# IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2

Elsa Berliocchi, Cercina Onesto, Gilles Pagès, Philippe Lenormand, Roser Buscà

► To cite this version:

Elsa Berliocchi, Cercina Onesto, Gilles Pagès, Philippe Lenormand, Roser Buscà. IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2. Methods, 2025, 243, pp.1-15. 10.1016/j.ymeth.2025.08.004. hal-05230298

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# IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2☆

Elsa Berliocchi<sup>a,c</sup>, Cercina Onesto<sup>a,b</sup>, Gilles Pagès<sup>a</sup>, Philippe Lenormand<sup>a</sup>, Roser Buscà<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

IF-CRIB

IF-Express protocol

Immunofluorescence

3D-printing

ERK2

PROTAC

dTAG

dTAG-13

FKPB12<sup>F36V</sup>

ERK quantity

## ABSTRACT

Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a knock-in strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose-response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models.

## 1. Introduction

The detection of cellular proteins using specific antibodies conjugated to fluorescent dyes (immunofluorescence), followed by fluorescence microscopy, is an essential approach for studying protein expression, subcellular distribution, colocalization with other cellular components, and dynamic changes under specific conditions [1,2]. This technique is widely applied to both cultured cells and tissue samples. Primary antibodies can be directly conjugated to fluorochromes or indirectly, requiring an additional incubation step with fluorochrome-labeled secondary antibodies.

For immunofluorescence studies on adherent cells, they are typically cultured on specially treated surfaces such as coated plastic or glass

coverslips. Commercially available devices, including plastic multi-well plates [3,4] and lab-on-a-chip systems [5], facilitate cell culture at different scales and are specifically designed for immunofluorescence assays. These systems enable direct detection and mounting from the culture surface, allowing for multiplexed analyses on the same slide, including differential drug treatments, transfections, or infections with various DNA constructs. However, such devices can be costly, particularly for large-scale experiments.

An alternative approach for performing multiple immunofluorescence assays with adherent cells involves culturing them on round glass coverslips placed within standard cell culture dishes. Glass is an optimal surface for cell growth due to its biocompatibility, ease of sterilization, and availability in different sizes at a reasonable cost. Additionally,

<footnote><sup>a</sup> Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France</footnote>

<footnote><sup>b</sup> Polytech Nice Sophia, Bioengineering Department, Sophia Antipolis, France</footnote>

<footnote><sup>c</sup> Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, Quebec, Canada</footnote>

<footnote>* This article is part of a special issue entitled: 'Editors Collection - YMETH' published in Methods.</footnote>

<footnote>* Corresponding author.
E-mail address: roser.busca@univ-cotedazur.fr (R. Buscà).</footnote>
individual coverslips can be handled separately, they can be placed in cell culture dished of several diameters, providing flexibility in experimental design, whether for simple or complex conditions. Because of these advantages, we routinely use glass round coverslips in our laboratory for immunofluorescence experiments on adherent cells [6–9].

Various protocols exist for immunofluorescence detection in tissue and adherent cells [10]. In this study, we introduce a rapid and streamlined protocol, termed IF-Express, specifically optimized for highly expressed proteins. This method reduces processing time while preserving cellular morphology through a gentle permeabilization approach. A critical step in immunofluorescence protocols involves multiple washing steps, which typically require the careful transfer of individual coverslips between washing wells or compartments using forceps. The most efficient washes require large volumes, and it is better to wash all samples equally, which is challenging when treating the coverslips one by one. This manual handling demands precision and focus to prevent misidentification, loss of sample order, or accidental breakage of coverslips.

To tackle these challenges, we have created the IF-CRIB, a 3D-printable washing rack designed for efficient handling and organization of round coverslips during washing steps thereby ensuring consistent and standardized processing. The rack has been named IF-CRIB for its shape resemblance to a baby crib. We provide 3D-printable files, enabling users to produce IF-CRIBs as needed, along with detailed methodological guidelines. Although primarily designed for streamlining washes, the IF-CRIB is also ideal for incubation in procedures such as staining protocols or short-term treatments and fixation of numerous coverslips. Additionally, we present an optimized immunofluorescence protocol, named IF-Express, which substantially decreases the overall experiment time.

As an illustrative example of IF-CRIB's application, we focused on an aspect of our ongoing research project investigating the role of ERK quantity in ERK signaling. This involved generating cells that express a degradable ERK2 protein variant, designated ERK2-dTAG, through an *in cellulo* knock-in procedure. The system PROteolysis-TTargeting Chimera (PROTAC) system [11,12] enables the degradation of the target protein upon treatment with a specific compound, dTAG-13 [13]. Obtaining these cells requires the screening of numerous cell clones, followed by their meticulous characterisation, which we efficiently conducted using immunofluorescence experiments with the IF-Express protocol and the newly developed IF-CRIB device.

ERK1 and ERK2, collectively known as ERK1/2, are isoforms of ERK-MAP kinases that play crucial roles in cell biology and development, including cell proliferation, adhesion, migration, differentiation, and survival [14–18]. As effector kinases in the ERK signaling cascade, ERK1/2 have hundreds of identified substrates [19,20]. This pathway is activated by extracellular ligands, such as various growth factors, which bind to specific receptors, subsequently triggering the activation of a cascade involving Ras, Raf and MEK1/2 which phosphorylate ERK1/2 resulting in their activation [21]. Upon phosphorylation, ERK1/2 translocate into the nucleus, where they access numerous nuclear substrates, including transcription factors [22].

Dysregulation of the ERK signaling pathway is commonly observed in a variety of cancers, where its overactivation contributes to tumorigenesis. Consequently, targeted inhibition of ERK has become a key therapeutic strategy, particularly in the treatment of melanoma [23]. However, cancer cells frequently acquire resistance to ERK inhibitors, which ultimately highly contributes to disease relapse [24].

While ERK kinase activity is the principal driver of its downstream signaling, emerging evidence suggests that ERK protein levels also play a critical role in modulating ERK pathway function. For example, amplification of the ERK2 gene has been associated with lung cancer progression [25], and elevated ERK2 expression levels have been linked to drug resistance in lung cancer cells [26]. Our recent findings demonstrate that increased ERK levels potentiate ERK signaling and that the alanine-rich N-terminal domain of ERKs, referred to as NTARs, are

crucial for translation initiation, thereby regulating ERK protein abundance, as revealed through a reporter gene system [27].

Our next objective is to first establish a cellular model that allows precise regulation of ERK protein levels and subsequently investigate the combined effect of ERK quantity reduction and ERK kinase inhibition. This approach aims to determine whether lowering ERK protein quantity decrease potentiates the efficacy of ERK inhibitors currently used in cancer therapy.

