Journal: Biomacromolecules

Article Title: Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

doi: 10.1021/acs.biomac.1c00884

Figure Lengend Snippet: (A) α-Synuclein zeta potential in the presence of carbosilane dendrimers: spherical I (red), dendron II (green), and bow-tie III (blue). (B) Determination of the number of dendritic system molecules attached to α-syn.

Article Snippet: Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4 °C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences).

Techniques:

Journal: Biomacromolecules

Article Title: Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

doi: 10.1021/acs.biomac.1c00884

Figure Lengend Snippet: CD spectra of α-synuclein ( c = 1 μM) alone and in the presence of dendritic systems I–III .

Article Snippet: Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4 °C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences).

Techniques:

Journal: Biomacromolecules

Article Title: Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

doi: 10.1021/acs.biomac.1c00884

Figure Lengend Snippet: CD spectra of α-synuclein alone and in the presence of dendritic systems I–III after 48–72 h of incubation.

Article Snippet: Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4 °C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences).

Techniques: Incubation

Journal: Biomacromolecules

Article Title: Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

doi: 10.1021/acs.biomac.1c00884

Figure Lengend Snippet: (A) Schematic representation of the differentiation protocol implemented for the generation of DAns from iPSCs generated from two PD patients (LRRK2-PD) and two healthy age-matched controls (CTRL). (B) Analyses of cell viability in an acute exposure (48 h) testing two different doses of dendrimer III (1 and 3 μM). Bars represent means with SEM as error bars. ( n = 4, using 2 LRRK2-PD-DAn lines from two independent experiments for each condition). (C–E) Cytoplasmic accumulation of α-syn in LRRK2-PD-DAns and LRRK2-PD-DAns treated with 1 μM III for 48 h. Quantitative analyses of α-syn intensity ( n = 42–45, using 2 LRRK2-PD-DAn lines from two-independent experiments, and **** p < 0.0001) (C). Representative immunofluorescence images showing diffuse cytoplasmic accumulation of α-syn in LRRK2-PD-DAns (D) and LRRK2-PD-DAns treated with 1 μM III for 48 h (E). Scale bars, 25 μm.

Article Snippet: Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4 °C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences).

Techniques: Generated, Immunofluorescence

Journal: Biomacromolecules

Article Title: Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson’s Disease Patient-Specific Dopamine Neurons

doi: 10.1021/acs.biomac.1c00884

Figure Lengend Snippet: α-Syn accumulation in DAns after 30 days of differentiation. (A,B) Quantitative analyses of α-syn-positive DAn (A) and α-syn aggregates (B). Bars represent means with SEM as error bars. ( n = 4, using 2 LRRK2-PD-DAn lines and 2 CTRL-DAn lines from two independent experiments). The differences in both, diffuse cytoplasmic α-syn staining and α-syn aggregates accumulation, are statistically significant (* p < 0.05) comparing LRRK2-PD-DAn + 1 μM III or CTRL-DAns to untreated LRRK2-PD-DAns. No significant differences (ns) were observed between LRRK2-PD-DAns + 1 μM III and CTRL-DAns. (C–E) Representative immunofluorescence images showing α-syn aggregates accumulation in LRRK2-PD-DAns; scale bar, 7.5 μm (C), LRRK2-PD-DAns treated with 1 μM III for 48 h; scale bar, 10 μm (D), and CTRL-DAns; scale bar, 25 μm (E).

Article Snippet: Cells were blocked and permeabilized with TBS with low triton (0.01% Triton X-100) and 3% donkey serum for 2 h. Subsequently, cells were incubated for 48 h at 4 °C with the following primary antibodies: mouse anti-TUJ1 (1:500, T8660, Sigma), rabbit anti-TH (1:250, sc-14007, Santa Cruz), and mouse anti-α-syn (1:500, 610787, BD Biosciences).

