IGFBP5 protein, human

The following mouse crosses were used to determine the % coverage of LTMR-RZ by the genetically labeled interneuron mouse lines (Figure S2D). For each cross at least three animals were analyzed with at least 100 GFP⁺ cells counted per animal. For tamoxifen regimens when CreER lines are used see Table S1B. For antibody species and dilution when immunohistochemistry is used see Key Resources Table. Excitatory matrix: CCK^CreER;Igfbp5-GFP; R26^LSL-Tom(Ai9). CCK^CreER;5HTr6-GFP;R26^LSL-Tom(Ai9). CCK^CreER;Cbln2-GFP; R26^LSL-Tom(Ai9). CCK^CreER;PV-Tom;R26^LSL-YFP(Ai3). CCK^CreER;NeuroD4-GFP;R26^LSL-Tom(Ai9). CCK^CreER; R26^LSL-Tom(Ai9) with PKCγ immunohistochemistry. 5HTr6-CreER;Cbln2-GFP;R26^LSL-Tom(Ai9). 5HTr6-CreER;NeudoD4-GFP;R26^LSL-Tom(Ai9). 5HTr6-CreER;Igfbp5-GFP;R26^LSL-Tom(Ai9). 5HTr6-GFP with PKCγ and PV immunohistochemistry. NeuroD4-GFP with PKCγ and PV immunohistochemistry. Cbln2-GFP with PKCγ and PV immunohistochemistry. Igfbp5-GFP with PKCγ and PV immunohistochemistry. WT tissue with PKCγ and PV immunohistochemistry. Inhibitory matrix: Cdh3-GFP with PV immunohistochemistry. Rorβ^GFP with PV immunohistochemistry. Kcnip2-GFP with PV immunohistochemistry. Kcnip2-CreER;Cdh3-GFP; R26^LSL-Tom(Ai9). Rorβ^CreER;Kcnip2-GFP;R26^LSL-Tom(Ai9). Rorβ^CreER;Cdh3-GFP;R26^LSL-Tom(Ai9).

Human Samples: Human skin was obtained from corrective plastic surgery. All tissues were obtained according to the guidelines of the University of Pittsburgh and under a protocol approved by the Institutional Review Board of the University of Pittsburgh. Subcutaneous fat tissue was removed and skin tissue was cut into 1.5 cm x 1.5 cm sections. Adenoviral (Ad) constructs were injected intradermally in a volume of 100 µl 1x PBS. Explants containing complete epidermal and dermal layers were cultured in an air liquid interface with the epidermal and keratin layers side up and exposed to air. The culture medium was replaced daily and consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% FBS (Sigma-Aldrich, St Louis, MO), penicillin, streptomycin, and anti-mycotic agent (Invitrogen Life Technologies, Carlsbad, CA). At the indicated time points, skin tissue was harvested and fixed in 10% formalin prior to embedding in paraffin. Skin punch biopsies were obtained from the clinically affected and unaffected skin of patients with SSc as we have previously described [9 (link), 11 (link)].
Adenoviral Constructs: Replication deficient adenoviruses serotype 5 encoding human IGFBP-3, IGFBP-4, or IGFBP-5 were generated as previously described [10 (link)]. Adenovirus serotype 5 lacking cDNA was used as a control. Adenoviruses (1 x 10⁸ pfu) were injected intradermally in a 100 µl volume.
Immunohistochemistry (IHC): Six µm sections of paraffin embedded tissues were deparaffinized and endogenous peroxidases were quenched with 3% H₂O₂. Sections were blocked with 5% serum and incubated with polyclonal anti-IGFBP-5 antibody (Gropep Ltd, Adelaide, Australia) or IgG control antibody (Lab Vision Corporation, Fremont, CA). Sections were washed and incubated with biontinylated secondary antibody (Vector Laboratories, Burlingame CA). Bound secondary antibody was detected using the Vectastain ABC kit (Vector Laboratories) and Zymed AEC Red kit (Zymed, San Francisco CA). A light hematoxylin counterstain was used to identify nuclei using Hematoxylin QS (Vector Laboratories). Images were taken on a Nikon Eclipse 800 microscope (Nikon Instruments Inc., Huntley, IL) using identical camera settings.
Measurement of Skin Dermal and Collagen Bundle Thickness:Six µm sections of paraffin-embedded skin tissue were stained with hematoxylin and eosin (H & E). Images were taken on a Nikon Eclipse 800 microscope. The thickness of the dermis and of individual collagen bundles was measured using Microsuite™ Software (Olympus America Inc.) as we previously described [11 (link)]. Thickness was measured in 5 random fields in each sample. Data are shown in arbitrary units.
Statistical Analysis:Dermal and collagen bundle thickness were analyzed using the Mann-Whitney U test.