To develop a cellular system in which ERK levels can be precisely regulated, we used NIH3T3 cells, a well-characterized model widely used in cell cycle research [28] and employed in PROTAC (PROteolysis-TTargeting Chimera) approach [12,29] (see Scheme 1). In this system, the protein of interest is selectively degraded because it is fused to a degradation tag (dTAG) which binds to the dTAG-13 compound (see Experimental Procedures and Results sections for detailed description of the concept. Upon addition of dTAG-13 to the culture medium, the recombinant tagged-protein undergoes rapid proteosomal degradation [12].

Here we describe the use of the IF-CRIB device and IF-Express protocol for screening and characterization of NIH3T3 cell clones expressing ERK2-dTAG through a knock-in strategy, as an example-case. Immunofluorescence experiments were performed at multiple steps. Both, the IF-CRIB and protocol were highly suitable for handling a large number of coverslips. Additionally, we analyzed ERK2-dTAG nuclear translocation in response to ERK pathway activation.

By providing the 3D-printable design of the IF-CRIB as part of this publication, we aim to facilitate its adoption and independent use by the broader scientific community.

## 2. Results and discussion

### 2.1. The IF-CRIB system conception

Immunofluorescence (IF) is a core technique in our research for analyzing both endogenous and overexpressed proteins in adherent cells. To support these experiments, we routinely utilize multiple glass coverslips, which provide an optimal surface for cell adhesion and growth while minimizing background fluorescence. These coverslips are typically placed within standard culture dishes during cell preparation and staining procedures.

The use of a large number of coverslips serves multiple experimental purposes, including the screening of numerous cell clones, as demonstrated in this study, as well as the testing of various antibodies, assessment of different transfection conditions, and optimization of drug treatments, among other applications. They can be manipulated independently in various experimental setups and are more affordable than other commercially available devices, such as individually wrapped slides from different manufacturers where each well (coverslip/condition) cannot be handled independently.

To ensure reproducibility and statistical reliability, experiments are typically performed in duplicate or triplicate, further increasing the number of coverslips required. Moreover, thorough washing with large volumes of buffer is a critical component of any immunofluorescence protocol, as it helps to minimize non-specific labeling and improve signal specificity.

To streamline the washing process and facilitate precise handling of a high large number of coverslips without altering their order, we developed a specialized device termed IF-CRIB. This plastic rack is robust device designed to hold multiple round glass coverslips in a defined arrangement, analogous to immunohistochemistry slide racks that accommodate rectangular slides for parallel processing. Unlike traditional racks, the IF-CRIB is smaller and specifically engineered to support round coverslips ranging from 12 to 20 mm of diameter, with 14 mm coverslips being used routinely in our experiments. The IF-CRIB is placed into a small container filled with the 75 mL pf washing buffer, such as phosphate-buffered saline (PBS) or water, but it can also contain
<figure><img src="image_4.png" /><figcaption>**Scheme 1.** The ERK2 PROTAC degradation system. Schematic representation of the ERK2-dTAG degradation system using the dTAG-13 compound, which binds ERK2-dTAG on one hand and recruits the E3 ligase on the other hand.</figcaption></figure>

treatment solutions for live-cell assays, or staining reagents for fixed-cell protocol. Additionally, the IF-CRIB facilitates synchronized treatment of multiple live-cell samples with a defined stimulus, followed by immediate fixation in the same container, making it particularly useful for time-sensitive experimental setups. Previously, in our laboratory, each coverslip was individually washed using forceps in a large volume of PBS or putting the coverslips back into the initial wells. This procedure frequently led to errors in sample order, as well as loss or breakage of the coverslips. The use of the IF-CRIB significantly reduced these issues.

The design of the IF-CRIB was inspired by a previously used custom-made laboratory tool, itself modeled after conventional slide racks employed in immunohistochemistry workflows in research laboratories and clinical settings. We developed a user-friendly, 3D-printable plastic model optimized for modern lab use. The device consists of two flat square supports connected by two parallel cylindrical rods, each featuring 1.2 mm notches that securely hold round coverslips in place, forming a structured and efficient washing rack (Fig. 1A).

The IF-CRIB can accommodate up to 15 round coverslips with diameters ranging from 8 to 20 mm. The version used in our experiments is tailored for 14 mm coverslips, but coverslips from 12 to 18 mm diameter can be used. The rack is 3D-printed as a single unit, requiring minimal post-processing beyond separating the components for final assembly (Fig. 1B). Using the provided STL file, the entire device can be printed in one step with advanced 3D printers. Once assembled, the IF-CRIB fits into a compact container with minimum internal dimensions of 7 × 5 × 3.5 cm, which is subsequently filled with washing buffer for experimental use (Fig. 1C).

An important contribution of this manuscript is the provision of printable files for the IF-CRIB, a tool that is highly useful in our laboratory. The 3D-printable files, including details of the 3D printer specifications and the plastic material used, are available in the Experimental section. The 3D printing files are in the STL format (provided STL files 1 and 2, and represented in supplementary data 3), STL format is compatible with various types of 3D printers and plastic materials, allowing laboratories to print their own IF-CRIBs in any desired

color (Fig. 1D). The use of different colors can aid in experimental organization, particularly when handling multiple coverslips under different conditions such as live-cell treatments with multiple drugs, kinetic studies, different transfection or infection conditions, or varying fixation methods. The IF-CRIB is designed for easy manipulation with appropriate forceps, allowing efficient handling of multiple coverslips while preserving their order throughout the procedure. It is noteworthy that any facility that ensures order preservation and avoids coverlips losses, breakings or mistakes, can lead to a gain in experiment time.

Alternative 3D designs could be developed to increase the number of notches, although the current CRIB size is optimal for ease of handling in our case.

The IF-CRIB avoids the need to insert and remove each coverslip from individual culture wells during washing or treatment steps. Concerning the washing buffer container, it is slightly larger than the IF-CRIB and could range from old slide boxes to tip boxes commonly available in the lab, or it can emerge from a 3D printed file also conceived and described in this manuscript (STL file 3 and Supplementary data 3). 75 mL of washing buffer is used for every washing step, it corresponds to the largest volume fitting in the washing container and which completely covers the IF-CRIB with the positioned coverslips.

In a nutshell, this washing procedure is designed for round glass coverslips and offers a more economical alternative to commercially available, more sophisticated devices for immunofluorescence. For high-throughput screening, many research labs or industrial applications use 96-well plates that require automatic slide washers. In contrast, the IF-CRIB is ideal for the conditions of research laboratory analyses, such as clone screening, evaluating the localization of immunofluorescence signals, colocalization with other markers. With IF-CRIB washes are very homogeneous across experiments.