Techniques: Staining, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Characterization of the scrambling domain of the TMEM16 family

doi: 10.1073/pnas.1703391114

Figure Lengend Snippet: Ca2+-dependent phospholipid scrambling. (A) Expression of TMEM16F. Cell lysates were prepared from wild-type BaF3 cells (+/+), TMEM16F−/− BaF3 cells (−/−), and TMEM16F−/− BaF3 cells transformed with TMEM16F (16F). Proteins (5 µg for +/+ and −/−; 0.1 µg for 16F) were separated by 7.5% SDS/PAGE and analyzed by Western blotting with anti-TMEM16F (Upper) or anti–α-tubulin (Lower) antibodies. (B) Ca2+-dependent PtdSer exposure by TMEM16F. Wild-type BaF3 cells (BaF316F+/+), TMEM16F−/− BaF3 cells (BaF316F−/−), and TMEM16F transformants (BaF316F−/−16F) were incubated at 20 or 4 °C with 3 µM A23187 and 1 mM CaCl2 in the presence of Cy5-Annexin V and PI. Annexin V-staining profiles in the PI-negative population at the indicated time after A23187 addition are shown.

Article Snippet: After incubating at room temperature for 1 h in blocking buffer [TBS (25 mM Tris⋅HCl, pH 7.5, 150 mM NaCl) containing 10% skim milk, 0.01% Tween 20], membranes were incubated at 4 °C overnight with a rabbit anti-mouse TMEM16F antibody ( 14 ) in Can Get Signal Solution 1 (Toyobo Life Science) or with a mouse anti–α-tubulin monoclonal antibody (Merck Millipore), rabbit anti-GFP antibody-HRP-DirecT (MBL), or rabbit anti-human ATP11C antibody ( 5 ) in blocking buffer.

Techniques: Expressing, Transformation Assay, SDS Page, Western Blot, Incubation, Staining