To assess proteolytic inhibition, the proteolytic activity of PAPP-A against IGFBP-4 was analyzed as previously described in detail [49 (link)]. Briefly, purified IGFBP-4 was labeled with 125I (Amersham Biosciences), and cleavage reactions (t = 60 min) were carried out in 50 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5, in the presence of a molar excess of IGF-II (Diagnostic Systems Laboratories) at 37 °C. Concentrations were: PAPP-A, 200 pM; IGFBP-4, 10 nM; IGF-II, 100 nM. In some reactions, purified antibody was added in known amounts as specified. Reactions were terminated by the addition of SDS-PAGE sample buffer supplemented with 25 mM EGTA. For kinetic analysis, reaction times were 0, 30, 60, and 120 min. Cleavage products were separated by 10-20% SDS-PAGE and visualized by autoradiography. The degree of cleavage was determined by quantification of band intensities using a Typhoon imaging system (GE Healthcare), and background levels (mock signals) were subtracted. Relative initial velocities (V_i/V₀) were determined by linear regression assuming no substrate depletion. Quantitative analyses for inhibitory constant (K_i) determination were carried out with GraphPad Prism 5.0 software using the Morrison K_i model (competitive inhibition). For semi-quantitative experiments, unlabeled IGFBP-4 (R&D Systems) was used, and proteolytic activity was assessed by the separation of cleavage products in Western blotting. Briefly, samples were separated by 4-20% SDS-PAGE, blotted onto a PVDF membrane (Millipore), blocked with LI-COR blocking buffer, and probed for the N- and C-terminal fragment of IGFBP-4 using specific antibodies (ab92625 and ab153654, Abcam) by incubation for 20 h at 4°C. Fluorescently labeled secondary antibodies (LI-COR) were used for detection of intact and cleaved IGFBP-4, images were captured using a LI-COR Odyssey scanner, and intensities measured using the Image J software. Cleavage (t = 30 min at 37 °C) of IGFBP-5 by human PAPP-A2 was carried out as previously detailed [27 (link)], using 10 nM IGFBP-5, 100 nM IGF-II, 100 pM PAPP-A2 and a variable concentration (0-1000 nM) of mAb 1/41.

The TroIGFBP5b sequence, except for the signal peptide (SP) domain, was amplified with primer TroIGFBP5b-F2/TroIGFBP5b-R1, using the pEASY-T-TroIGFBP5b plasmid as a template by PCR, and named TroIGFBP5b-ΔSP. Subsequently, two HBM-deleted mutants (²¹⁵RKGFFKRKQCKPSRGRKR²³² to ²¹⁵RKGFFPSRGRKR²²⁶, delete ²²⁰KRKQCK²²⁵) of TroIGFBP5b were amplified with full-length TroIGFBP5b or TroIGFBP5b-ΔSP using the primer pairs TroIGFBP5b-F1/TroIGFBP5b-R3 or TroIGFBP5b-F2/TroIGFBP5b-R3 and TroIGFBP5b-F3/TroIGFBP5b-R1 by overlap PCR assay and named TroIGFBP5b-ΔHBM and TroIGFBP5b-Δ(HBM+SP), respectively.
To construct a eukaryotic expression vector for overexpressing IGFBP5 in vivo, TroIGFBP5b and TroIGFBP5b-ΔHBM were inserted into the pCN3 vector, which expresses the human cytomegalovirus immediate-early promoter, at the EcoR V site, resulting in pTroIGFBP5b and pTroIGFBP5b-ΔHBM (34 (link)). pEGFPX-N3 was used for subcellular localization and was reformed from the pEGFP-N3 vector (35 (link)). Recombinant GFP plasmids were constructed by connecting TroIGFBP5b, TroIGFBP5b-ΔSP, TroIGFBP5b-ΔHBM, and TroIGFBP5b-Δ(HBM+SP) to pEGFPX-N3 at the SmaI site, resulting in pTroIGFBP5b-WT-N3, pTroIGFBP5b-ΔHBM-N3, pTroIGFBP5b-ΔSP-N3, and pTroIGFBP5b-Δ(HBM+SP)-N3, respectively. To obtain biologically active recombinant TroIGFBP5b-ΔSP and TroIGFBP5b-Δ(HBM+SP) proteins, pET-32a, which could express a His-tag and a thioredoxin protein (Trx), was used and linearized at the EcoRV site. All of the above-mentioned positive constructs were confirmed by colony PCR and sequencing. The plasmids used in the cell-related experiments, as well as those injected into the fish body, were endotoxin-free plasmids harvested using Plasmid Extraction Kit (TransGen, China) according to the supplier’s instructions.
The siRNA synthesis followed the instructions of RiboMAX™ Express RNAi System (Promega, USA) as described (36 (link)). Briefly, two pairs of primers, siTroIGFBP5b-P1/siTroIGFBP5b-P2 and siTroIGFBP5b-P3/siTroIGFBP5b-P4, were designed to obtain two DNA oligonucleotides after incubation at 95°C for 5 min. The templates were allowed to cool slowly to room temperature (RT). Next, these two DNA oligonucleotides were used to separately synthesize the sense strand RNA or the antisense strand RNA templates at 37°C for 2 h. Afterwards, the DNA template was removed from the separate short RNA strands by digestion with DNase, and then the two RNA strands were mixed to synthesize the siRNA. Finally, the synthesized siRNA was purified following the manufacturer’s instructions. The control siRNA (siTroIGFBP5b-C) was synthesized with siTroIGFBP5b- C-P1/P2 and siTroCCL4-C-P3/P4 as described above. The primers used in this study are listed in Supplementary Table S1.

