Input: How many people attend the 233rd ACS national meeting?

Context:
The major actions taken by the board of directors and council during the national meeting in Chicago were reported in C&EN, April 30 (page 32).
The Society Committee on Budget & Finance met on Saturday, March 24, to review the society's 2006 financial performance. The society ended 2006 with a net contribution from operations of $12.2 million, on revenues of $424.0 million and expenses of $411.8 million. This was $7.8 million favorable to the approved budget.
After including the results of the Member Insurance Program and new ventures, the society's overall net contribution for 2006 was $11.5 million, which was $7.4 million favorable to the approved budget. The favorable variance was primarily attributable to higher than budgeted electronic services revenue and investment income, as well as expense savings from lower than budgeted health care costs and reduced IT spending. In addition, the society ended the year in compliance with the board-established financial guidelines.
The Society Committee on Education (SOCED) received an update from President Catherine Hunt on the thematic programming featured in Chicago focusing on the sustainability of energy, food, and water. President-Elect Bruce Bursten solicited input from the committee pertaining to the central role of education in his agenda. SOCED received a presentation from the Membership Affairs Committee on its white paper on membership requirements.
Committee members strongly support the proposal to include undergraduates as members of the society, but they requested that financial arrangements be clearly spelled out in the petition to ensure that the highly successful Student Affiliates program remains intact. The committee discussed the Education Division programs that were reviewed in 2006 and those that will be reviewed in 2007, under the auspices of the Program Review Advisory Group. SOCED received an update from the Committee on Professional Training regarding the draft ACS guidelines for approval of bachelor's degree programs in chemistry.
Committee members discussed the report prepared by the Globalization Task Force, focusing on those sections relevant to education. The committee suggested initiatives related to the new ACS strategic plan, including a potential program that would engage retired chemists in the K-12 classroom. SOCED created a task force to consider the role of online, or "virtual," simulations in the chemistry laboratory, recognizing the value of online/virtual experiences as a supplement to, but not a replacement for, hands-on laboratory experiments.
The committee ratified two interim actions taken since the Dec. 3, 2006, meeting: to remove the financial restriction of the ACS Petroleum Research Fund (ACS PRF) Supplement for Underrepresented Minority Research Programs (SUMR) and to contact nominators whose nominations for the Volunteer Service Award had expired and to invite them to reactivate their nomination packet for the 2008 Volunteer Service Award.
Acting under delegated authority, the committee voted to accept the recommendations of the ACS Petroleum Research Fund Advisory Board (February 2007 meeting) for funding grants totaling $5.2 million; voted to recommend to the board a screened list of six nominees (due to a two-way tie for fifth place) for the 2008 Priestley Medal; voted to recommend to the board a screened list of five nominees for the 2008 Award for Volunteer Service to ACS; on the recommendation of the ACS Committee on Frasch Foundation Grants, voted to recommend to the board that it recommend to the trustee (US Trust) of the Frasch Foundation 12 grants for research in agricultural chemistry for the period of 2007–12; voted to recommend to the ACS Board of Directors that a new national award be established, the "ACS Award for Affordable Green Chemistry," sponsored by Rohm and Haas; and voted to recommend to the ACS Board of Directors that a new endowment be established, the "Affordable Green Chemistry Endowment Fund," to support the award.
The committee also reviewed the final report from the Special Board Task Force on the Review of the ACS National Awards Program, chaired by Ronald Breslow; established a Canvassing & Selection Subcommittee; and reviewed a list of external awards for which ACS may want to nominate candidates. The committee agreed to include the list of significant external awards in the awards locator database that is being developed.
The committee was updated on efforts to reconcile ACS's technical divisions' desires to leverage national meeting content using the Internet with our journal editors' concerns about prior publication issues. A conference call on this issue was scheduled for April 21, 2007.
The committee received a presentation on the recent actions of the ACS Board of Directors International Strategy Group (ISG). The group's charge is to develop recommendations for a short- and long-term international strategy for the society.
The committee was updated on the status of the activities of the Board Oversight Group on Leadership Development (BOG). Potential solutions for the unexpectedly high cost of facilitator training and transitioning from the current Leaders Conference format to the newly designed curriculum were presented to the committee.
The committee reviewed plans for conducting the 2007 Membership Satisfaction Survey. Preliminary results are expected in May or June with a final report to be delivered to the board at the 2007 Boston national meeting.
The committee received a briefing on the status of the MORE Project: Multidisciplinary Opportunities though Resource Enhancement. Twenty-eight proposals were received, and a decision on which proposals to support will be made in early May.
The chair led a discussion on draft 2007 committee goals, and committee members offered several suggestions related to successfully meeting them. One suggestion was to modify a communications goal to make it more completely reflect the duties of the committee outlined in the board regulations. The chair and committee members will examine the suggestion and revisit the question after the board retreat where board committee duties will be examined.
ACS President Hunt discussed her 2007-08 Presidential Task Force on Enhancing Science & Technology, which is charged with developing advocacy best practices that can enhance ACS's attainment of its public policy priorities. The task force is composed of a diverse set of ACS members as well as former U.S. Representative and chairman of the House Science Committee, Sherwood Boehlert, who will cochair the task force.
• Results of the 2007 Public Policy Priorities Survey, which resulted in a four-tiered ranking of ACS's 2007 public policies. The ranking will help focus staff resources in conducting outreach and advocacy on behalf of ACS members.
• The hiring of a communications consulting firm for 2007 to assist ACS in implementing the initial phase of the ACS Strategic Communications Plan.
• Creation of a pilot ACS state government affairs advocacy program. Committee members agreed to the creation of a pilot, and staff will propose an initial list of states, policy focus, and a budget to carry out the program.
The committee met in executive session on March 23 and in open session jointly with the Joint Board-Council Committee on Publications and the Division of Chemical Information on March 26.
The committee heard from Chemical Abstracts Service (CAS) management on a range of issues including a report on continuing database building efforts, product enhancements, and CAS's centennial celebration plans.
The Committee on Chemical Safety (CCS) provides advice on the handling of chemicals and seeks to ensure safe facilities, designs, and operations by calling attention to potential hazards and stimulating education in safe practices.
CCS has several publications (many downloadable), including the flagship publication, "Safety in Academic Chemistry Labs" (SACL). Work has recently started on the translation of SACL into Arabic. This is in addition to the online Spanish version of SACL. Also online are the "Student Lab Code of Conduct for Secondary Science Programs" and a security vulnerability analysis checklist. A K-12 restricted hazardous substances list is under development. The third edition of the "Chemical Safety Manual for Small Businesses" will be ready soon.
The committee's Task Force on Laboratory Environment, Health & Safety is working on a new edition of "Laboratory Waste Management." Task force members also commented on the recent Environmental Protection Agency Proposed Rule for Hazardous Waste in Academic Laboratories. Our Video Safety Resources Task Force is developing video resources to be distributed over the Web.
CCS has been involved in collaborations for the updating of publications like "Prudent Practices in the Laboratory" and "ACS Guidelines for the Teaching of High School Chemistry." Along with other ACS units, CCS is exploring participating in the EPA's School Chemicals Cleanout Campaign.
