Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 1. DNA hypomethylation results in the activation of L1TD1 expression and loss of L1TD1 affects cell viability in HAP1 cells. (A) Quantification of DNA methylation levels at the L1TD1 promoter in HAP1 wildtype (WT), DNMT1 KO, and DNMT1/L1TD1 DKO cells using the MethyLight assay. DNA methylation is shown as percentage of methylation ratio (PMR). (B) qRT-PCR analysis of L1TD1 mRNA expression in HAP1 WT, DNMT1 KO, and DNMT1/ L1TD1 DKO cells. GAPDH was used as a normalization control and relative L1TD1 mRNA levels in DNMT1 KO cells were set to 1. Data are shown as a

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Control

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 3. L1TD1 cross-talk with its ancestor L1 ORF1p. (A) Volcano plot displaying the comparison of the proteomes of HAP1 DNMT1 KO and DNMT1/ L1TD1 DKO cells determined by mass spectrometry. Differentially abundant proteins were plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1, adj. p-value<0.05 [red] and log2FC ≤ –1, adj. p-value<0.05 [blue]). (B) Volcano plot illustrating the DESeq2 analysis of RNA-seq performed with HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells. Differentially expressed genes are plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1,

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Comparison, Mass Spectrometry, RNA Sequencing

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 4. L1TD1 promotes L1 retrotransposition. (A) Schematic representation of plasmids used for retrotransposition (figure modified from Kopera et al., 2016 and generated with BioRender.com). The pJJ101/L1.3 construct contains the full-length human L1.3 element with a blasticidin deaminase gene (mblast) inserted in antisense within the 3’UTR. The mblast gene is disrupted by an intron and mblast expression occurs only when L1 transcript is expressed, reverse transcribed, and inserted into the genome. The pJJ105/L1.3 plasmid contains a mutation in the reverse transcriptase (RT), resulting in defective retrotransposition. The backbone plasmid pCEP4 was used as additional negative control. The blasticidin deaminase gene containing plasmid pLenti6.2 was used as transfection/selection control. (B) Workflow of retrotransposition assay. DNMT1 KO and DNMT1/L1TD1 DKO cells were separately transfected with pJJ101 and control plasmids. Equal number of cells were seeded for each condition. Blasticidin selection (10 µg/ml) was started at day 4 and resistant colonies were counted on day 13. This panel was created using BioRender.com. (C) Bar graph showing the average number of retrotransposition events per 106 cells seeded in three independent experiments. Blasticidin-resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of three independent experiments, ****p≤0.0001. (D) Representative pictures of bromophenol blue stainings of blasticidin-resistant colonies for each genotype and each transfection.

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Modification, Generated, Construct, Expressing, Reverse Transcription, Plasmid Preparation, Mutagenesis, Negative Control, Transfection, Selection, Control

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: Nature chemical biology

Article Title: Diabetes reversal by inhibition of the low molecular weight tyrosine phosphatase

doi: 10.1038/nchembio.2344

Figure Lengend Snippet: (a) Surface representation of human LMPTP-A showing phosphate (P) non-covalently bound in the active-site. Residues are colored according to magnitude of shift in the HSQC 15 N- 1 H spectrum upon Compd. 18 titration (red>orange>green). Gray residues had negligible shifts or could not be assigned. (b) Crystal structure of bovine LMPTP W49Y/N50E bound to orthovanadate and Compd. 18 (cyan and blue sticks; Q=quinoline; Pip=piperidine; BN=benzonitrile; L=linker), with selected side-chains (yellow=carbon; red=oxygen; blue=nitrogen; pink=vanadium) and H-bonds/ionic interactions (dashed green/gray lines) shown. (c) Inhibition of phosphatase activity of LMPTP-A/mutants by Compd. 18 using 0.4 mM OMFP substrate. Mean±SD % activity is shown. Data is representative of 3 independent experiments. (d) Compd. 18 modeled into the crystal structure of phosphate-bound human LMPTP, based on an overlay with the bovine ternary complex crystal structure (RMSD=0.33 Å). Selected residues are colored by NMR shift as in (a) . Dashed red line depicts predicted clash between apical oxygen (“A”) of phosphate and Q. (e–f) Structural rationale for SAR data, with atoms at 66% of their true radii. (e) “Side” view of pocket, rotated ~90° about a horizontal axis. The molecular surface has been sliced through the active-site to reveal the tight fit of Q in the pocket. Atoms with a formal charge (±) are labeled. BN is highly polarized, as indicated (δ±); arrows labeled “S” indicate solvent exposure of ring substitutions. (f) “Top” view looking down at the active-site pocket filled by Q. Arrow above atom N1 locates the “saddle-point” at pocket exit.

Article Snippet: cDNAs encoding mouse and human LMPTP-A (protein reference sequences NP_067305.2 and NP_004291.1) and bovine LMPTP (protein reference sequence NP_776403.1) were codon-optimized for E. coli , synthesized, and cloned into the pGEX-4T vector using BamHI/EcoRI by Genscript.

Techniques: Titration, Inhibition, Activity Assay, Labeling, Solvent