Placental tissue was mechanically homogenised in Trizol Reagent (Invitrogen™ Thermo Fisher Scientific, Dublin, Ireland) using TissueLyser II (Qiagen, Manchester, UK) and RNA was isolated which was subsequently purified using the Qiagen RNeasy^® Plus Mini (#74134) clean-up protocol according to the manufacturer’s instructions. cDNA synthesis was performed using the Superscript IV Double-Stranded cDNA Synthesis Kit (Invitrogen™ Thermo Fisher Scientific, Ireland). Initial assessment for senescence-associated genes were performed using the RT2 Profiler PCR Array Gene Expression for senescence-associated genes in 96-well plates (Cat no. PAHS-050Z, Qiagen, Ireland). The samples from each group were pooled into and loaded on each 96-well plate. In total, there were 4 groups: PE, PE associated with IUGR, IUGR, and controls, and 4 plates were subjected to Real Time qPCR on the Applied Biosciences StepOne Plus Detection System. The qPCR Assays used in PCR Arrays enables 96 genes in a 96-well plate to be simultaneously analysed using the RT² SYBR Green q PCR Array System protocol. Relative quantification was performed using data analysis web portal (GeneGlobe, Germantown, MD, USA, https://geneglobe.qiagen.com/us/ (accessed on 17 January 2023)) to calculate the fold change using 2^−ΔΔCT formula, where ΔCT is calculated between gene of interest (GOI) and an average of reference genes (HKG), followed by ΔΔCT calculations. Five HKG genes, namely ACTB, B2M, GAPDH, RPLP0, and HPRT1 were included in each PCR plate as internal controls. Eleven genes were selected for validation based on the significant differences between groups when compared with controls, obtained from the RT2 profiler PCR Array: CHEK1, CCNB1, PCNA, PTEN, CDKN2A, ATM, TBX2, GLB1, ID1, IGFBP5, and SOD2. Taqman probes were used for genes of interest (Applied Biosciences, Westmeath, Ireland). The Tata box protein (TBP) gene was used as an internal control with SYBR green primers (Supplementary Table S1). Standard relative PCR quantification was performed to validate the fold change using 2^−ΔΔCT) formula. Taqman probes were used for genes of interest (Applied Biosciences, Ireland). The Tata box protein (TBP) gene was used as an internal control (Eurofins, Dublin, Ireland; Supplementary Table S1). TBP has been shown to be most stable in placental tissue, ensuring sensitivity and specificity of gene expression profile [65 (link)]. Real-time PCR was performed on the Applied Biosciences StepOne Plus Detection System. Standard relative PCR quantification was performed using the manufacturer’s instruction, and fold change was calculated based on relative change between GOI and the internal standard.