The Committee on Chemists with Disabilities (CWD) met at the 233rd ACS national meeting, Chicago, on Monday, March 26. Judy Summers-Gates reported on the Joint Subcommittee on Diversity meeting. This subcommittee is made up of representatives of the five committees that support people in chemistry (as opposed to a category of the profession): CWD, Committee on Minority Affairs, Committee on Technician Affairs, Women Chemists Committee, and Younger Chemists Committee, and its goal is to develop ways to coordinate the efforts of the five groups.
The CWD Ambassador Program that was announced at CWD's 25th anniversary celebration at the Washington, D.C., meeting was discussed. Zelda Wasserman reported on the status of the letter from CWD to the ACS Board regarding captioning of ACS video materials. Janelle Kasper-Wolf, of ACS staff, discussed adding new questions to the ACS annual employment salary survey to obtain information for the committee.
At the Chicago national meeting, the Committee on Community Activities (CCA) partnered with the ACS Education Division and the Office of the President to host "Chemistry In Action—It's Easy Being Green" at the Peggy Notebaert Nature Museum on Saturday, March 24. More than 250 children participated in the hands-on activities focused on recycling. ACS President Hunt presented a Salutes to Excellence plaque to the museum for its dedication to community outreach.
The Chemists Celebrate Earth Day celebration occurred in 120 local sections with 138 coordinators leading the efforts within their communities. This represents an increase of more than 30% in local section and coordinator participation from 2006.
CCA was featured in C&EN's April 16th issue on page 53. A shortcut to CCA's homepage was created: chemistry.org/committees/cca.html.
During the Boston national meeting, CCA and the Office of Community Activities will celebrate National Chemistry Week's 20th Anniversary and its theme, "The Many Faces of Chemistry." A special outreach event is being planned for Sunday, Aug. 19. Hands-on activities will focus on health and wellness.
The Committee on Corporation Associates (CCA) advises and influences ACS to ensure that its products and services are of value to industrial members and their companies. CCA vice chair, Roslyn White (SC Johnson), provided an overview of recent interactions between Corporation Associates and the U.K.-based Society of Chemical Industry (SCI).
CCA gave feedback to a recommendations report from the ACS Board Committee on Professional & Member Relations Task Force on Globalization. Presentations were also received from the ACS Green Chemistry Institute and SCI.
Staff reported on the Department of Industry Member Programs' activities since the San Francisco meeting. The report covered the Regional Industrial Innovation Awards, the World Congress on Industrial Biotechnology, the Analytical Pavilion sponsored by C&EN, and the ACS/Pharma Leaders Meeting.
The Awards/Finance & Grants Subcommittee reported that CCA received two funding proposals that total $7,500. Funding was provided to the following: The Committee on Economic & Professional Affairs at $3,000 for the Chicago symposium on "Benefits Trends for the Chemical Workforce" and the Office of Graduate Education and the Department of Career Development & Management at $4,500 for a workshop on "Preparing for Life after Graduate School," to be held in conjunction with the 39th Central Regional Meeting.
The subcommittee also requested that ACS staff provide CCA with an official annual statement of Corporation Associates' financial reserves as of Jan. 1 of each year.
The Programs Subcommittee reported on planned programming activities in 2007 and beyond between CCA and SCI. The subcommittee gave an update on a Boston symposium cosponsored by Corporation Associates and the Medicinal Chemistry Division featuring past ACS Heroes of Chemistry from the pharmaceutical industry.
By request of the subcommittee, representatives from Chemical Abstracts Service gave an overview on AnaVist—a tool with potential applications for CCA's efforts to provide member companies with industry-relevant information and reports. The subcommittee also requested that CCA earmark approximately $20,000 of Corporation Associates funds in 2008 for its activities.
The Educational Outreach Subcommittee reported on its decision to collaborate with the Graduate Student Symposium Programming Committee of the Chemical Education Division on a graduate student industry roundtable program in Boston.
The subcommittee requested $5,000 in support of this effort. The subcommittee also discussed a request for corporate executive support of an American Association of Physics Teachers initiative to promote undergraduate research.
The Committee on Environmental Improvement (CEI) continues to be focused on the sustainability of the chemical enterprise. In Chicago, the committee introduced a multiyear effort to make ACS meetings more sustainable. This effort is designed to take advantage of the large size and diverse programs of the society to lead in the sustainability arena by "walking the talk."
The committee held a dialogue with representatives of the U.S. Environmental Protection Agency who are trying to "green" federal conferences and work with the travel and tourism industry to change practices and shrink the environmental footprint of meetings. The committee also used advertising and student volunteers to engage individual meeting participants in a campaign to increase recycling by asking "Are you sustainable?" Moving forward, CEI looks forward to working closely with the Committee on Meetings & Expositions to advance this agenda.
CEI was also pleased to participate in the meeting theme on sustainability through the ACS presidential programming. CEI cohosted the Monday presidential luncheon to discuss sustainability issues with the Committee on Science and is leading the follow-up to that luncheon, which will include recommendations on advancing sustainability in the three focal areas of the meeting—energy, food, and water.
The committee also continued its dialogue with the Committee on Corporation Associates about a collaborative workshop. This activity, tentatively slated for the New Orleans meeting, will seek additional insights from chemical and allied products companies about public policy barriers that limit adoption of more sustainable products and practices as well as policy incentives that would lead to increased sustainability in the chemical enterprise.
At its Chicago meeting, the committee welcomed the president of the Jordanian Chemical Society and the past-president of the Arab Union of Chemists.
The committee was briefed on Pittcon 2007, where, with financial support from the Society of Analytical Chemists of Pittsburgh, ACS coordinated participation of a scientific delegation from Adriatic nations.
The committee heard reports on the 2007 Frontiers of Chemical Science III: Research & Education in the Middle East meeting; the 2007 Transatlantic Frontiers of Chemistry meeting, which was jointly sponsored by ACS, the German Chemical Society, and the Royal Society of Chemistry; planned workshops to engage U.S. and Chinese early-career scientists in chemical biology, supramolecular, and new materials chemistry; and ACS Discovery Corps U.S./Brazil Research Collaboration Project in Biomass Conversion to Biofuels, Biomaterials & Chemicals.
The committee discussed Latin American engagement opportunities created through Puerto Rico's involvement in three key chemical science events there: the 2009 ACS Southeast Regional Meeting, the 2008 Federation of Latin American Chemical Associations (FLAQ) meeting, and the proposed IUPAC 2011 Congress & General Assembly.
The committee heard reports on letter-writing efforts by the ACS president to government officials in Libya and Mexico expressing concerns about challenges to the scientific freedom and human rights of scientists there.
The Committee on Minority Affairs (CMA) approved new vision, mission, and values statements at the Chicago national meeting. The mission of CMA is to increase the participation of minority chemical scientists and influence policy on behalf of minorities in ACS and the chemical enterprise.
