Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 1. CAPERα and TBX3 directly interact via the TBX3 repressor domain. (A) Representative spectrum for CAPERα identified in anti-TBX3 co-IP of HEK293 cell lysates. Mass spec analysis identified six specific CAPERα peptides, providing 8.5% sequence coverage of the protein. This spectrum shows fragmentation of one of these peptides, C*PSIAAAIAAVNALHGR, with diagnostic b- and y-series ions shown in red and blue, respectively. * indicates Figure 1. Continued on next page

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Sequencing, Diagnostic Assay

Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 2. Knockdown of endogenous CAPERα and TBX3 in primary human fibroblasts and mouse embryos induces premature senescence and disrupts expression of cell cycle and senescence regulators. (A–C) Representative bright field images of senescence associated β-galactosidase (SA-βG) assays of HFFs transduced with control, TBX3 shRNA A or CAPERα shRNA A. Only occasional cells in the control transduction have detectable lacZ staining (blue) Figure 2. Continued on next page

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Knockdown, Expressing, Transduction, Control, shRNA, Staining

Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 3. RB and p16 mediate senescence after CAPERα/TBX3 loss of function and CAPERα/TBX3 regulates chromatin structure of CDKN2A-p16. (A–F) SA-βgal assays of HFFs stably transduced with control (Ctl) or p53 (Masutomi et al., 2003) or RB (Boehm et al., 2005) shRNAs subsequently transduced with CAPERα or TBX3 shRNAs. (G) % Quantitation of A–F from three replicate experiments. * indicates p<0.05 relative to Control or p53 shRNAs. (H) Cell proliferation assayed by crystal violet incorporation (OD units) in HFFs treated as in A–F. * indicates p<0.001 relative to Ctl or p53 shRNAs. (I–L) SA-βgal Figure 3. Continued on next page

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Stable Transfection, Transduction, Control, Quantitation Assay

Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 4. CAPERα/TBX3 directly represses expression of the long noncoding RNA UCA1. (A–C) Gel showing RT-PCR analysis of TBX3, CAPERα, and HPRT expression in control, TBX3 and CAPERα siRNA-transfected HEK293 cells. The siRNAs effectively decreased transcript levels of their targets. (D) Differential display: representative PAGE gel of cDNAs derived from random primed, RT-PCR'd mRNAs from CAPERα, TBX3 and control siRNA transfected HEK293 cells. Blue arrowheads denote upregulated transcripts subsequently identified by sequencing as DUSP4 and UCA1. (E and F) qPCR analysis of TBX3 and CAPERα transcript levels in control and TBX3 or CAPERα shRNA transduced HFFs (repeat of experiment shown in Figure 2—figure supplements 1A and 2A). (G) RT-PCR analysis of UCA1 and HPRT gene expression in control, TBX3 or CAPERα shRNA-transduced HFFs. (H) qPCR analysis of UCA1 transcript levels in control, TBX3 or CAPERα shRNA transduced HFFs. Results confirm differential display result that KD of TBX3 or CAPERα results in increase in UCA1 transcript levels. (I) Schematic representation of the UCA1 locus with primer sets employed for ChIP-PCR amplifica tion of denoted regions 5′ of gene (A1, A2, A3). (J) Anti-TBX3 ChIP-PCR of regions of the UCA1 promoter in HFFs; only A3 is ChIP'd by TBX3 (lane 18, red arrowhead). (K) Anti-CAPERα ChIP-PCR of regions of the UCA1 promoter in HFFs; only A3 chromatin is ChIP'd (lane 18, red arrowhead). (L) ChIP-PCR analysis of UCA1/A3 chromatin from in HFFs transduced with control (C) or TBX3 (KD) shRNA; ChIP antibodies are listed at top. Note decreased CAPERα binding after TBX3 KD (lane 17, red arrowhead), gain of activating mark H3K4me3 and loss of repressive marks H3K9me3 and H3K27me3. (M) ChIP-PCR analysis of UCA1/A3 with antibodies listed at top of panel in HFFs transduced with control (C) or CAPERα shRNAs. Note continued TBX3 binding despite CAPERα KD (lane 11, red arrowhead) and changes in chromatin marks parallel those seen in with TBX3 KD in panel L. TBX3, CAPERα = human; Tbx3, Caperα = mouse. DOI: 10.7554/eLife.02805.014 The following figure supplements are available for figure 4:

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Derivative Assay, Random Primed, Sequencing, shRNA, Gene Expression, Transduction, Binding Assay

Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 5. UCA1 expression is sufficient to induce senescence and required for normal execution of oncogene-induced senescence. (A) UCA1 and HPRT transcripts assessed by RT-PCR in control and UCA1-overexpressing HFFs. (B and C) Representative bright field images of SA-βgal assay of cultured HFFs transfected with control and UCA1 overexpression plasmids. (D) % quantitation of SA-βgal cells from five replicates in control and UCA1 overex pressing HFFs. * indicates p<0.05. (E and F) Immunohistochemical analysis reveals co-localization of H3K9me3 and DAPI in SAHFs in HFFs transfected with UCA1 overexpression plasmid (F) but not control plasmid (E). (G) Cell count of control and UCA1 overexpressing HFFs 3 days post transfection. Mean ± SD of 3 plates is shown at each time point. * indicates p<0.005 relative to control. (H) Crystal violet assay of cell growth in control and UCA1 overexpressing HFFs transfected with 2 μg of expression or control vector and assayed daily for 3 days post- transfection. * indicates p<0.01 relative to control. (I) Crystal violet assay of HFFs cultured for 3 days after transfecting 0, 1, 2, or 4 μg of control or UCA1 overexpression plasmid. * indicates p<0.01 relative to control. (J) Transcript levels assessed by qPCR; values reflect fold change in UCA1-overexpressing HFFs relative to control after normalization to HPRT levels. * indicates p<0.05 relative to control. (K) qPCR analysis of UCA1 expression in untransduced, presenescent (PS) HFFs and HFFs transduced with constitutively active G12VRAS (RAS). * indicates p<0.05 relative to PS. (L) Efficient knockdown of UCA1 transcripts in RAS HFFs with UCA1 shRNA (quantitated in panel T). (M–P) SA-βgal assays of RAS HFFs transduced with either control or UCA1 shRNA at 3 (M and O) and 5 (N and P) days post transduction. (Q) % quantitation of SA-βgal cells from six replicate experiments as represented in panels M–P. * indicates p<0.001 relative to control. (R) % quantitation of Ki67 + cells from three replicates in control vs UCA1 shRNA transduced RAS HFFs. * indicates p<0.001 relative to control. (S) RT-PCR for UCA1 transcripts shows persistent knockdown of UCA1 in RAS shRNA cells with increasing passage (P0–P2). (T) qPCR analysis of fold changes in transcript levels of cell cycle and senescence genes after UCA1 shRNA knockdown in RAS HFFs. * indicates p<0.05 relative to control. (U) ChIP-PCR analysis of UCA1 region A3 with antibodies listed at top in PS and RAS HFFs. Note gain of activating (H3K4me3, H3K9ace, H4K5ace) and loss of repressive marks (H3K9me3, H3K27me3) at the UCA1 locus after oncogene-induced senescence by RAS. TBX3, CAPERα = human; Tbx3, Caperα = mouse. DOI: 10.7554/eLife.02805.016 The following figure supplements are available for figure 5:

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Cell Culture, Transfection, Over Expression, Quantitation Assay, Immunohistochemical staining, Plasmid Preparation, Cell Counting, Crystal Violet Assay, Transduction, Knockdown, shRNA

Journal: eLife

Article Title: Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

doi: 10.7554/elife.02805

Figure Lengend Snippet: Figure 7. Disruption of the CAPERα/TBX3 repressor by OIS activates CDKN2A-p16 and UCA1 to trigger a senescence transcriptional response. (A) ChIP-PCR of regions upstream of the CDKN2A-p16 transcriptional start site (position relative to TSS in parentheses) in PS and RAS HFFs; the −-3706–3308 amplicon is a negative control. OIS disrupts binding of p16 regulatory elements (initially identified in Figure 3O) by TBX3 and CAPERα. (B) ChIP-PCR of p16 -4855 element shown in A. Decreased TBX3 and CAPERα binding in RAS correlates with loss of repressive chromatin marks and gain of activating Figure 7. Continued on next page

Article Snippet: Amylose bound MBP and MBP-tagged TBX3 affinity columns were prepared as per the procedure (E8022S, NEB) described in the manufacturer's protocol.