We can also use the CRIBS for treating multiple coverslips with drugs for short period of time at 37 °C in growing medium or for organizing the coverslips after multiple cell transfections and detecting overexpressed genes.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** The IF-CRIB The design of the two distinct components of the IF-CRIB, as visualized from the «STL» file and the corresponding technical drawing, consists of an assembly of two square parts and two cylindrical elements (A). The process of 3D printing of the IF-CRIB from the loaded «Stl» file, along with the fully printed structure upon completion (B). Images of the assembled IF-CRIB, incorporating round glass coverslips and a small 3D-printed cuvette filled with PBS for washing procedures (C). IF-CRIB. Design image for 3D printing of the washing cuvette and three IF-CRIBs in different colors are presented, allowing for the differentiation of experimental conditions (D).</figcaption></figure>

## 2.2. The IF-Express protocol

Immunofluorescence (IF) protocols vary across laboratories, often tailored to specific antigens and fixation methods [30]. Some are complex and time-consuming. The IF-Express protocol presented here offers two key advantages: reduced processing time and improved reproducibility in our hands, making it ideal for high-throughput clone screening and subsequent characterization.

In this study, we applied IF-Express to paraformaldehyde (PFA)-fixed

cells expressing well-characterized, easily detectable antigens, such as HA-tagged proteins. Although this report focuses on these specific conditions, we have successfully extended the protocol to other fixation methods, including methanol and methanol-acetone (at -20 °C for 2 min), as well as to additional antibodies targeting epitope-tags such as Myc and FLAG. Furthermore, the protocol has been used with antibodies against endogenous proteins, including ERK, phospho-ERK, tubulin, and E-cadherin, among others (not shown). IF-Express has also been applied across a variety of cell systems, including HeLa, HEK-293, renal cell
carcinoma, breast cancer, lung cancer, and melanoma cell lines (data not shown). A schematic overview is shown in Flowchart 1, and detailed conditions are provided in the Experimental Section. (legend of the flow chart to be added: Detailed protocol of the IF-Express procedure, out-lining all steps from cell fixation to the mounting of round coverlips onto slides. Illustrations of selected steps are shown on the right.) Briefly, cells are cultured on sterilized round coverslips within dishes, fixed with 4% PFA (or alternatively, methanol or methanol/acetone at -20 °C), and washed thoroughly within dishes with PBS to remove fixative residues.

Primary antibody incubation is performed in a Block-Perm solution (3 % BSA, 0.1 % saponin in PBS) by depositing coverslips up-side-down onto drops on parafilm, combining blocking and permeabilization in one step. This greatly reduces experiment duration while maintaining effi-ciency. In our protocol we incubate the primary antibody for 20 min, but this incubation time needs to be optimized according to the antibody used, for example anti-tag antibodies used in overexpressed proteins might only require a 10-minute incubation. After two washes using a large PBS volume (75 mL in the designed container) thanks to the IF-CRIB system, fluorophore-conjugated secondary antibodies are applied via drops on parafilm, followed by additional rinses with abundant PBS and a final the same volume volume distilled water wash to enhance clarity for microscopy. Each time that a washing step is carried out (the number of washing steps might be modified according to the antibody used), the IF-CRIB is gently shaken in the washing container, the washing buffer is completely changed once. To improve washing

efficiency, residual antibody solution on each coverslip is dried onto absorbent laboratory paper after each incubation step, prior to placing the coverslip into a IF-CRIB notch. Secondary antibody incubation time can be reduced if high affinity antibodies are used, always verifying the lack of background due to this antibody. Here the conditions are opti-mized for our purpose. Other antigen proteins and antibodies might require different fixation methods and antibodies incubation times. Nuclei are counterstained with DAPI during mounting.

The full protocol, from fixation to mounting takes approximately two hours, allowing rapid assessment of positive clones for downstream applications. In practice, cells are grown in 35-, 60-, or 100-mm dishes with two glass coverslips. Glass coverslips with fixed cells are processed for IF or kept in cold PBS to reproduce the experiment later, while the remaining culture is used for Western blotting, freezing, or expansion. Only IF-positive clones are proceeded to Western blot confirmation.

The IF-Express protocol involves a minimum of six washing steps, making it particularly well-suited to the IF-CRIB system, which facili-tates high-throughput handling. In this study, we analyzed 90 clones in duplicate, demonstrating the efficiency and scalability of this approach. As a result of its reliability and practicality, this optimized workflow has been adopted as a standard procedure in our laboratory.

We would like to emphasize that, in this study, only one primary antibody (anti-HA tag antibody) was used. Consequently, the same secondary antibody was applied in all cases. When processing multiple coverslips stained with different antibodies, using several IF-CRIB

<figure><img src="image_6.png" /><figcaption>Flowchart 1. IF-Express protocol.</figcaption></figure>
devices differentiated by distinct colors may be advantageous to prevent antibody cross-contamination during the washings. The number of washing steps as well as the washing volume can be adapted to the antibody used.

### 2.3. Applications of the IF-CRIB and the IF-Express for the screening and characterization of in cellulo knock-in cells expressing a degradable form of ERK2 (ERK2-dTAG)

One of our overreaching research goals is to test whether lowering ERK protein levels can improve the effectiveness of kinase inhibitors targeting the ERK pathway in cancer treatment. Hence, we constructed a cell system endogenously expressing a degradable form of ERK2 (ERK2-dTAG). In this process, the immunofluorescence approach was continuously employed (e.g., for cell clone screening and characterization). It is worth noting that we used the application on ERK2 as an example of a process requiring multiple immunofluorescence steps and thereby the IF-CRIB and protocol.

To initially achieve this, we chose to use the PROTAC (PROteolysis-TTargeting Chimera) system which uses the bifunctional molecule dTAG-

13 [11,12,29]. This molecule binds the FKPB12<sup>F36V</sup> dTAG protein fused to the target protein and simultaneously recruits an E3 ubiquitin ligase via its CRBN binding domain, leading to ubiquitination and proteasomal degradation [11,13] (Scheme 1). The PROTAC system has already been used to target the BRAF<sup>V600E</sup> mutant, a common oncogenic driver in melanoma, thereby indirectly affecting the ERK pathway [31]. However, it has not yet been directly applied to the ERK kinases themselves.