An aggressive new strategic plan was approved by CMA to guide its activities over the next three years. By the end of 2009, CMA will increase the number of ACS Scholars that graduate to 100 per year, add 100 new minorities to leadership positions in ACS, engage in several collaborations, and increase the number of minority members of ACS by 5,000. CMA will focus initially on increasing minorities in ACS leadership. In working toward this goal, CMA began work on two new leadership-development programs for minority chemists.
CMA continues to support the work of the Joint Subcommittee on Diversity (JSD) in developing programs, products, and services to ensure full participation of all members in ACS. In Chicago, JSD premiered a diversity booth at the meeting exposition hall and cosponsored symposia.
The Committee on Patents & Related Matters (CPRM) discussed proposed legislative and regulatory changes to the U.S. patent system as well as open-access legislation and the potential effects such matters might have on industry and academia as well as on ACS.
CPRM also continued its work on several new educational tools to assist and inform members on patent issues and other intellectual property matters important to a successful career in the chemical enterprise. Many of these tools are now available on the committee's expanded website, membership.acs.org/C/CPRM/.
At the March 2007 meeting, the Committee on Professional Training (CPT) reviewed 42 new and additional information reports from ACS-approved chemistry programs. CPT held conferences with four schools seeking approval, discussed three updates and five site visit reports, and approved three new schools. The total number of ACS-approved chemistry programs is now 642.
The committee released the full draft of the ACS guidelines for review and comment. Copies of the draft were distributed to the department chairs at all ACS-approved schools, the chairs of all ACS committees, and the chairs of all ACS technical divisions.
Several CPT members met with the ACS technical divisions during the Chicago meeting to present an overview of the draft and obtain feedback. The draft guidelines document is available on the CPT website, and the committee invites any comments to be sent to cpt@acs.org.
In other business, the committee continued development of the two workshops with minority-serving institutions that will be held in 2007. The committee reviewed the editorial policies for the 2007 edition of the ACS Directory of Graduate Research, which is using a new protocol for retrieving research publication titles in an effort to improve the accuracy of the directory.
C&EN finished 2006 with an exceptionally strong editorial package. The first months of 2007 are proving to be equally successful in fulfilling the magazine's mission of keeping its readers informed. On the advertising side, revenues in 2006 increased for the second year in a row, and the early months of 2007 show continuing positive signs. The most significant editorial achievement was the successful launch of the redesign of the print edition of C&EN with the Oct. 16, 2006, issue.
The Subcommittee on Copyright has successfully updated the Copyright Module on the ACS Publications website. The subcommittee is looking into the possibility of conducting copyright programs at future ACS national and regional meetings.
The final monitoring reports for Chemistry of Materials, Journal of Agricultural & Food Chemistry, and Molecular Pharmaceutics were presented and accepted by the committee. Journal of Chemical Information & Modeling, Organic Letters, Accounts of Chemical Research, and the Journal of Chemical Theory & Computation will be monitored next.
3. Examining the scientific basis of public policies related to the chemical sciences and making recommendations to the appropriate ACS units.
In the first of these areas, ComSci partnered with President Hunt and the Committee on Environmental Improvement in planning and hosting a sustainability luncheon that featured roundtable discussions centering on a key sustainability question. At the Boston national meeting, ComSci will deliver a full-day program on the subject of "Partnerships in Innovation & Competitiveness."
Regarding the second thrust, ComSci will present two programs in Boston: a box lunch that will feature two speakers taking opposing sides on the subject of "Genetic Screening & Diagnostic Testing: Do You Really Want to Know?" and a symposium titled "Creating & Sustaining International Research Collaborations."
In support of the last thrust, ComSci is planning two events for 2008: "Balancing Security & Openness" will gather data to determine if the recent emphasis on security is hindering scientific progress and "Transitioning Chemical Science to Commercially Successful Products."
The Women Chemists Committee (WCC) hosted more than 70 attendees at its open meeting recently in Chicago, where representatives from Iota Sigma Pi, Women in Science & Engineering, the Association of Women in Science, and the Chicago local section helped WCC celebrate the committee's 80th anniversary.
The Women in Industry Breakfast was also highly successful with a new format of speed networking. More than 100 participants had the opportunity to practice their elevator speeches and make several professional connections. A related workshop will be offered by WCC in Boston.
In Chicago, WCC sponsored two symposia, "Women Achieving Success: The ACS as a Platform in Leadership Development" in honor of Madeleine Joullié's 80th birthday and the ACS Award for Encouraging Women into Careers in the Chemical Sciences: Symposium in Honor of Bojan H. Jennings.
More than 225 ACS meeting attendees were present for the biannual WCC Luncheon and heard the keynote speaker Laura Kiessling, 2007 Francis P. Garvan-John Olin Medal Recipient. Twelve women presented their research at this meeting with funding by the WCC/Eli Lilly Travel Grant Award. WCC members also spent time educating expo attendees on programs offered by the ACS Office of Diversity Programs at its new booth.
In Chicago, the Younger Chemists Committee (YCC) welcomed its new committee members with an information session centered on YCC's charter as well as on its strategic plan: to make ACS relevant to younger chemists, to involve younger chemists in all levels of the society, and to integrate younger chemists into the profession.
In January, YCC again hosted a Leadership Development Workshop during the ACS Leaders Conference. There were more than 80 applications for the 15 awards, which covered travel and registration for the conference. YCC plans to again fund the travel awards and provide leadership training for young chemists in 2008. YCC also solicited applications and selected a new graduate student representative on the Graduate Education Advisory Board.
During the Chicago meeting, YCC programs included "Starting a Successful Research Program at a Predominantly Undergraduate Institution," "Career Experiences at the Interface of Chemistry & Biology," and "Chemistry Pedagogy 101."
In addition to these programs, YCC cosponsored five programs with various committees and divisions. YCC continues to reach out to ACS committees and divisions and has initiated liaisonships with 11 technical divisions to encourage technical programming that highlights the contributions of younger chemists. Looking forward to Boston, YCC is planning symposia including "The Many Faces of Chemistry: International Opportunities for Chemists"; "Being a Responsible Chemist: Ethics, Politics & Policy"; and "Changing Landscapes of the Bio-Pharma Industry."
The Committee on Committees (ConC) conducted its annual training session for new national committee chairs at the ACS Leaders Conference in January 2007. ConC's interactive session for committee chairs in Chicago served as an opportune follow-on and a forum for informative interchange among seasoned and new chairs.
ConC began developing its recommendations for the 2008 committee chair appointments for consideration by the president-elect and chair of the board. ConC continues to focus efforts to identify members with the skills and expertise specified by the committee chairs using the councilor preference form.
The form will be sent to councilors in May. ConC also seeks the names of noncouncilor members for consideration for service on council-related committees, especially those with no prior appointment.
As part of ongoing activities with the joint CPC-Board Governance Review Task Force, ConC has collected data on committee liaisons to other committees. This information will be distributed to committee chairs. The number of liaisons indicates that unofficial but strong communication channels exist within the ACS committee structure.
