive quality control substrate ubiquitination

doi: 10.1038/s42003-023-04763-9

Figure Lengend Snippet: A Mouse genomic locus illustration of RNF149 . Forward and reverse primer binding sites (475, 492 and 477) are shown as well as scissor icons indicating the designed Cas9 cleavage sites. Expected PCR product size of the primer sets are indicated. Genotyping examples of RNF149 + / + and RNF149-/- animals using the designated primer sets shown on bottom. PCR products were sequenced and confirmed the deletion of the 19 kb RNF149 genomic fragment. B RT-PCR results of cDNA samples from the indicated genotypes were performed on MEF cells from the respective mice. RNA levels were normalized to a housekeeping gene (HPRT1). C Blood cells extracted from the indicated RNF149 genotype were evaluated at the age of 6 month. WBC-white blood cells; Neu-neutrophils; Mon-monocyte; Lym-lymphocyte. * p < 0.05; ** p < 0.01.

Article Snippet: Quantitative expression of RNF149 transcript was performed using R.T. PCR predesigned primers for mouse RNF149 and HPRT1 genes (Mm01234645 and Mm00446968) according to manufacturer conditions (Applied Biosystems).

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Communications Biology

Article Title: The role of RNF149 in the pre-emptive quality control substrate ubiquitination

doi: 10.1038/s42003-023-04763-9

Figure Lengend Snippet: A Left- Illustration of VCAM-1 containing the Flag signal sequence (FSS), the glycosylation sites, transmembrane domain (TM), and C-terminus HA tag. Right-Cells expressing Flag-VCAM1-HA were untreated or subjected to velcade and CAM741 treatment. The HA immunoblot reveals both ER and cytosol species of VCAM1 whereas the Flag immunoblot reveals only the pEQC non-translocated species that harbors the signal sequence. The lower band that is not recognized by the Flag immunoblot is an ER translocated species whose signal sequence has been cleaved. Proteasomal stabilization of this species (also seen in Fig. with only velcade) indicates that a small percentage of VCAM1 is also subjected to ERAD. B RNF149 wildtype and knockout MEF cells stably expressing VCAM1 were treated with CAM741 and velcade as indicated. Steady state levels of VCAM1 were revealed by a HA immunoblot and ratios of pEQC or ERAD were quantified and standard deviations from three repetitions are presented.

Article Snippet: Quantitative expression of RNF149 transcript was performed using R.T. PCR predesigned primers for mouse RNF149 and HPRT1 genes (Mm01234645 and Mm00446968) according to manufacturer conditions (Applied Biosystems).

Techniques: Sequencing, Glycoproteomics, Expressing, Western Blot, Knock-Out, Stable Transfection

Journal: Communications Biology

Article Title: The role of RNF149 in the pre-emptive quality control substrate ubiquitination

doi: 10.1038/s42003-023-04763-9

Figure Lengend Snippet: A Live cell imaging of cells expressing RNF149-GFP and ER translocon marker Sec61beta-Cherry under control and ER stress conditions. The partial co-localization of RNF149 with Sec61 seems to be induced during ER stress as apparent from the enhanced co-localization (yellow) observed under thapsigargin (Tg) conditions (120 min, 400 nM). Representative images are presented, scale bars are 10um. B Immunofluorescence of cells expressing RNF149-GFP, VCAM-HA and ManIA-Cherry show a disperse pattern of VCAM and a partial co-localization of RNF149 and ManIA. Treatment with CAM741 caused accumulation of VCAM at the QCVs, together with ManIA and RNF149. Nuclear staining was performed by DAPI staining and added to the merged figure. Scale bars are 10um. C Half-life evaluations of RNF149 were evaluated by treating 293 cells expressing RNF149 with CHX for the indicated time points. The evaluation was performed in the presence or absence of velcade or bafilomycin A (top) or in the presence or absence of a catalytic inactive form of ATG4b (bottom). A quantification of the RNF149 fraction remaining along the four hour time course, is indicated (right).

Article Snippet: Quantitative expression of RNF149 transcript was performed using R.T. PCR predesigned primers for mouse RNF149 and HPRT1 genes (Mm01234645 and Mm00446968) according to manufacturer conditions (Applied Biosystems).

Techniques: Live Cell Imaging, Expressing, Marker, Control, Immunofluorescence, Staining