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: Characterization of OsTir1-F74G and Cep192 mAID-mNG mice. (A) Kaplan-Meier-plot showing symptom onset after i.p. injection of OsTir1 Tir/Tir mice with 5mg/kg 5-Ph-IAA (N = 3), 400mg/kg IAA (N = 5), or 800mg/kg IAA (N = 5). (B) Symptoms including spasms and paralysis were graded by severity, 0 = symptom free, 5 = near complete paralysis, humane endpoint. 5mg/kg 5-Ph-IAA (N = 3), 400mg/kg IAA (N = 5), or 800mg/kg IAA (N = 5). (C) Schematic showing the targeting strategy to integrate the lox-stop-lox (LSL)-OsTir1-Myc construct at the Rosa26 locus. (LSL)-OsTir1-Myc animals were crossed to Sox2-Cre expressing mice to achieve whole-body expression of OSTIR1-F74G-Myc. (D) Immunoblot probed with an antibody detecting Myc-tagged OSTIR1 in the noted organs isolated from an OsTir1 f/f (f) and an OsTir1 Tir/Tir (T) mouse. SI – small intestine, Pan – Pancreas, Liv – Liver, Lu – Lung, Spl – Spleen, Br – Brain, He – Heart, Kid – Kidney, Sto – Stomach, SM – Skeletal Muscle. Ponceau-S-staining is shown as a loading control. A biological replicate with a second pair of genotypes is shown in Supplemental Fig. 1G. (E) Schematic illustration showing the strategy to target exon 48 of Cep192 to endogenously tag the C-terminus of Cep192 with mAID and moxNeonGreen (Cep192 AID ). (F) Representative confocal immunofluorescence images of the indicated organs showing CEP192 AID signal (NeonGreen; cyan), and immunostained γ-tubulin (yellow) and CEP135 (magenta); Scale bar 10 µm. (G) Quantification of the CEP192 AID NeonGreen (NG) signal intensity in the noted tissues was measured by fluorescence microscopy and is shown relative to the small intestine (SI). Col – Colon, Sto – Stomach, Pan – Pancreas, Liv – Liver, Lu – Lung, He – Heart, Spl – Spleen, Br – Brain. N = 3 mice. (H-I) Graphs showing the NeonGreen (NG) signal quantified in the SI (H) and the spleen (I) of untreated Cep192 AID mice that were either wt/wt, wt/Tir, or Tir/Tir for OsTir1. N = 3 per genotype. Data is shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with Sidak’s multiple comparisons test (G-I). ns p≥0.05, * p<0.05, ** p<0.01.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Injection, Construct, Expressing, Western Blot, Isolation, Staining, Control, Immunofluorescence, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: Characterization of the novel OsTir1 Tir and Cep192 AID mice. (A) Symptom grades of the individual OsTir1 Tir/Tir mice injected either with 5mg/kg 5-Ph-IAA (N = 3), 400mg/kg IAA (N = 5), or 800mg/kg IAA (N = 5) as in . Symptoms including spasms and paralysis were graded by severity over time; 0 = symptom-free, 5 = near complete paralysis, humane endpoint. (B) Pictures of OsTir1 Tir/Tir mice 30min after injection of 800 mg/kg IAA. Red arrows mark cramping paws and limbs. (C) Representative confocal Immunofluorescence images showing CEP192 AID signal (NeonGreen; green), and immunostained Centrin (red) and CEP135 (cyan) in the SI of OSTIR1 expressing mice that were left untreated or injected with 400mg/kg for 5h or 800mg/kg IAA for 1h. Scale bar 10 µm. (D) Graph showing the body weight of 9-30 weeks old male and female mice of the indicated genotype combinations. Cep192 : A/A – AID/AID, OsTir1 : f/f – floxed/floxed, T/T – Tir/Tir. N = 3-8 mice per genotype. (E) Litter sizes of breedings with the indicated genotype combinations of homozygous (hom) or heterozygous (het) Cep192 AID and OsTir1 Tir alleles. n = 6-18 litters as noted in the figure. 2xhet: Cep192 wt/AID ; OsTir1 wt/Tir . 2xhom: Cep192 AID/AID ; OsTir1 Tir/Tir . (F) Genotype distribution of the offspring of OsTir1 Tir heterozygous breedings. Dashed lines mark the expected Mendelian distribution. N = 9 litters. (G) Immunoblot probed with an antibody detecting OSTIR1-F74G-Myc in the indicated organs of an OsTir1 f/f (f) and an OsTir1 Tir/Tir (T) mouse. This experiment is a biological replicate of the immunoblot shown in . SI – small intestine, Pan – Pancreas, Liv – Liver, Lu – Lung, Spl – Spleen, Br – Brain, He – Heart, Kid – Kidney, Sto – Stomach, SM – Skeletal Muscle. Ponceau- S-staining is shown as a reference for the amount of protein loaded. (H) Graph showing the genotype distribution of the offspring of heterozygous Cep192 AID breedings. Dashed lines mark the expected Mendelian distribution. N = 11 litters. (I) Representative confocal immunofluorescence microscopy images of the indicated tissues as in (H) showing CEP192 AID signal (NeonGreeen, cyan), and immunostained γ-tubulin (yellow) and CEP135 (magenta). Scale bar 10 µm. (J) Graph showing the individual data points of the quantification in . The CEP192 AID NeonGreen (NG) signal was measured as raw integrated density with background subtraction of N = 3 mice across tissues. SI – Small intestine, Col – Colon, Sto – Stomach, Pan – Pancreas, Liv – Liver, Lu – Lung, He – Heart, Spl – Spleen, Br – Brain. (K) Quantification and representative confocal immunofluorescence image of the CEP192 AID NeonGreen (NG) signal in hepatocytes (1) and non-parenchymal cells (2, non-Hep) in the liver as in (I-J). Scale bar 10 µm. Data is displayed as mean ± SEM. Statistical significance was determined by one-way ANOVA with Sidak’s multiple comparisons test. In (A), male and female groups were analyzed separately. ns p≥0.05, * p<0.05, **** p<0.0001.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Injection, Immunofluorescence, Expressing, Western Blot, Staining, Microscopy