On Sunday evening, the Committee on Nominations & Elections (N&E) sponsored its fifth successful Town Hall Meeting for President-Elect Nominees. An estimated 200 people attended this session. This forum facilitated communication among the 2008 president-elect nominees, councilors, and other members. N&E will hold another Town Hall Meeting featuring the candidates for director-at-large at the fall meeting in Boston.
Now that voting over the Internet has become an accepted procedure for ACS national elections, the ACS technical divisions and local sections have expressed strong interest in using this method for their elections. N&E has developed protocols for elections for local sections and divisions. This document will be forwarded to the appropriate committees for their review and distribution.
N&E is responsible for reviewing annually the distribution of member populations within the six electoral districts to ensure that the districts have equitable representation. According to bylaw V, section 4(a), the member population of each electoral district must be within 10% of the result of dividing by six the number of members whose addresses lie within these districts. The committee is happy to report that the six electoral districts are in compliance.
The committee has developed a petition on election procedures for president-elect and district director. The proposed election mechanism provides for a preferential (ranked) ballot and an "instant runoff." N&E continues to address the areas of campaigning and the timing of our national election process. Between the Chicago and Boston meetings, the committee plans to sponsor online forums for input from councilors and other interested members on these issues.
In response to member concerns regarding the collection of signatures for petition candidates, N&E reviewed the society's bylaws. The bylaws state that an endorsement is required, but does not stipulate the method of endorsement. N&E has determined that original or electronic signatures are acceptable and will establish appropriate procedures for receipt of electronic signatures.
The Committee on Constitution & Bylaws (C&B), acting for the council, issued new certified bylaws to the Corning Section, the Portland Section, the Division of Colloid & Surface Chemistry, and the Division of Chemical Education. The committee reviewed new proposed amendments for the Division of Medicinal Chemistry, the Columbus Section, the Detroit Section, and the Southern Arizona Section.
Three petitions were presented to council for action at this meeting. Regarding the "Petition on Election Procedures 2006," a motion to separate the petition was approved, and the petition was divided. Provisions affecting bylaw V, sec. 2d, bylaw V, sec. 3c, and bylaw V, sec. 4f, which deal with election procedures and the timing of run-off elections, were approved by council and will become effective following confirmation by the board of directors.
The second part of the petition regarding bylaw V, sec. 2c, and bylaw V, sec. 3b, which deal with signature requirements for petition candidates for president-elect and director-at-large respectively, was recommitted to the Committee on Nominations & Elections, which has primary substantive responsibility for the petition.
The Committee on Nominations & Elections was asked to reconsider the signature requirements, procedures for acceptance of electronic signatures, and recommendations from the Governance Review Task Force on election procedures.
The second petition presented to council for action was the "Petition on Rules for Nominating Members of N&E for National Offices." This petition was not approved by council. The third petition, the "Petition on Multiyear Dues," was amended by incidental motion on the council floor, calling for the petition to become effective when technical components are instituted to track payments, but no later than Jan. 1, 2010. Council approved the incidental motion and then approved the petition.
The committee reviewed one petition for consideration, the "Petition on Local Section Affiliations," which will be submitted to council for action at the fall 2007 meeting in Boston.
The committee met with representatives of the Committee on Membership Affairs and the Governance Review Task Force to continue discussions on proposals currently being formulated on membership requirements and student membership. In addition, the committee discussed election issues of concern to the Southern California Section.
We hope you enjoyed the presidential and division thematic program, "Sustainability of Energy, Food & Water" in Chicago. A small, dedicated group of volunteers and staff labored tirelessly to create and coordinate this programming; to them the Committee on Divisional Activities (DAC) offers sincere thanks.
DAC has committed to transfer the process of choosing and organizing future national meeting themes to a body that represents all divisions. We made substantial progress in Chicago, where division, secretariat, and committee representatives convened to discuss national meeting program concepts. They proposed themes for the 2008 Philadelphia national meeting as well as a framework for a future national programming group.
Divisions have successfully served their members fortunate enough to attend national meetings. To maximize benefits to division members, DAC encourages divisions to consider extending the reach of the content they deliver at national meetings through Internet-based distribution channels and will support worthy efforts in this direction via Innovative Program Grants.
The committee voted in Chicago to propose modifications to the division funding formula that will more greatly reward interdisciplinary programming. The new formula will also be simpler and more transparent to divisions. DAC will present the revised plan to council for action in Boston.
The Committee on Economic & Professional Affairs (CEPA), working with ACS staff in the Departments of Career Management & Development and Member Research & Technology, continues to update and implement its strategic plan to address the career needs of society members.
Specifically, the committee reviewed and revised existing workshops and materials to help ACS members get jobs. CEPA is developing new programs to address the needs of mid- and late-career chemists to ensure their continued competitiveness in the workplace and to ease their career transitions. New initiatives in these areas include the development of workshops, online training, surveys to assess member needs, suggested changes to public policies, and updates to professional and ethical workplace guidelines. As a result of discussions at the Public Policy Roundtable, which was held in San Francisco, a background paper is being developed on trends in health care issues.
The newly revised "Chemical Professional's Code of Conduct" was presented to council, which approved it. The Standards & Ethics Subcommittee is preparing a revision of the "Academic Professional Guidelines" to be presented to council for consideration in Boston.
CEPA reviewed the Globalization Task Force Report. As our science diffuses around the globe, we want to make sure that our members are aware of the economic and professional challenges they will face and that they have the tools they need to succeed. Therefore, CEPA made a commitment to work with other committees, divisions, and ACS staff to develop programs and policies that position our membership to compete in the global workforce.
CEPA heard and discussed a presentation on the proposal from the Membership Affairs Committee on broadening the requirements of membership. CEPA supports the spirit of this proposal and encourages further detailed studies to assess financial impacts on local sections and student affiliates chapters.
The Local Section Activities Committee (LSAC) recognized local sections celebrating significant anniversaries in 2007, including Savannah River (50 years), Northeast Tennessee (75 years), and the St. Louis and Syracuse local sections (both celebrating 100 years).
LSAC hosted the local section leaders track in conjunction with the ACS Leaders Conference in Baltimore on Jan. 26–28. A total of 135 delegates from 124 local sections participated in the weekend leadership conference.
LSAC also hosted a Local Section Summit on March 2–4 in Arlington, Va. The summit focused on practical operational issues that will support local sections' long-term success. Specific areas that were discussed include the development of a multiyear plan to expand or develop programming for local sections, opportunities to encourage innovation and experimentation within and among local sections, and capitalizing on existing opportunities to facilitate partnerships between local sections and other ACS groups.
Following the San Francisco national meeting, LSAC launched a local section Science Café minigrant program. Fifty-five local sections accepted LSAC invitation to host Science Cafés in 2007.
A DVD entitled "ACS Close to Home: Local Sections Connecting Chemistry & the Community" was released earlier this year. The video provides a seven-minute overview of the many outreach and educational programs sponsored by local sections and the critical role they play in positively influencing the public's perception of chemistry and its practitioners. Copies of the DVD were sent to all local section officers.
