Microseal b seal

SCAP cells (5.0 × 10⁵) were seeded in a 4-well plate and harvested 6 h after PBM treatment. The mRNA was extracted (RNeasy Mini kit, Qiagen, Germany), and the cDNA was synthesized using commercial kits (Rever Tra Ace qPCR Master Mix, Toyobo, Japan). mRNA was quantified with a plate reader (Take 3 at Synergy HT, Biotek, USA), reverse transcription was conducted using a thermal cycler (Qiagen, Germany), and real-time PCR was performed using a CFX96 Real-Time System (BioRad, USA). For real-time PCR, Hardshell 96-Well PCR Plates (BioRad, USA), Microseal ‘B’ seals (BioRad, USA), and SYBR green master mix (BioRad, USA) were used. All the processes followed a protocol provided by the manufacturers. The sequence of primers used in quantitative real-time PCR is given in Table 2.Table 2

Sequence of primers used for real-time PCR.

GAPDH forward	5′-CGACCACTTTGTCAAGCTCA-3′
GAPDH reverse	5′-GAGGGTCTCTCTCTTCCTCT-3′
caspase3 forward	5′-GACTCTAGACGGCATCCAGC-3′
caspase3 reverse	5′-CCAGTGAGACTTGGTGCAGT-3′
JNK1 forward	5′-TGTCACACCTGGAAACCTGA-3′
JNK1 reverse	5′-GAAAGGAGAGGGAACGGAAC-3′
RIPK1 forward	5′-CCATGACCCTTTTGCACAGC-3′
RIPK1 reverse	5′-TGGCTGCACTGGAATAAGCA-3′
SOD2 forward	5′-GTTGGCCAAGGGAGATGTTA-3′
SOD2 reverse	5′-TAGGGCTGAGGTTTGTCCAG-3′
SOD3 forward	5′-CCAACAGACACCCTCCACTC-3′
SOD3 reverse	5′-AAGGATGGTGGGTCTCGGTA-3′
PRDX3 forward	5′-GTTGTCGCAGTCTCAGTGGA-3′
PRDX3 reverse	5′-GAGTGCGATGTTCATGTGGC-3′

The expression levels of RBP6, VEX1, and mVSG genes was analysed by RT-qPCR using Luna Universal qPCR MasterMix (NEB) with 500 nM of primers. All primer pairs are listed in Table 1. RNA was extracted using a Qiagen RNeasy Kit and the samples were treated with DNase 1 for 1 h according to manufactures instructions and eluted in 30 µL of RNase free water. The samples were quantified using a Nanodrop (ThermoFisher). cDNA was prepared using SuperScript IV (ThermoFisher) following the supplier instructions from 1–2 µg RNA with a polyT primer. For each pair of primers (used at 500 nM), triplicates of each sample were run per plate (Hard-shell PCR Plates 96 well, thin wall; Bio-Rad), which were sealed with Microseal “B” Seals (BioRad). All experiments were run on a CFX96 Touch Real-time Detection system with a C1000 Touch Thermal cycler (Bio-Rad), using the following PCR cycling conditions: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s (fluorescence intensity data collected at the end of the last step). Data was then analysed by relative quantification using the ΔΔCt method (CFX Maestro software—Bio-Rad) and Cq determination regression was used. In all cases, product abundance was determined relative to an actin control locus.

Nanobodies were diluted to 1 mg/ml in 20 μl 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 1x SYPRO Orange (Life Technologies). Samples were pipetted into a Hard‐Shell® 96‐well plate (Bio‐Rad). The plate was sealed with transparent MicroSeal® “B” Seal (Bio‐Rad) and briefly centrifuged to remove air bubbles. Experiments were performed using the CFX96 Real‐Time System (C1000 Thermal Cycler, Bio‐Rad). The samples were incubated for 5 min at 20°C before the temperature was gradually increased to 95°C with 1‐K increments and 45 s for each incubation step. At the end of each step, the SYPRO Orange fluorescence was measured using the HEX channel. Melting temperatures are defined as the inflection point of the melting curve before reaching the first melting peak. VHH variants were considered hyperthermostable if they produced no melting peak.

qPCR was performed using a CFX96 Connect apparatus (Bio-Rad, Japan). Hard-Shell® 96-Well PCR Plates (Cat # HSP 9601, Bio-Rad) sealed with optically clear adhesive seals (Microseal® ‘B’ seal, Cat # MSB1001, Bio-Rad) were used in all experiments. The thermocycler program consisted of an initial hot start cycle at 95 °C for 3 min, followed by 33 cycles at 95 °C for 10 s and 59 °C for 30 s. Mouse actin beta (Actb) was amplified using the following primers: F-5′-AACCCTAAGGCCAACCGTGAA-3′, R-5′-ATGGCGTGAGGGAGAGCATA-3′ (with estimated product length 194 bp). The primers were used at a concentration of 300 nM. SYBR Green-based PCR supermix (Bio-Rad) was used for all reactions according to manufacturer’s instructions. Each reaction was performed in a final volume of 8 μL. To confirm product specificity, a melting curve analysis was performed after each amplification, and agarose gel analysis was performed to ensure the amplification of the right product (Additional file 1: Figure S1).

DSF experiments were performed according to the previously described protocol [49 (link)]. Briefly, ADP-ribose was dissolved in H₂O to a final concentration of 200 mM, and serial 1:2 dilutions created a ten-point concentration series from 200 to 0.39 mM. DSF buffer was prepared by diluting SYRPO orange (Thermo Fisher Scientific, S6650) from 5000x to 10x in 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Triton X-100. The ADP-ribose concentration series was then diluted 1:100 with DSF buffer (2 μL + 198 μL DSF buffer) and 10 μL was aliquoted into a 384-well white PCR plate (Axygen, PCR-384-LC480-W-NF) and the plates were incubated in the dark for 20 minutes. Purified Mac1 or buffer was then added to each well (10 μL at 4 μM). Each well had a total of 20 μL that contained 2 μM protein (or buffer only), 5x SYPRO orange and 1 to 0.002 mM ADP-ribose (or water only). Each condition was repeated four times, while the no-ADP-ribose conditions were repeated eight times. The PCR plate was sealed with a Microseal ‘B’ seal (Bio-Rad, MSB1001) and spun to remove air bubbles. The temperature ramp was performed using a BioRad CFX 384 qPCR instrument with fluorescence monitored using the FRET channel from 25 to 95°C at a rate of 1°C/min. Plots of raw RFU values are shown in S10 Fig. T_ma values were calculated online using DSFworld using fitting model 1 [66 ].