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: The AID2 system efficiently degrades CEP192 AID in primary cultures. (A) Quantification of NeonGreen (NG) intensity in immunofluorescence microscopy images relative to DMSO in MEF lines of the indicated genotypes treated with 5-Ph-IAA for various times. N = 3-5 MEF lines per genotype. (B-C) Cep192 AID/AID MEFs that were either wt/wt or Tir/Tir for OsTir1 were treated with 5-Ph-IAA (+) or DMSO (-) for 5h. (B) Representative images of OSTIR1 expressing MEFs immunostained for γ-tubulin (yellow) and CEP135 (red). CEP192 AID is shown in cyan. Insets show centrosomes in mitotic cells. Scale bar 5 µm. (C) Graph showing the number of mitotic spindle poles quantified in immunofluorescence images as in (B); n = 10-30 mitotic cells in N = 4 MEF lines per genotype. (D-E) Cep192 AID/AID MEFs with or without OSTIR1 expression were treated with 5-Ph-IAA (+) or DMSO (-) and live imaged to determine the duration and fate of mitosis. (D) Quantification of the time spent in mitosis from rounding up until completion of cell division or cell death. (E) Graph showing the cell fate after mitosis: cells completed mitosis by successful division (blue), died (black), or re-adhered without division (green). N = 4 MEF lines per genotype. All data is displayed as mean ± SEM. Statistical significance was determined using one-way (D) or two-way (A, C, E) ANOVA with Sidak’s multiple comparisons test. ns p≥0.05, *** p<0.001, **** p<0.0001.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Immunofluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: Complete CEP192 AID in MEFs impairs mitosis. (A) Graph showing the CEP192 AID NeonGreen signal intensity measured relative to timepoint t = 0 min in MEF lines of the indicated genotypes. 5-Ph-IAA was added at timepoint t = 0 min. Cep192 wt / AID ; OsTir1 wt/wt N = 1 MEF lines, n = 4 cells; Cep192 wt / AID ; OsTir1 wt/Tir N = 2, n = 3-8; Cep192 wt / AID ; OsTir1 Tir/Tir N = 3, n = 3-11. (B) Cep192 AID / AID ; OsTir1 Tir/Tir and Cep192 AID / AID ; OsTir1 wt/wt MEF lines treated with 5-Ph-IAA for 5h were immunostained with an antibody raised against mouse CEP192 and an antibody recognizing γ-tubulin. Signal intensities of CEP192 AID (NeonGreen), and immunostained CEP192 and γ-tubulin were quantified relative to the DMSO condition. N = 3-4 MEF lines per genotype. (C) Representative live imaging brightfield stills of mitotic Cep192 AID/AID MEFs with or without OSTIR1 treated with 5-Ph-IAA or DMSO. MEFs without OSTIR1 or 5-Ph-IAA undergo complete mitosis with visible metaphase plates (yellow *), anaphase (magenta *), and cytokinesis (cyan*). MEFs expressing OSTIR1 and treated with 5-Ph-IAA round-up and re-adhere to the plate without undergoing cell division (red arrow). All data is shown as mean ± SEM. Statistical significance was measured by two-way ANOVA ANOVA with Sidak’s multiple comparisons test (B). ns p≥0.05, **** p<0.0001.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Imaging, Expressing