The Committee on Meetings & Expositions (M&E) reported that the 233rd ACS national meeting hosted 14,520 attendees. This included 7,152 chemical scientists, 5,059 students, 1,283 exhibitors, 119 precollege teachers, 573 exposition visitors, and 453 guests. The exposition had 424 booths with 268 companies.
The 10 2006 regional meetings set a new standard for excellence with attendance exceeding 8,000, a 30% increase in average meeting attendance compared to the 2005 meetings. A total of 4,717 abstracts were submitted. A region summit was held in February at which the final report of the ReACT study group was reviewed.
The practice of tracking the number of presenter no-shows continues. M&E will collaborate with the Committee on Divisional Activities to study options for addressing this problem. Suggestions will be presented at the Boston meeting for implementation in 2008.
It is the intent of M&E to pursue the goal of making our meetings "greener." We will communicate with staff and governance units to identify actions for both the short and long term.
The American Institute of Chemical Engineers (AIChE) and ACS will hold their 2008 spring meetings simultaneously in New Orleans. An ad hoc working group consisting of members from M&E, DAC, and AIChE are actively exploring joint programming opportunities for this meeting.
The Committee on Membership Affairs (MAC) met in executive session on Saturday and Sunday in Chicago and reported that the ACS closed 2006 with 160,491 members, our highest year-end membership count since 2002. Of the 17,857 applications processed in 2006, more than 1,000 came from the Member-Get-a-Member campaign in which many councilors participated. The society's retention rate in 2006 remained strong at 92%. The committee also reported that recruitment for the first two months of 2007 netted 2,844 new applications—729 more than for the same time period last year.
MAC continues to work with deliberate speed on the proposed new bylaw language for members, student members, and society affiliates-the three ways to connect to the society. The committee received input from the Governance Review Task Force and its action teams, the Council Policy Committee, the board of directors, the Committee on Constitution & Bylaws, and several other committees between the San Francisco and Chicago meetings. These interactions have resulted in the current bylaw change recommendations.
In Chicago, representatives from MAC attended several committee meetings and all seven councilor caucuses to summarize the current proposal for membership changes, answer questions, and seek input. In addition, all committee chairs were invited to have their respective committees review these bylaw changes and respond to MAC—if possible—before council met on Wednesday. MAC received 11 responses: eight supported the proposed changes as is, and three supported the proposed language with specified changes or considerations.
The comprehensive petition will likely represent the most significant and voluminous change in the ACS bylaws that has occurred in decades, and MAC is proud to be among the leaders in its development and in efforts to get it right the first time. Hundreds of individuals have contributed to this major effort, since MAC began such discussions at the spring 2004 national meeting.
The Committee on Ethics met in Chicago and discussed the possibility of organizing and scheduling a committee retreat in the near future to enable the committee to move from the current stage of exploring the needs and interests of ACS members to setting priorities for the next few years.
The Project SEED program offers summer research opportunities for high school students from economically disadvantaged families. Since its inception in 1968, the program has had a significant impact on the lives of more than 8,400 students. At the selection meeting in March, the committee approved research projects for 340 SEED I students and 98 SEED II students for this summer in more than 100 institutions.
The 2006 annual assessment surveys from 300 students indicate that 78% of the Project SEED participants are planning to major in a chemistry-related science, and 66% aspire to continue to graduate education. This program is made possible by contributions from industry, academia, local sections, ACS friends and members, the ACS Petroleum Research Fund, and the Project SEED Endowment.
The committee formally submitted a request to ConC to amend the Project SEED acronym and the committee duties described in the Supplementary Information of the "ACS Charter, Constitution, Bylaws & Regulations."
In Chicago, the committee's agenda focused on the ACS Strategic Plan and how Project SEED fits into it, the Program Review Advisory Group (PRAG) review of the Project SEED program, the committee's review of an online application form, and planning of the 40th anniversary celebration to be held at the Philadelphia meeting in the fall of 2008. The committee selected a task force to review the criteria for selection of the Project SEED ChemLuminary Award.
3. Making ACS relevant to technicians.
Last year, CTA, along with the Division of Chemical Technicians, the Committee on Economic & Professional Affairs, and ChemTechLinks, started the Equipping the 2015 Chemical Technology Workforce initiative. This year, the initiative awarded six $500 minigrants to activities and programs that support the educational and professional development of chemical technicians.
We are pleased to announce that the winners of the minigrants are the ACS Division of Environmental Chemistry; the Chemical Technician Program Chair for the 39th ACS Central Regional Meeting in Covington, Ky.; Delta College, University Center, Mich.; Grand Rapids Community College, in Michigan; Mount San Antonio College, Walnut, Calif.; and Southwestern College in Chula Vista, Calif.
The winners are collaborating with industry, academia, and ACS local sections on such activities as chemical technology career fairs for high school students, discussion panels on employability skills for technicians, and technical programming at regional and national meetings on the vital role technicians have in the chemical enterprise.
Because of the enthusiastic response to the minigrants, Equipping the 2015 Chemical Technology Workforce will be supporting another round of minigrants to be distributed in the fall. Details will be available on the website. For more information, go to www.ChemTechLinks.org and click on "Equipping the 2015 Chemical Technology Workforce."
CTA has also joined with the Joint Subcommittee on Diversity, formerly known as the Collaboration of Committees Working Group. Because this group is focused on increasing diversity in ACS and the chemical enterprise, we believe that this is an opportunity to raise awareness of the value of technicians. CTA looks forward to collaborating on the promotion of traditionally underrepresented chemical professionals.
In 2007, CTA will be placing renewed focus on distribution of the ACS Chemical Technology Student Recognition Award. The award recognizes academic excellence in students preparing for careers as chemical technicians. For more information on the award, please visit the CTA website at chemistry.org/committees/cta.

Answer: There are 14,520 attendees, including 7,152 chemical scientists, 5,059 students, 1,283 exhibitors, 119 precollege teachers, 573 exposition visitors, and 453 guests.


Input: Is the ISR necessary for transgene reactivation?

Context:
Current address: Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom.
In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.
Copyright: © 2018 De Vito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. RNAseq data are available in the ArrayExpress database under the accession number E-MTAB-6452.
Funding: This study was funded by the Ajinomoto Innovation Alliance Program, (AIAP; https://www.ajinomoto.com/en/rd/AIAP/index.html#aiap) (to M.V.S and D.G), which is a joint research initiative of Ajinomoto Co., Inc., Japan. One of the authors [M.B.] is an employee of Ajinomoto Co., and his specific roles are articulated in the ‘author contributions’ section. The commercial funder provided support in the form of salary for author [M.B.] and some of the necessary research materials (medium for cell culture), but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript, and the authors had unrestricted access to the data. Due to a confidentiality agreement, the commercial funder participated only in the decision to publish the data obtained during the study, without any restriction.
Competing interests: This study was funded by Ajinomoto Co., Inc., Japan and one of the authors [M.B.] is an employee of this commercial funder. No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study. The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials.