Techniques: Disruption, Amplification, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: | Preparation and structure determination of bt TMEM206 using MISO and conventional purification approach . a , Schematics of the protein construct and MISO purification on a single 3 μl microfluidics streptavidin column. b , Complete MISO purification chromatogram of btTMEM206-YFP from cleared detergent-solubilized HEK cells. Fluorescent signal was detected at WL 525 nm and originates from YFP. c , Elution peak was fractionated into 1 μl fractions used for protein characterization. d , SDS-PAGE from bt TMEM206-YFP purified on MISO (left) and bt TMEM206 purified by conventional approach (right). The conventionally purified TMEM206 was gel-filtered (panel h) and the peak fraction was used for SDS-PAGE. e , Negative stain micrograph from MISO- purified sample shows homogeneous protein particles. f and i , Cryo-EM micrographs of MISO-purified bt TMEM206-YFP and conventionally purified TMEM206 on holey EM grids. h , SEC from bt TMEM206 purified by the conventional method. g, j-n , Comparison of 2D class averages and 3D reconstructions from bt TMEM206-YFP (g, k, m) and bt TMEM206 (j, l, n). Additional diffuse density from YFP in 2D classes is marked with white asterisk (panel g). o , Structural alignment between bt TMEM206 (grey) and bt TMEM206-YFP (coloured) models shows that YFP does not modify the structure.

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Purification, Construct, SDS Page, Staining, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: | De termination of m TMEM16F structure using half a 10 cm dish of HEK cells. a , Schematics of the protein construct and MISO purification on a single 3 μl streptavidin column. b , Complete MISO purification chromatogram from a cleared lysate of HEK cells. Fluorescent signal from YFP was detected at WL 525 nm. c , Elution peak was fractionated into 1 μl fractions used for protein characterization. d , SDS-PAGE from m TMEM16F-YFP purified on MISO. e , Negative stain micrograph from MISO-purified sample shows homogeneous protein particles. f, Cryo-EM micrographs of MISO-purified m TMEM16F-YFP on holey EM grids. g, 3D reconstruction of TMEM16F-YFP to a resolution of 3.5 Å . High-resolution map is overlayed with a low-pass filtered map to show the detergent micelle. h , Close-up view on the Ca 2+ binding site with bound Ca 2+ ion. i , Details of density map for a trans-membrane helix and for a β-hairpin. j , Structural alignment between MISO purified m TMEM16F-YFP and m TMEM16F prepared by conventional approach (PDB 6P46).

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Construct, Purification, SDS Page, Staining, Cryo-EM Sample Prep, Binding Assay, Membrane

Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: a , Protein constructs and schematics of the experiment. 80 μl of extract was purified on a 3 μl streptavidin column. b, Elution peak detected at WL of 525 nm was fractionated into 1 μl fraction used for negative stain EM (grey in the chromatogram) while subsequent 80 nl fractions were spotted on holey gold grids (blue) and cryo-plunged. c , Negative stain micrograph and an example of cryo-EM micrograph from MISO device. Panel below shows 2D classes from the cryo-EM dataset. d, 3D cryo-EM maps of the of the complete membrane protein complex solved to resolution of 3.5 Å. e, 3D reconstruction of intracellular domain (ICD) only resolved to 3.4 Å. f, Density for two distinct Ca 2+ ions bound to calcium binding sites (CBS) with fitted structure of human TRPC6 (PDB 6UZ8). g, Experimental scheme for purification of TRPC6 on a 2-column MISO chip. h, Protein purified on 1 μl streptavidin column was further polished on a 10 μl Superose 6. i, SDS-PAGE imaged using fluorescence at WL 488 nm. j, Negative stain EM micrographs from the selected fractions.

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Construct, Purification, Staining, Cryo-EM Sample Prep, Membrane, Binding Assay, SDS Page, Fluorescence