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: CEP192 is not required for primary or motile cilia (A-B) MEF lines of the indicated genotypes were serum starved in the presence of 5-Ph-IAA for 24h. (A) Quantification of the number of cells with a primary cilium; N = 2 MEF lines per genotype from 2 separate passages. (B) Representative confocal image of MEFs expressing CEP192 AID NeonGreen (green), and immunostained for the primary cilia marker Arl13B (red). Yellow boxes show insets of a primary cilium. Scale bar 5 µm. (C) Schematic illustration showing isolation and differentiation of mTECs. 5-Ph-IAA was added to the basal media of air-liquid-interphase cultures (ALI). (D) Representative confocal images of mTECs with (wt/ Tir ) or without (wt/wt) OSTIR1 expression at ALI day 5 expressing CEP192 AID (green) and immunostained for PCNT (red) and CEP164 (gray). Yellow circle marks the CEP192 AID signal at the centrosome in non-differentiating cells. Yellow boxes mark the CEP192 AID signal in differentiating cells with numbers denoting early (1), mid (2), and late (3), and complete (4) differentiation. Scale bars 5 µm. (E) Representative confocal images of mTECs at ALI day 5 were treated with 5-Ph-IAA for 24h. CEP192 AID signal is shown in green and tissues were immunostained for PCNT (red) and CEP164 (gray). Scale bars 5 µm. (F) Time course showing CEP192 AID intensity at newly amplified centrioles following 5-Ph-IAA treatment in differentiating cells without (wt/wt) or with OSTIR1 expression (wt/ Tir ). The signal intensity was normalized to the untreated (UT) condition. N = 2 mice per genotype indicated by different symbols, n=6 fields of view. (G-I) mTECs expressing CEP192 AID either wt or heterozygous for OsTir1 were treated with 5-Ph-IAA for ALI days 0-7. Cells were fixed on ALI day 7 and immunostained for the differentiation marker FOXJ1 and for acetylated tubulin to label motile cilia (AcTub). (G) Graph showing the percentage of cells with motile cilia in mTEC cultures in the absence or presence of Verapamil. (H) Quantification of the percentage of cells in mTEC cultures with FOXJ1+ nuclei with or without Verapamil. (G-H) N = 3 mice per genotype indicated by different symbols, n=6 fields of view per genotype and condition. (I) Representative confocal image of mTEC cultures on ALI day 7 expressing CEP192 AID (green) immunostained for FOXJ1 (red) and AcTub (gray). Scale bars 5 µm. All data is displayed as mean ± SEM. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test (F), or a two-tailed, unpaired Student’s t-test (A, G-H). ns p≥0.05, ** p<0.01.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Expressing, Marker, Isolation, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: CEP192 is not needed for primary or motile cilia maintenance (A) Quantification of the NeonGreen signal of serum-starved Cep192 AID / AID MEFs with or without OSTIR1 normalized to the mean of Cep192 AID / AID ; OsTir1 wt/wt MEFs. n = 39-42 cells per genotype. (B-C) Quantification of Cep192 AID / AID MEF cells wt/wt or wt/Tir for OsTir1 with a primary cilium. Cells were serum starved for 24 h before 5-Ph-IAA treatment for (B) 24h or (C) 48h. N = 2 MEF lines per genotype from 1 or 2 separate passages. (D) Time course of 5-Ph-IAA treatment of mTEC cultures showing CEP192 AID (NeonGreen) intensity at the centrioles of non-multiciliated cells without or with OSTIR1 (wt/ Tir ) relative to untreated control. N = 2 mice per genotype indicated by symbol shape, n = 6 fields of view. (E-G) mTECs were cultured at an air-liquid-interphase (ALI) for 7 days to allow motile cilia formation. 5-Ph-IAA was added to the basal media for ALI days 7-9. (E) Quantification of the percentage of cells with motile cilia marked by acetylated tubulin (AcTub) and (F) the fraction of cells positive for the differentiation marker FOXJ1. N = 3 mice per genotype indicated by symbol shape, n = 6 fields of view. (G) Representative confocal images of mTEC cultures of the indicated genotypes expressing CEP192 AID (green) and immunostained for AcTub (gray) and FOXJ1 (red). Scale bars 10 µm. All data is shown as mean ± SEM. Statistical significance was measured by two -way ANOVA with Sidak’s multiple comparisons test (D), and two-tailed, unpaired Student’s t-test (A-C, E-F). ns p≥0.05, **** p<0.0001.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Control, Cell Culture, Marker, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: The AID2 system induces rapid and reversible CEP192 AID degradation in mice. (A) Representative confocal images of the small intestine and the spleen of CEP192 AID mice expressing OSTIR1 that were left untreated (UT), or analysed at various times after i.p. injection of 5-Ph-IAA. CEP192 AID (cyan), γ-tubulin (yellow), CEP135 (magenta); scale bars 5 µm. (B) Graph showing the CEP192 AID intensity (NeonGreen, NG) in the small intestine (SI, blue) and the spleen (red) measured by fluorescence microscopy relative to the untreated (UT) control at 15 min, 30 min, 45 min, 1.5 h, 3 h, and 5 h after i.p. injection. Maximum degradation (Dmax) was reached by 5h. (C-H) Quantifications of the CEP192 AID (NeonGreen, NG) signal intensity in the indicated organs are shown relative to the untreated controls (UT) 30 min after treatment. (G) Since CEP192 AID was not expressed in hepatocytes, the signal was quantified in non-parenchymal cells in the liver (Non-Hep). (I) Table shows the degree of degradation at 30 min for the organs in (C-H). (J) The CEP192 AID signal (NG) was quantified at the indicated time points up to 72h after 5-Ph-IAA injection. Data points for UT and time points up to 5h are the same as in (B). The dashed line indicates 100%; N = 3-6 mice per time point. Data is shown as mean ± SEM. Statistical significance was determined using a two-tailed, unpaired Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Expressing, Injection, Fluorescence, Microscopy, Control, Two Tailed Test

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: Different doses of 5-Ph-IAA alter the degradation dynamics. (A) Representative immunofluorescence images of organs from Cep192 AID / AID mice expressing OSTIR1 that were untreated (UT) or injected 5-Ph-IAA. Analysis was performed 30 min after 5-Ph-IAA administration or following repeat 5-Ph-IAA administration every 12 h for 3 days. CEP192 AID signal (cyan), and immunostained γ-tubulin (yellow) and CEP135 (magenta); scale bar 5 µm. (B-C) Quantification of the CEP192 AID NeonGreen (NG) signal relative to the untreated control (UT) of (B) the small intestine (SI) and (C) the spleen of mice injected with 1 mg/kg or 5 mg/kg 5-Ph-IAA at the indicated timepoints. All data is shown as mean ± SEM.