In animals, excessive, insufficient, or imbalanced nutrient availability is known to strongly impact on phenotype and health, both short and long-term, and across generations [1, 2]. In particular, studies in yeast, animal models and humans have shown that reduced food intake, reducing either overall calories, or only sugars, proteins, or even single amino acids (AA), such as Methionine (Met), may extend lifespan and healthspan, and reduce the risk of cancer and other age-related diseases [3–9]. In addition, fasting or specific AA deprivation have shown potential therapeutic applications, owing to their ability to directly reduce the growth of some tumor types [10, 11], sensitize cancer cells to chemo- or immunotherapy [12, 13], and allow efficient hematopoietic stem cell engraftment . However, little is known about the specific processes and molecular mechanisms mediating the roles of nutrient restriction in human health and longevity.
A properly balanced diet in metazoans contains optimal amounts of a subset of AA, which cannot be synthetized de novo and are therefore named essential amino acids (EAAs). In humans these include Met, Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Phenylalanine (Phe), Threonine (Thr), Tryptophan (Trp), and Valine (Val), while a few others are considered as semi-essential, such as Glutamine (Gln) and Tyrosine (Tyr) [15, 16]. Consistently, EAA deprivation triggers a cell-autonomous adaptive response, characterized by extensive metabolic and gene expression modifications, implementing biosynthetic, catabolic, and plasma membrane transport processes, aimed at reconstituting the full AA complement [17, 18]. The best known and conserved pathways responding to AA deprivation are triggered by mechanistic Target of Rapamycin Complex 1 (mTORC1) and General amino acid Control Non-derepressible 2 (GCN2) protein kinases [15, 19, 20]. Activation of mTORC1 requires in particular the presence of Gln, Arg and Leu, but also Met , which activate the kinase through sensors mainly acting upstream of Rag GTPases at lysosomal membranes . In turn, mTORC1 promotes cell growth, proliferation and anabolism upon activation, and translational attenuation and autophagy upon inhibition [19, 20].
By contrast, GCN2 is activated by deprivation of any individual EAA, by means of its histidyl-tRNA synthetase-related domain, which binds uncharged tRNAs accumulating during AA limitation [23, 24]. Upon activation, GCN2 phosphorylates and inhibits its only known downstream target, namely the eukaryotic Initiation Factor 2 α (eIF2α), thereby initiating the Integrated Stress Response (ISR). This leads to attenuation of general translation, and induction of a transcriptional/translational program, aimed at increasing stress resistance and restoring cell homeostasis, by upregulating a specific subset of genes, including Activating Transcription Factor 4 (ATF4) and C/EBP-Homologous Protein (CHOP) [25–27]. Thus, inhibition of mTORC1 and activation of GCN2 by AA restriction cooperate to attenuate general translation at the initiation step, increase catabolism and turnover, and enhance stress resistance to promote adaptation . However, how these processes eventually induce protective mechanisms against the alterations associated with aging, which include pervasive epigenetic and transcriptional changes [28, 29], remains largely unknown.
We previously reported the unexpected observation that prolonged deprivation of either Tyr, or of both Methionine and Cysteine (Met/Cys), triggers the selective and reversible reactivation of exogenous transcriptional units, including plasmids, retroviral vectors and proviruses, integrated into the genome and transcriptionally repressed by defensive mechanisms against non-native DNA sequences [30, 31]. This phenomenon was observed both in HeLa epithelial and ACH-2 lymphocytic human cells, and was independent of the transgene or provirus (Ocular Albinism type 1, OA1; Green Fluorescent Protein, GFP; Lysosomal-Associated Membrane Protein 1, LAMP1; Human Immunodeficiency Virus-1, HIV-1), or of the exogenous promoter driving their transcription, either viral (cytomegalovirus, CMV; Long Terminal Repeat, LTR) or human (Phospho-Glycerate Kinase 1, PGK1; Elongation Factor-1α, EF-1α) . Furthermore, this transgene reactivation response was not reproduced by serum starvation, activation of p38, or pharmacological inhibitors of mTOR (PP242 or rapamycin), sirtuins and DNA methylation. By contrast, it was induced by pan histone deacetylase (HDAC) inhibitors, and by selective inhibitors of class II HDACs . Consistently, we found that the mechanism responsible involves epigenetic modifications at the transgene promoter, including reduced nucleosome occupancy and increased histone acetylation, and is mediated in part by reduced expression of a class II HDAC, namely HDAC4 .
These findings indicate that AA deprivation induces a specific epigenetic and transcriptional response, affecting the expression of newly-integrated exogenous transgenes and proviruses, and suggesting that endogenous sequences sharing similar structural and functional features may represent a transcriptional target as well [30, 31]. In particular, transposable elements, such as LTR-retrotransposons (or endogenous retroviruses, ERVs), are genomic “parasites” anciently-integrated into the genome, and silenced by epigenetic mechanisms of mammalian cells against the spreading of mobile elements, eventually becoming "endogenized" during evolution [32, 33]. This raises the question of whether their expression is also sensitive to AA restriction. In addition, it remains unclear whether or not the transgene reactivation response is related to specific AA deprivations, and most importantly which is the AA sensing/signaling pathway involved, in particular whether the GCN2 kinase is implicated. Thus, here we used the reactivation of silenced transgenes in cultured cells, as a model to investigate a novel molecular pathway induced by imbalanced EAA starvation, implicated in the epigenetic/transcriptional regulation of exogenous non-native DNA sequences and possibly of other endogenous anciently-integrated genomic elements.
HeLa human epithelial carcinoma, HepG2 human hepatocellular carcinoma and C2C12 mouse skeletal muscle cells were maintained in DMEM containing glutaMAX (Invitrogen) and supplemented with 10% FBS (Sigma), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), at 37°C in a 5% CO2 humidified atmosphere. Cell lines carrying integrated and partially silenced transgenes were also maintained in 600–1000 μg/ml G418.
The C2C12 cell line was provided by ATCC. HeLa and HepG2 cells were obtained by Drs. F. Blasi and G. Tonon at San Raffaele Scientific Institute, Milan, Italy, respectively, and were authenticated by Short Tandem Repeat (STR) profiling, using the Cell ID System kit (Promega), according to the manufacturer’s instructions. Briefly, STR-based multiplex PCR was carried out in a final volume of 25 μL/reaction, including 5 μL Cell ID Enzyme Mix 5X, 2.5 μL Cell ID Primer Mix 10X and 3 ng of template DNA. The thermal cycling conditions were: 1 cycle at 96°C for 2 min, followed by 32 cycles at 94°C for 30 sec, 62°C for 90 sec, and 72°C for 90 sec, and 1 cycle at 60°C for 45 sec. The following STR loci were amplified: AMEL, CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA. Fragment length analysis of STR-PCR products was performed by Eurofins Genomics, using standard procedures of capillary electrophoresis on the Applied Biosystems 3130 XL sequencing machine, and assessment of the STR profile was performed at the online STR matching analysis service provided at http://www.dsmz.de/fp/cgi-bin/str.html.