We engineered a homologous recombination vector encoding FKPB12<sup>F36V</sup> (that we will name dTAG), fused to two HA-tags for detection, along with an intron and an IRES-driven neomycin resistance gene. This construct was integrated into ERK2's 8th exon (Scheme 2 A). Simultaneously, NIH3T3 cells were co-transfected with a CRISPR-Cas9 plasmid (Supplementary data 1) to increase knock-in efficiency, as the CRISPR-Cas9 guided nuclease introduced double-strand breaks in the genomic DNA (Scheme 2A). This facilitated dTAG (FKPB12<sup>F36V</sup>-2HA sequence integration into the endogenous *erk2* locus (see Scheme 2A and Supplementary data 1 for CRISPR sequences). Since there is no promoter driving the expression of the neomycin resistance gene (Neo<sup>R</sup>) in this plasmid, NeoR is only expressed when the insert is integrated downstream of the ERK2 endogenous promoter. NIH3T3 cells were chosen for

## A Genomic *erk2* fragments cloned in the Empal cloning vector

<figure><img src="image_7.png" /></figure>

## B The FKPB<sup>F36V</sup>-2HA and neomycin resistance fragments into mouse *erk2* gene

<figure><img src="image_8.png" /><figcaption>**Scheme 2.** Simplified map (not drawn to scale) of the homologous recombination system designed to introduce the FKPB12<sup>F36V</sup>-2HA and neomycin resistance (Neo<sup>R</sup>) insert into the 8th exon of mouse *erk2* locus. A) Schematic representation of the genomic *erk2* fragments cloned in the Empal cloning vector. Mouse *erk2* pre-mRNA is represented in green, mouse *erk2* gene in blue and the homologous recombination left and right arms in black. Green boxes represent the 8th *erk2* exon (126 bp, including 9 bp of 3'UTR) and the 3'UTR is represented by a green striped box. Red arrow indicates the stop codon at which the recombinant fragment shown in B will be introduced. Back arrow indicates this same site. B) Schematic representation of the FKPB12<sup>F36V</sup>-2HA and Neomycin resistant (Neo<sup>R</sup>) fragments introduced into the mouse *erk2* gene. The left and right homologous recombination arms shown in A of 1100 bp and 758 bp respectively, are represented. Mouse *erk2* pre-mRNA is represented in green, the dTAG-2HA and Neo<sup>R</sup> cassette inserted into the genome after recombination is represented in blue.</figcaption></figure>
their well-characterized ERK expression and activation control, ease of cell cycle modulation, and large cytoplasm, which aids for immunofluorescence analysis. It is important to note that the construct encoding the protein of interest fused to the FKBP12<sup>F36V</sup> dTAG is inserted into the endogenous locus of the gene of interest via a knock-in strategy, ensuring physiological expression levels.

## 2.4. Screening of clones expressing ERK2-dTAG-HA in NIH3T3 cells

Following three weeks of neomycin selection, 90 clones were screened via immunofluorescence with the IF-Express protocol and the IF-CRIB, using an anti-HA antibody. HA immunofluorescence confirmed ERK2-dTAG-HA expression in selected clones, predominantly localized in the cytoplasm in serum-cultured conditions. Fig. 2 illustrates HA

<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Selection of clones expressing ERK2-dTAG-HA in NIH3T3 cells, using the IF-CRIB and the IF Express protocol. Immunofluorescence images of four independently grown NIH3T3 cell clones (clones 1–4 out of 90 selected) following neomycin selection. Cells were labeled with an anti-HA antibody (1/100 dilution) and a secondary antibody conjugated to Alexa-488 (green 1/400). Nuclei were stained with DAPI incorporated in the mounting medium Scale bar = 80 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
detection in two positive (clones 1 and 4) and two negative clones (clones 2 and 3).

The experiment was performed on all 90 neomycin-selected clones, revealing a range of fluorescence intensities. Only clones with stronger HA signals, such as clones 1 and 4 (representing approximately one-third of the total) were retained and cryopreserved for further characterization. Another one-third exhibited intermediate labeling, lower than the high-intensity clones but higher than the one-third with absent or very low HA detection, potentially indicating cells with only one recombined allele (not shown). We then assessed the functional degradation of ERK2-dTAG-HA in selected high-expressing clones following treatment with the PROTAC molecule dTAG-13. We analysed the positive clones in duplicate, and we observed indistinguishable signals for each clone duplicate.

We consider that the use of the IF-CRIB, which enables the simultaneous washing of multiple coverslips in a container with a large volume (75 mL) of PBS, contributes both to improve washing efficiency and to save handling time. This is very useful when processing a large number of conditions and ensures that all coverslips are washed under identical conditions and with equal volumes. In all positive clones ERK was mainly localized in the cytoplasm, as no proliferative stimulation pulse or ERK dephosphorylation treatment had been applied (Fig. 2).

## 2.5. Characterization of ERK2-dTAG expressing cells: Dose-response and time kinetics of target protein degradation upon dTAG-13 treatment

To evaluate the functionality of the PROTAC system, we assessed the efficacy of the dTAG-13 drug and determined its effective dose by treating the previously identified positive clones (clones 1 and 4) with increasing concentrations of dTAG-13 (from 3 nM to 300 nM) in the culture medium for 1 h, as recommended in the literature [29]. Control cells were treated with DMSO, the drug solvent. Subsequently, cells were fixed and subjected to the IF-Express immunofluorescence protocol, utilizing the IF-CRIB, and the anti-HA antibody (Fig. 3).

Fig. 3A presents the immunofluorescence images of clone 1 as a representative example, stained with the anti-HA antibody. The same experiment with clone 4 gave very similar results (not shown). Under DMSO control conditions, HA labeling was strong, as well as in the 3 nM dTAG-13 condition. A reduction in HA signal was observed at 10 nM, and the protein was markedly less visible at 30 nM. At 100 nM and 300 nM, the HA signal was nearly undetectable. The quantification of this representative experiment is shown in the corresponding graph on the right, where fluorescence intensity is plotted according to the dTAG-13 drug concentration. Cell extracts from similarly treated samples were analyzed by Western blotting using both an anti-phospho-ERK antibody after a 15-minute treatment with pervanadate (PerV = Sodium orthovanadate 100 µM + 3 mM H₂O₂) to maximize ERK2 phosphorylation [32], and an anti-HA antibody. Signals obtained with the anti-HA and anti-phospho-ERK antibodies were similar, indicating that the knocked-in ERK2-dTAG protein is phosphorylated, and can thus be activated. The Western blot results (Fig. 3B) confirmed the findings obtained from the immunofluorescence experiment using the IF-Express (Fig. 3A). To note, ERK2-dTAG appeared at around 55 kDa resulting from the addition of ERK2 (42 kDa) and the dTAG (13 kDa). It is worth mentioning that clone 1 and 4 only presented ERK2-dTAG-HA expression and not that of endogenous ERK2 (not shown).

In conclusion, treatment with 30 nM dTAG-13, resulted in a marked reduction of ERK2-dTAG levels, while 100 nM led to near-complete depletion of the protein. Immunofluorescence staining using the rapid IF-Express protocol including IF-CRIB was extremely reproducible across experiments. The ability to uniformly wash and manipulate coverslips with ease was a key factor contributing to the consistency of the results. These findings could be extended to all the clones analysed in duplicate by rapid IF-Express protocol including IF-CRIB use.