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Immunofluorescence, Expressing, Injection, Control

Journal: bioRxiv

Article Title: The AID2 system offers a potent tool for rapid, reversible, or sustained degradation of essential proteins in live mice

doi: 10.1101/2024.06.04.597287

Figure Lengend Snippet: Chronic degradation of CEP192 AID causes cell death in the gastrointestinal tract and weight loss. (A) Schematic illustration of the experiment designed to test the effects of prolonged CEP192 depletion in mice. Mice were i.p. injected with PBS or 5-Ph-IAA every 12h and analyzed after 3 and 8 days. (B) Representative confocal immunofluorescence images of the small intestine (SI) and the spleen of Cep192 AID/AID ; OsTir1 wt/ Tir mice left untreated (UT) or subjected to repeated 5-Ph-IAA injections for 3 or 8 days. CEP192 AID signal (cyan), γ-tubulin (yellow), and CEP135 (magenta); scale bars 5 µm. (C-D) Graphs showing the CEP192 AID signal (NeonGreen, NG) in immunofluorescence images of the (C) SI and the (D) spleen relative to the untreated control (UT) at the indicated time points. (E-F) Quantifications of the γ-tubulin signal intensity in immunofluorescence images in the (E) SI and (F) the spleen after 3 and 8 days. (G) Bodyweight changes of Cep192 AID/AID ; OsTir1 wt/ Tir mice repeatedly injected with 5-Ph-IAA over 8 days. N = 5 mice per genotype (8 days); N = 3 mice per genotype (3 days). (H-I) Cell death was measured by immunohistochemistry for cleaved caspase-3 (C3) in the crypts of the small intestine (SI). Mouse tissues were analysed 8 days after treatment. (H) Quantification of cleaved caspase-3 positive cells per crypt. (I) Representative images of the SI. Black box: Cep192 wt/wt ; OsTir1 Tir/Tir mice injected with PBS; Blue box: Cep192 wt/wt ; OsTir1 Tir/Tir mice injected with 5-Ph-IA: Orange box: Cep192 AID / AID ; OsTir1 wt/ Tir mice injected with 5-Ph-IAA (orange box). Scale bars 100 µm. (J) Quantification of the spleen size after 8 days of repeated 5-Ph-IAA injections and in untreated Cep192 AID/AID ; OsTir1 wt/ Tir control mice (black). N = 3 mice per group. (K) Graph showing the number of mitotic cells per crypt quantified in H&E-stained paraffin sections of the small intestine (SI) of untreated Cep192 AID/AID ; OsTir1 wt/ Tir mice (black box) or from mice injected with 5-Ph-IAA for 8 days ( Cep192 wt/wt ; OsTir1 Tir / Tir mice, blue box; Cep192 AID/AID ; OsTir1 wt/ Tir orange box). (L) Representative H&E-stained paraffin sections as in (K). Insets highlight mitotic cells. Scale bars 50 µm. Data is shown as mean ± SEM. Statistical significance was measured using one-way ANOVA (C-E, H, J, K) with Sidak’s multiple comparisons test, or mixed-effects analysis with Dunnett’s multiple comparisons test. ns p≥0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Article Snippet: Primary antibodies used were CEP135 (rabbit polyclonal, homemade, Alexa555- conjugated, 1:500), γ-Tubulin (goat polyclonal, homemade, Alexa647-conjugated, 1:500), CEP164 (rabbit polyclonal, EMD Millipore Corp., ABE2621, 1:1000), mouse CEP192 (rabbit polyclonal, gift from Karen Oegema, 1:1000), FOXJ1 (mouse monoclonal, ThermoFisher #14- 9965-82, 1:1000), Acetyl-α-Tubulin (Rabbit polyclonal, Lys40 (D20G3), Cell Signaling #5335T, 1:1000), mouse anti-PCNT (1:250, BD Transduction Laboratories #611814), ARL13B (mouse monoclonal, N295B66, Antibodies Incorporated #75-287, 1:1000).

Techniques: Injection, Immunofluorescence, Control, Immunohistochemistry, Staining