Stable cell clones, expressing myc-tagged human OA1 (GPR143) or GFP transcripts, were generated using pcDNA3.1/OA1myc-His or pcDNA3.1/EGFP vectors . Briefly, HeLa, HepG2 and C2C12 cells were transfected using FuGENE 6 (Roche) and selected with 800, 1000, and 650 μg/ml of G418 (Sigma), respectively, which was maintained thereafter to avoid loss of plasmid integration. G418-resistant clones were isolated and analyzed for protein expression by epifluorescence and/or immunoblotting.
Full DMEM-based medium, carrying the entire AA complement, and media deprived of Met/Cys (both AAs), Met (only), Cys (only), Alanine (Ala), Thr, Gln, Val, Leu, Tyr, Trp, Lys and His were prepared using the Nutrition free DMEM (cat.#09077–05, from Nacalai Tesque, Inc., Kyoto, Japan), by adding Glucose, NaHCO3, and either all 20 AAs (for full medium) or 18–19 AAs only (for deprivations of two-one AAs). Single AAs, Glucose, and NaHCO3 were from Sigma. Further details and amounts utilized are indicated in S1 Table. All media were supplemented with 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin G (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and G418 as required. HBSS was from Invitrogen. Cells were seeded at 10–30% of confluency; cells to be starved for 48 h were plated 2–3 times more confluent compared to the control. The following day, cells were washed and cultured in the appropriate medium, with or without EAA, for 24–48 h.
L-Histidinol (HisOH), PP242, Integrated Stress Response Inhibitor (ISRIB), SP600125, Cycloheximide (CHX) were from Sigma; Salubrinal was from Tocris Bioscience; U0126 was from Promega. Drugs were used at the following final concentrations: HisOH at 4–16 mM; PP242 at 1–3 μM; ISRIB at 100 nM; SP600125 at 20 μM in HepG2 cells and 50 μM in HeLa cells; Cycloheximide (CHX) at 50 ug/ml in HepG2 cells and 100 ug/ml in HeLa cells; Salubrinal at 75 μM; U0126 at 50 μM. Vehicle was used as mock control. Treatments with drugs to be tested for their ability to inhibit transgene reactivation (ISRIB, SP600125 and U0126) were initiated 1h before the subsequent addition of L-Histidinol (ISRIB) or the subsequent depletion of Met/Cys (SP600125 and U0126).
Total RNA was purified using the RNeasy Mini kit (Qiagen), according to manufacturer’s instructions. RNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific). Equal amount (1 μg) of RNA from HeLa, HepG2 and C2C12 cells was reverse transcribed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) using oligo-dT as primers, and diluted to 5 ng/μl. The cDNA (2 μl) was amplified by real-time PCR using SYBR green Master Mix on a Light Cycler 480 (Roche), according to manufacturer’s instructions. The thermal cycling conditions were: 1 cycle at 95°C for 5 min, followed by 40–45 cycles at 95° for 20 sec, 56° for 20 sec and 72° for 20 sec. The sequences, efficiencies and annealing temperatures of the primers are provided in S2 Table. Data were analyzed with Microsoft Excel using the formula EtargetΔct target (control-sample) /EreferenceΔct reference (control-sample) . Reference genes for normalizations were ARPC2 (actin-related protein 2/3 complex, subunit 2) for HeLa and HepG2 cells; and Actb (actin beta) for C2C12 cells, unless otherwise indicated.
siRNA (Mission esiRNA, 200 ng/μL; Sigma) against ATF4 and GCN2 were designed against the targeted sequences NM_001675 and NM_001013703, respectively. Cells seeded in 6-well plates were transfected with 1 μg of siRNAs and 5 μL of Lipofectamine 2000 (Invitrogen), following manufacturer’s instructions, at day 1 post-plating for ATF4 and at day 1 and 2 post-plating for GCN2. At day 2 (ATF4) or 3 (GCN2) post-plating, cells were washed and cultured in medium in the absence or presence of HisOH 4 mM for 6 h. siRNAs against RLuc (Sigma), targeting Renilla Luciferase, were used as negative control. For CRISPR/Cas9 experiments, we used the “all-in-one Cas9-reporter” vector, expressing GFP (Sigma), which is characterized by a single vector format including the Cas9 protein expression cassette and gRNA (guide RNA). GFP is co-expressed from the same mRNA as the Cas9 protein, enabling tracking of transfection efficiency and enrichment of transfected cells by fluorescence activated cell sorting (FACS). The human U6 promoter drives gRNA expression, and the CMV promoter drives Cas9 and GFP expression. The oligonucleotide sequences for the three gRNAs targeting GCN2 exon 1 or 6 are listed in S2 Table. We transfected HeLa and HepG2 cells with these plasmids individually (one plasmid one guide) and sorted the GFP-positive, transfected cells by FACS. Screening GCN2-KO clones was performed by western blotting. In the case of HepG2-OA1 cells, two rounds of selection were necessary to obtain three GCN2-KO clones by using a guide RNA against exon 1. Compared to the original HepG2-OA1 cell line and to the clone resulting from the first round of selection (185#27), the selected clones E23, F22 and F27 showed a very low amount—if any—of residual GCN2 protein (see results).
Genomic DNA of HeLa and HepG2 cells was purified using DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer’s instructions. DNA concentration was determined by Nanodrop 8000 Spectrophotometer (Thermo Scientific). PCR conditions for amplification of GCN2 exon 1 and 6 were as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 40 sec, 56°C for 40 sec, and 72°C for 40 sec; and a final extension step of 5 min at 72°C. The primer sequences are provided in S2 Table.
For OA1, western immunoblotting was carried out as described . For GCN2, cells were lysed in RIPA buffer, boiled at 95°C for 5 min and resolved on a 7.5% polyacrylamide gel; immunoblotting was then performed following standard procedures. Primary Abs were as follows: anti-human OA1, previously developed by our group in rabbits ; anti-GCN2 (Cell Signaling, Cat. #3302).
Statistical analyses were performed using Microsoft Excel for Mac (version 15.32, Microsoft) for Student’s t-test; or GraphPad Prism (version 5.0d for Mac, GraphPad Software, Inc.) for one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons post-tests. T-test was used when only two means, typically sample versus control, were compared, as specified in the figure legends. One way ANOVA was used for multiple comparisons, followed by either a Dunnett’s (to compare every mean to a control mean), or a Tukey’s (to compare every mean with every other mean) post-test, by setting the significance level at 0.05 (95% confidence intervals). Both tests compare the difference between means to the amount of scatter, quantified using information from all the groups. Specifically, Prism computes the Tukey-Kramer test, allowing unequal sample sizes. P values in Figures are generally referred to comparison between a sample and the control (full medium/mock), and are indicated as follows: *P<0.05, **P<0.01, ***P<0.001. Comparisons not involving the control are similarly indicated, by a horizontal line at the top of the graphs, encompassing the two samples under analysis. Additional details regarding the specific experiments are reported in the Figure Legends.