To further investigate the temporal dynamics of ERK2-dTAG-HA protein degradation following dTAG-13 treatment, we selected 100

nM as the optimal concentration based on the previous results shown in Fig. 3. Clone 1 cells were incubated with 100 nM dTAG-13 for 10, 20, 30, and 45 min. After extensive PBS washes, cells were fixed and processed for the IF-Express protocol using the IF-CRIB for washings and the anti-HA antibody for detection. The images in Fig. 4A illustrate a progressive decline in ERK2-dTAG-HA expression, beginning at 10 min post-treatment, where labeling was comparable to the DMSO control. By 20 min, the HA signal was significantly reduced, and by 30 min, it had nearly disappeared. The signal at 45 min post-treatment remained indistinguishable from that at 30 min. The quantification of this experiment is shown in the corresponding graph on the right, as previously described. Immunofluorescence experiments with clone 4 revealed very similar results.

Additionally, cell extracts from similarly treated samples were analyzed by Western blotting using both an anti-phospho-ERK antibody after a 15-minute treatment with pervanadate to maximize ERK2 phosphorylation, and an anti-HA antibody. Signals obtained with the anti-HA and anti-phospho-ERK antibodies were similar, indicating that the ERK2-dTAG is phosphorylated and can thus be activated. The Western blot results corroborated the findings obtained by immunofluorescence (Fig. 4A).

To summarize, in ERK2-dTAG-HA-expressing cells, treatment with 100 nM dTAG-13 for 30 min to 45 min resulted in the near-complete depletion of the ERK2-dTAG protein, as confirmed by both immunofluorescence and Western blot analysis (Figs. 3 and 4).

It is important to emphasize that experiments performed using the IF-Express protocol with IF-CRIB system yielded results very similar to those obtained by western immunoblot blotting. While western blotting is considered as a highly quantitative method for measuring protein levels, immunofluorescence is generally considered as a more qualitative and less quantitative approach. However, our optimized immunofluorescence procedure using the IF-CRIB and protocol proved to be nearly as informative, as it offers reliability comparable to western blotting while significantly reducing experimental time.

## 2.6. ERK2-dTAG sub-cellular localization upon ERK activation

A defining characteristic of ERK proteins is their nuclear translocation upon activation by phosphorylation. Both ERK1 and ERK2 exhibit identical behavior [14,22]. Once inside the nucleus, ERKs phosphorylate nuclear substrates involved in various cellular processes [20].

To determine whether the incorporation of the dTAG-HA protein sequence into endogenous ERK2 affected the kinase's ability to translocate into the nucleus and carry out its function, we first serum-starved clones 1 and 4 for 24 h. Cells were then either left untreated (NS) or stimulated with pervanadate (PerV = sodium orthovanadate 100 µM + 3 mM H₂O₂) for 15 min prior to fixation. As mentioned, pervanadate is a potent phosphatase inhibitor that prevents ERK dephosphorylation, thereby maintaining kinase activation. This sustained activation has been shown to promote the nuclear translocation of ERKs [22].

Following fixation, cells were subjected to the IF-Express immunofluorescence protocol using IF-CRIB and an anti-HA antibody (Fig. 5). In serum-starved, non-stimulated cells (NS), ERK2-dTAG-HA was primarily localized in the cytoplasm, as indicated by a cytoplasmic HA signal and the absence of nuclear staining (Fig. 5A). In contrast, pervanadate treatment (PerV) led to HA labeling within multiple nuclei (Fig. 5, clone 1). Magnified images further confirm the nuclear accumulation of activated ERK2-dTAG (Fig. 5B). While the nucleus of serum-starved cells appears devoid of ERK2-dTAG, the protein is clearly localized in the nuclei of pervanadate-treated cells. These findings demonstrate that the endogenously expressed, degradable form of ERK2 fused to an HA tag retains its ability to translocate to the nucleus, a critical step for ERK functional activity. Thus, we successfully generated NIH3T3 cells expressing ERK2-dTAG, in which the newly synthesized endogenous ERK2-dTAG—capable of nuclear translocation—could be efficiently
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Dose-response of dTAG-13 drug to reduce ERK2-dTAG expression, using the IF-CRIB and the IF Express protocol. **A)** Immunofluorescence images of ERK2-dTAG NIH3T3 clone #1 cells after treatment with the dTAG-13 drug for 1 h at varying concentrations (3 nM, 10 nM, 30 nM, 100 nM, and 300 nM) diluted in DMSO and added to the culture medium (0.1 % DMSO final). The same volume of DMSO was used as a control condition without dTAG-13. The labeling was performed using an anti-HA antibody, followed by incubation with an Alexa-488-conjugated secondary antibody as in Fig. 2. Nuclei were stained with DAPI. (Scale bar = 80 µm). Global fluorescence of each HA-stained image was measured using the EVOS M-5000 imaging system, and relative fluorescence units (RFU) were plotted, with the Y-axis representing RFU and the X-axis representing the dTAG-13 drug concentration. The images and quantification are representative of several analysed clones such as clone #4. **B)** Western blot analysis of 30 µg of Laemmli extracts from clone #1 treated for 1 h with the indicated concentrations of dTAG-13. Cells were stimulated for 15 min with pervanadate (PerV) (100 µm) to promote ERK phosphorylation. The membrane was incubated with anti-HA and anti-phospho-ERK antibodies at respective concentrations of 1/2000 and 1/2000. Secondary anti-mouse and anti-rabbit-conjugated antibodies were at 1/7000 dilution each. Detection of HSP90 (dilution of 1/200) protein was used as a loading control. Molecular weight figures on the left. ERK2-dTAG appears at 55 kDa resulting from the addition of ERK2 (42 kDa) and the dTAG (13 kDa).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Kinetics of ERK2-dTAG disappearance upon dTAG-13 treatment, using the IF-CRIB and the IF Express protocol. **A)** Immunofluorescence images showing the time kinetics of dTAG-13 drug treatment at a 100 nM concentration in clone #1 ERK2-dTAG NIH3T3 cells. Treatment was performed at the following time points: 10, 20, 30, and 45 min. Treatment with the same volume of DMSO was used as a negative control. (Scale bar = 80 µm). Global fluorescence of each HA-stained image was measured using the EVOS M-5000 imaging system, and relative fluorescence units (RFU) were plotted, with the Y-axis representing RFU and the X-axis representing the time of dTAG-13 treatment. The images and quantification are representative of four analysed clones such as clone #4. **B)** Western blot analysis of 90 µg of Laemmli extracts from clone #11 treated with 100 nM dTAG-13 for various time points. DMSO was used as the negative control. Cells were stimulated for 15 min with pervanadate (PerV) to induce ERK phosphorylation. The membrane was incubated with anti-HA and anti-phospho-ERK antibodies as in Fig. 3B. Detection of HSP90 protein was used as a loading control. ERK2-dTAG appears at 55 kDa resulting from the addition of ERK2 (42 kDa) and the dTAG (13 kDa).</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 5.** ERK2-dTAG sub-cellular Localization Upon ERK Activation, using the IF-CRIB and the IF Express protocol. Immunofluorescence images of clone #1 ERK2-dTAG NIH3T3 cells that were serum-starved for 24 h and stimulated or not (NS) for 15 min with pervanadate (PerV) to induce ERK phosphorylation and nuclear translocation. For ERK2-dTAG detection, fixed cells were labeled with an anti-HA antibody followed by an Alexa-488-conjugated secondary antibody as in Fig. 2. DAPI dye was used to stain the nuclei. Scale Bar = 80 µm (panel A). Panel B shows higher magnification images of a single non-stimulated cell (NS, left) and two stimulated PerV-stimulated cells (middle and right). Arrows indicate the HA labeling corresponding to the nucleus. Scale bar = 20 µM.</figcaption></figure>

degraded within from 30 to 45 min following treatment with 100 nM of the degrader drug dTAG-13.

The aim of our study was to generate cells in which ERK quantity could be modulated to determine whether reducing ERK quantity increases the effectiveness of ERK inhibitors currently used as targeted cancer therapies. Approximately 90 % of cancers involve mutations in components of the ERK signaling pathway, including the tyrosine kinase receptor, Ras, or Raf proteins, which can be considered oncogenic [33]. Several pathway inhibitors, including the MEK inhibitor trametinib and B-Raf inhibitor dabrafenib, are used in targeted cancer therapy to inhibit ERK pathway activity. We have recently shown that elevated ERK levels potentiate ERK signaling, and that the alanine-rich N-terminal domains of ERKs (NTARs) play a pivotal role in translation initiation and, consequently, in regulating ERK protein abundance [27]. Thus, both ERK activity and quantity are key factors in ERK function and thereby cancer biology.

The obtained ERK2-dTAG transgenic NIH3T3 cells still express endogenous ERK1. Our lab and others have reported that ERK1 and ERK2 are functionally redundant [14,19,34,35]. We have previously shown that when one ERK isoform is knocked-down by specific shRNAs, the other isoform becomes overactivated, allowing for global ERK activation and proper cell proliferation [36]. For this reason, our proof of concept ought to be conducted in cells where endogenous ERK1 has been knocked out. We are currently working on obtaining ERK1-knockout cells, in which endogenous ERK2 will be replaced by ERK2-dTAG-HA using the same procedure described in this work. This tool will enable us to investigate whether reducing ERK levels potentiates the effect of targeted ERK pathway inhibitors used in cancer therapy.

In summary, this study highlights the successful application of the IF-CRIB washing device for the screening of NIH3T3 cell clones in which endogenous ERK2 was modified using knock-in technology. When combined with the IF-Express immunofluorescence protocol, the IF-
CRIB enables a substantial reduction in processing time, particularly advantageous when working with large numbers of coverslips. This increased efficiency makes the system especially well-suited for high-throughput clone analysis. As a result, both IF-CRIB and IF-Express have become standard tools in our laboratory for all immunofluorescence-based experiments.

### 3. Conclusions

In this study, we developed a novel device, the IF-CRIB, designed to facilitate rapid and efficient immunofluorescence experiments using the IF-Express protocol. This method was applied to characterize newly generated cells expressing a degradable form of ERK2, known as ERK2-dTAG. Our findings are highly relevant as they: (i) provide the scientific community with a new, cost-effective, robust and user-friendly, 3D-printable device optimized for immunofluorescence analysis on multiple round glass coverslips, (ii) establish a rapid and efficient immunofluorescence protocol that yields reliable results and helps to avoid coverslips losses, mis-ordering and breakings, particularly advantageous when processing multiple conditions, and (iii) validate a knock-in system for generating a degradable version of ERK2, which, once introduced into NIH3T3 cells, represents a valuable tool to investigate whether ERK protein levels influence ERK signaling, a finding that could prove critical in identifying compounds capable of regulating ERK quantity and potentially enhancing the efficacy of ERK-targeting inhibitors.

### 4. Experimental section

#### 4.1. Cell culture, transfection, and clone generation

NIH 3 T3 mouse fibroblasts (ATCC #CRL-1658) [36] were cultured in DMEM with GlutaMAX medium (GIBCO #31966-021), supplemented with 7% fetal calf serum (FCS) (Dutscher #S005I90003) and penicillin/streptomycin (GIBCO #15140-122) [100 IU (50 µg)/ml], under a humidified atmosphere of 5% CO₂ at 37 °C.

Fibroblasts were seeded in 100-mm diameter culture dishes containing two 14 mm round glass coverslips (previously sterilized by a microwave pulse of 2 min at maximum intensity and were transfected with 9 µg of plasmid DNA containing 1 µg of CRISPR-Cas9 construct targeting the end of *erk2* exon 8 and 8 µg of homologous recombination vector described in the cloning strategies part of the experimental section. The homologous recombination vector included a neomycin resistance cassette (Neo®) downstream of a powerfull IRES in absence of a promoter sequence.

Transfections were performed using a home-optimized polyethyleneimine (PEI) method, employing Polyethylenimine "Max" (Polysciences, Inc #24765) as the transfection reagent, prepared as a 1 mg/ml solution (pH 7.0) in water. PEI was complexed with plasmid DNA at a ratio of 2.5 µl PEI per 1 µg of DNA. The PEI/DNA complexes were formed by incubating PEI and plasmid DNA in 150 mM NaCl for 30 min at room temperature (RT). The final transfection mixture constituted 1/10 (v/v) of the total culture medium volume containing FCS. The PEI/DNA precipitate was added dropwise to the cell culture medium, and the medium was replaced 16 to 24 h post-transfection [27].

The DNA quantity and transfection mix volume were adjusted according to the surface area of the culture dish.

Cells were immediately and continuously treated with Neomycin (Gibco™, #10131027), diluted 1:100 in the culture medium. After several weeks, clones began to emerge. A total of 90 clones were selected and initially expanded in 48-well plates, followed by further expansion in 6-well plates for rapid immunofluorescence screening. Among these, 30 clones exhibited high positive labeling. To accelerate the effect of the Neomycin, cells were cultivated in 1% of FCS for 48 h at the beginning of the treatment.

For dose response and kinetic experiments, we selected two clones

from the high labelling group (clone 1 and clone 4). Clone 1 is shown in this study.

The highest HA-expressing clones (30% of total clones) were further validated by Western blot using an anti-HA antibody (Eurogentec, #16B12) and an anti-phospho-ERK antibody (Cell Signaling Technologies, #4370) following pervanadate treatment to enhance ERK phosphorylation. Pervanadate was made by adding extemporaneously H₂O₂ to a sodium orthovanadate solution (final concentration: Sodium orthovanadate 100 µM, Merck #450243 + 3 mM of H₂O₂, Merck #H1009). When a resistant clone was isolated and amplified with at least two coverslips in the plate, and achieved confluence, cells were fixed and left in the well filled with PBS, to wait for the rest of the clones to achieve confluence and be fixed. The selected clones were cryopreserved, and for subsequent experiments, clones 1 and 4 were used.

#### 4.2. The dTAG-13 molecule and the PROTAC system

As described in the results and discussion section above, in this work, the PROTAC molecule is named dTAG-13 drug [11,13]. It corresponds to a chemical compound used in the dTAG (Degradation Tag) system for targeted protein degradation. It is a heterobifunctional degrader composed of two key components: the CRBN-Binding Thalidomide moiety which binds to the ubiquitin ligase Cereblon (CRBN), and the FKPB12<sup>F36V</sup> Ligand (AP1867) which specifically binds to a mutant form of FKPB12, known as FKPB12<sup>F36V</sup>, without affecting the wild-type version. dTAG-13 facilitates the selective proteasomal degradation of target proteins that are fused with FKPB12<sup>F36V</sup>. This method provides a valuable alternative to genetic approaches for target validation in drug discovery [12,29]. The dTAG-13 molecule is currently commercially available (Sigma # SML2601).

In this work, the protein of interest is selectively degraded because it is fused to a degradation tag (dTAG) which binds to the dTag-13 compound, corresponding to the FKPB12<sup>F36V</sup> protein. Upon addition of dTAG-13 compound to the culture medium, the recombinant tagged-protein undergoes rapid degradation [12]. It is important to note that the construct encoding the protein of interest fused to the FKPB12<sup>F36V</sup> dTAG is inserted into the endogenous locus of the gene of interest via a knock-in strategy, ensuring physiological expression levels.

#### 4.3. Plasmids, vectors, genomic DNA obtention and CRISPR cloning

To obtain *in cellulo* knock-in cells containing the degradation tag (dTAG) FKPB12<sup>F36V</sup> together with 2HA tags (for easier detection) and the NeoR sequence (for clone screening) in the ERK2 locus, we used the following sequences and plasmids:

The FKPB12<sup>F36V</sup> sequence was obtained from the plasmid GW906 kindly provided by G. Winter [29]. The Plasmid paav sept acceptor to bring the IRES-Neomycin Resistance flanked by loxP sites was purchased from Addgene (Addgene # 25648) and used in Kim et al., in 2008 [37]. The FKPB12<sup>F36V</sup> fragment was amplified with PL-19-08 and PL-19-09 primers (listed in Supplementary data 2) which also reconstituted the beginning of the 3'UTR of mouse *erk2* downstream of the FKPB12<sup>F36V</sup> sequence. To increase knock-in efficiency, genomic DNA was cut on both strands by guided nuclease with CRISPR-Cas9. The guide sequences were cloned into the plasmid PX330 (Addgene #42230) [38]. The 19-nucleotide-long sequence of the guide RNAs was selected using the Broad Institute's website, now named CRISPick (https://portals.broadinstitute.org/gppx/crispick/public). Three guide RNAs were chosen around the TAA stop codon of mouse *erk2*. They allowed homologous recombination with similar efficiency. The CRISPR sequences are specified in Supplementary data 1 and were cloned as annealed primers in the plasmid PX330 cut by BbsI enzyme. All three CRISPR guide sequences demonstrated comparable efficiency, as indicated by the similar number of clones obtained following co-transfection with the homologous recombination vector in clonogenicity assays. For the purposes of this study, we present results obtained using the guide RNA CRISPR
mE2stop-01.

The mouse genomic DNA was purified by lysis of a confluent 10 cm plate of NIH-3 T3 cells with 500 µl of lysis buffer containing 100 mM tris HCl pH = 8, 5 mM EDTA, 0.2 % SDS, 200 mM NaCl and with the addition of 1 % of proteinase K (Sigma #P2908). Buffer containing proteinase K was incubated at 50 °C overnight prior to precipitation with same volume of isopropanol, the pellet was rinsed twice in 70 % EtOH and resuspended in TE. 100 ng of genomic DNA from mouse NIH3T3 cells, were used to isolate an 1865 bp DNA fragment amplified with KOD hot start enzyme (Sigma #71086) in the presence of the primers PL-18–159 and PL-18–162 (Supplementary data 2), according to manufacturer conditions with annealing at 63 °C for with cycles for 2 min. The genomic fragments for the left homology arm containing the position of the m-erk2 stop codon (5') was amplified with primers PL-19–06 and primer 19–07 (Supplementary data 2) which removed the stop codon and generated 3 mismatches in the 16 nucleotides upstream of this stop codon to avoid the recombined sequence to be cut by any CRISPR (red characters on the sequence in Scheme 3). The genomic fragment for homology on the right of m-erk2-stop codon (3') was amplified with primers PL-19–10 and 19–11 (Supplementary data 2).

The golden-gate plasmid acceptor: Empal-ampiR- (BsmBI CTAA-AAGT) has the following sequence in the MCS: CTAAAGAGACGaga-tctCGTCTCGAAGT, the internal BsmBI sites generating CTAA and AAGT cohesive ends. In this plasmid, assembly was performed at once by Golden-Gate cloning with BsmB1 enzyme and T4-DNA ligase as described by Engler et al. [39]. Four PCR fragments previously blunt-cloned in Empal-KanaR [27] were assembled at once: the left genomic arm (primers PL-19-06 and PL-19-07, size 1107 bp), the FKPB12<sup>F36V</sup> fragment (by using the primers primers PL-19-08 and PL-22-49, size 400 bp), the loxP-IRES-EMCV-neo<sup>R</sup>-loxP-SV40polyA, amplified from adggene (Adgene# 25648) with primers PL-22-17 and PL-22-19 (obtaining a size of 1694 bp), and the right genomic arm (primers PL-19-10 and PL-19-11, size 722 bp). The cohesive ends sequences of each fragment were the following: left-arm (CTAA;GGTA), FKPB12<sup>F36V</sup> (GGTA;TGAA), loxP-IRES-NeoR-loxp-SV40polyA (TGAA;CTCC), right-arm (CTCC;AAGT).

4.4. The IF-CRIB

The IF-CRIB was 3D-printed as a single piece containing 4 parts to be
assembled, using the provided STL file 1 (F1 image in Supplementary
data 3) as shown in Fig. 1. This piece consists of two square parts, each
containing two square notches, and two cylindrical components
designed to fit into these notches. Each cylindrical piece contains 15
evenly spaced notches of 1.2 mm. After 8 h of printing (generally
overnight) each of the four components was separated from the printed
block and assembled as shown in Fig. 1. The size of the whole piece was
60 mm Long X 32 mm Large X 28 mm Depth.

The 3D printer used for fabrication was the Ultimaker S5 FDM; however, any 3D printer compatible with «STL» files should work. The software used to create the whole piece for printing was the Cura 4.10 (now the 5.8 is available). The plastic filaments used were PLA/PHA with a 2.85 mm diameter with the print Core AA0.4, available in multiple colors (Fig. 1). Regarding printer settings, the quality setting of 0.1 mm (by default) was used, and infilling was of 70 % and the support was activated with the Extruder 1.

In case of possessing or having access to higher performance 3D
printers, the CRIB was also designed as a whole piece already mounted.
The «Stl» file for this model is also provided as the STL file 2 and the
image is shown in Supplementary data 3 (F2) and it was designed to be
printed from the «Stl» file using the Bambu Studio software. The Bambu
Lab 3D printer was the Bambu Lab PAS model. Modeling was performed
using the Tinkercad site. The printing speed was of 200 mm/s. For this
printing the Bambu Lab ABS filament with a 1.75 mm (with the density
of 1.05 g/cm3) diameter (ref# 40100) was used and density for printing
the object was also of 70 %. The whole piece was printed in 30 min.

The container used was sourced from a recycling box (size: 73 mm long, 53 mm large and 37 mm depth) but it can be also printed as an entire piece by using the Bambu Lab 3D printer as described above. The «Stl» file for this box, (also called IF cuvette) is provided as the STL file 2 and the image is shown in Supplementary data 3 (F3). The filament was a waterproof PLA filament from Bambu Lab of 1.25 g/cm3 and the printing density was also of 70 %. The whole box was printed in 20 min.

4.5. Immunofluorescence experiments (IF-Express)

ERK2-dTAG-HA-expressing NIH3T3 cells were cultured on glass coverslips in 6-well plates. The cells were treated with the dTAG-13 drug, diluted in the culture medium at the desired concentration and for the specified times. Following treatment, the cells were washed three times with PBS in the culture wells and then fixed for 20 min in 3 % paraformaldehyde, as outlined in Flowchart 1, which specifies the steps of the IF-Express protocol. After three additional PBS washes directly in the plate, the cells were incubated with the primary anti-HA antibody (1:200 dilution) (Eurogentec #16B12), diluted in PBS containing 0.5 % bovine serum albumin (BSA) and 0.02 % saponin (both from Sigma), for 1 h on drops placed on parafilm. Then the coverslips are delicately taken out from the parafilm-covered plate and placed inside the IF-CRIB, with cells facing in the same direction. The cells were then washed twice with PBS for 1 min each using the IF-CRIB inserted in the container. Then the coverslips are taken from the IF-CRIB to be deposited onto drops of medium spotted on parafilm for an additional hour containing the anti-mouse Alexa Fluor 488-conjugated secondary antibody (Invitrogen #A11017). After washing with the IF-CRIB distilled water, the coverslips were mounted on glass slides using a DAPI-containing mounting medium (Sigma Fluoroshield with DAPI #F6057). All over the protocol, coverslips are always manipulated by using inverted microforceps. The samples were viewed and photographed using the EVOS M5000 microscope. Quantification of each experiment was performed using the EVOS M5000 quantification system.

4.6. Immunoblotting

Cells were lysed in Laemmli sample buffer prior to boiling for 10 min (200 µl per 10 cm diameter plates). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (10 % acrylamide-bisacrylamide [29:1] gels), with 15 µg to 40 µg of protein loaded per lane, depending on the gel size. Proteins were transferred to BioTrace NT nitrocellulose membranes (PALL, #66435). Membranes were blocked by incubation with 2.5 % BSA in PBS containing 0.12 % cold-water fish gelatin and 0.1 % casein (all from Sigma references). Antibodies were incubated in PBS containing 0.1 % cold-water fish gelatin and 0.08 % casein. HA-tagged constructs were visualized using the monoclonal antibody HA11 (1:1000; Eurogentec, #16B12). Phosphorylated ERK1/2 was detected using the mouse monoclonal anti-phospho-ERK1/2 antibody (1:9000; Sigma, #M8159), with identical results obtained using the rabbit monoclonal anti-phospho-ERK antibody (1:1000; Cell Signaling Technologies, #4370). Total ERK was detected using the rabbit monoclonal anti-ERK antibody (1:2000; Cell Signaling, #4695) or the rabbit anti-rat ERK1 antibody (1:4000; Fisher, #1019–9152). Secondary antibodies were IRDye anti-rabbit 800CW (1:7000) and anti-mouse 680RD (1:7000) from LI-COR. Band detection and quantification were performed using the LI-COR Odyssey Imaging System. All images are presented with a gamma value of 1 during image processing.

Funding sources

This work was supported by CNRS (Centre National de la Recheche
Scientifique), Université Côte d'Azur, the Research fund from the Can-
ceropôle PACA, ANR (Agence Nationale de la Recherche), INCA (Institut
national du Cancer), the Ligue Nationale contre le Cancer (bearing the
"Ligue" label in 2019), Fondation ARC de la Recherche contre le Cancer Programme (bearing the ARC label from 2022: program ARCAGE-ING2023020006332), the Fondation Estée Lauder and the Pink Ribbon award.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors made limited use of Instatext Premium and ChatGPT by OpenAI for occasional writing and grammar checks. They also used Perplexity AI to verify a few old references from PubMed. All content was subsequently reviewed and edited by the authors, who take full responsibility for the final version of this work.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We are indebted to Olivier Dalmau from the Université Côte d'Azur UniCA-Campus Valrose Fab-Lab for the Multimaker printing advice and coaching. We greatly thank Alexis Casile and the new "#Alexis-3D.com" start-up for the conception improvement and printing of the IF-CRIB device with the Bambu lab printer and for providing the filaments. We thank Dr G. Winter for providing the GW906 Plasmid containing the FΚΡΒ12<sup>F36V</sup> sequence. We express our gratitude to all the funders that are listed above in Funding sources.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymeth.2025.08.004.

## Data availability

The data of this study are available within the article and its supporting information (Supplementary data Supplementary data).

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