To examine the expression behavior of genomic repeats upon AA starvation, we performed a transcriptomic analysis taking advantage of an intramural sequencing facility. HeLa-OA1 cells were cultured in normal medium (for 6-30-120 hours) or in absence of Met/Cys (for 6-15-30-72-120 hours). Total RNA was prepared using Trizol (Sigma) to preserve transcripts of both small and long sizes (from Alu, of about 0.3 kb, to Long Interspersed Nuclear Elements, LINEs, and ERVs, up to 6–8 kb long), DNase treated to avoid contamination of genomic DNA, and processed for NGS sequencing by Ovation RNA-Seq System V2 protocol and HiSeq 2000 apparatus. Raw sequence data (10–20 M reads/sample) were aligned to the human genome (build hg19) with SOAPSplice . Read counts over repeated regions, defined by RepeatMasker track from UCSC genome browser , were obtained using bedtools suite . Normalization factors and read dispersion (d) were estimated with edgeR , variation of abundance during time was analyzed using maSigPro package , fitting with a negative binomial distribution (Θ = 1/d, Q = 0.01), with a cutoff on stepwise regression fit r2 = 0.7. Read counts were transformed to RPKM for visualization purposes. The expression of the OA1 transgene and HDAC4, which are progressively up- and down-regulated during starvation, respectively , were used as internal controls.
For genomic repeat analysis, reads belonging to repetitive elements were classified according to RepeatMasker and assigned to repeat classes (total number in the genome = 21), families (total number in the genome = 56) and finally subfamilies (total number in the genome = 1396), each including a variable number of genomic loci (from a few hundred for endogenous retroviruses, up to several thousand for Alu). Repeat subfamilies were then clustered according to their expression pattern in starved vs control cells, by maSigPro using default parameters, and repeats classes or families that are significantly enriched in each cluster, compared to all genomic repeats, were identified by applying a Fisher Exact test (using scipy.stats, a statistical module of Python). Alternatively, differentially expressed repeat subfamilies were identified by averaging three time points of starvation (15-30-72 h) and controls. Repeats significantly up- or downregulated (104 and 77, respectively) were selected based on a P value <0.05 (unpaired two-tailed Student’s t-test, assuming equal variance), and analyzed for their class enrichment by a Fisher Exact test as described above.
For gene set enrichment analysis of Met/Cys deprived vs control HeLa cells, differentially expressed genes were selected considering three time points of starvation (15-30-72 h) and controls, based on a P value <0.05 (unpaired two-tailed Student’s t-test, assuming equal variance) and a fold change >2. This led to a total of 2033 differentially expressed genes, 996 upregulated and 1037 downregulated. The enrichment analysis was performed separately for up and down regulated genes, or with all differentially expressed genes together (both), using the KEGG database. The analysis was performed with correction for the background of all expressed genes (about 13600 genes showing an average expression over 3 starvation and 3 control samples of at least 5 counts) and by using default parameters (adjusted P value and q-value cut-off of <0.05 and 0.2, respectively). Differentially expressed genes were also selected considering all starvation time points, as with genomic repeats, by maSigPro using default parameters, and a fold change of at least 1.5, leading to similar enrichment results (not shown). RNAseq gene expression data are available in the ArrayExpress database under the accession number E-MTAB-6452.
To provide proof-of-principle that AA starvation may affect the expression of transposable elements, we performed an RNAseq analysis of the previously described HeLa-OA1 cells, carrying an integrated and partially silenced OA1 transgene . Since the reactivation of the transgene by starvation is a progressive phenomenon , we performed a time-course experiment, where each time point represents one biological sample, rather than a biological triplicate of a single time point. To this aim, cells were cultured either in normal medium, or in absence of Met/Cys for different time points (6-15-30-72-120 hours), resulting in the progressive upregulation of the OA1 transgene during starvation (Fig 1A and 1B), consistent with previously published results . The expression of genomic repeats was determined according to RepeatMasker annotation and classification into classes, families, and subfamilies. Repeat species were then subjected to differential expression and enrichment analyses in starved vs control conditions. Out of 1396 annotated repeat subfamilies, 172 species displayed a differential expression profile during starvation.
Fig 1. Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells.
(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium. Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs. control (full medium at 6 h = 1) (B). (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile. (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column). Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs. LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated. *P<0.05, ***P<0.001 (Fisher exact test).
As shown in Fig 1C, the clustering of differentially expressed repeats, according to their expression pattern, reveals profiles comparable to the behavior of the transgene in the same conditions, i.e. upregulation upon starvation and no change in regular medium (Cluster 1 and 2). In particular, Cluster 1 contains sequences that, similarly to the OA1 transgene, are progressively upregulated upon starvation (Fig 1A and 1C) , while Cluster 2 contains sequences that are upregulated at early time points. Interestingly, repeat families that are significantly enriched in these two clusters belong mostly to the group of LTR-retrotransposons, including ERV1, ERVK, ERVL, ERVL-MaLR and other LTR sequences (Fig 1D; S1A and S2A Figs). By contrast, DNA transposons (such as TcMar-Tigger) and L1 non-LTR retrotransposons are enriched among repeats that are downregulated during starvation, particularly at late time points (Clusters 3 and 4) (Fig 1D; S1A and S2A Figs). Consistent results were obtained by selecting significantly up- or downregulated genomic repeats (overall 181 species), based on their average expression out of three time points of starvation (15-30-72 h, when the transgene upregulation is more homogeneous) and controls, and on a P value <0.05 (S1B and S2B Figs). These findings suggest that EAA starvation induces genome-wide effects involving repetitive elements, and that—among major repeat classes—it upregulates in particular the expression of ERVs.
In addition, to obtain a general overview of main gene pathways changing their expression together with the transgene during AA starvation, we performed gene expression and enrichment analyses of regular genes, by considering three time points of starvation (15-30-72 h) and controls. Differentially expressed genes were selected based on a P value <0.05 and a fold change between means of at least 2, and analyzed with the EnrichR tool . As shown in Fig 2 and S1 File, enrichment analyses against the KEGG and Reactome databases reveals a predominance of downregulated pathways, namely ribosome and translation, proteasome, AA metabolism, oxidative phosphorylation and other pathways related to mitochondrial functions, which are affected in Huntington, Alzheimer and Parkinson diseases (http://www.genome.jp/kegg/pathway.html). In particular, a large fraction of ribosomal protein mRNAs is downregulated upon Met/Cys starvation (Fig 2A and 2C; S1 File), consistent with the notion that their genes–despite being scattered throughout the genome—are coordinately expressed in a variety of conditions . This reduced expression may depend on multiple pathways that control ribosome biogenesis in response to external stimuli, including the downregulation of Myc activity , the downregulation of mTORC1 [42, 44], or possibly the activation of the ISR, as described in yeast . By contrast, upregulated genes show a significant enrichment for transcription and gene expression (Fig 2B). Similar results were obtained by the Gene Ontology Biological Process (GO-BP) database (S1 File), overall indicating a general downregulation of translation and metabolism, and upregulation of transcription, during the time interval of Met/Cys starvation corresponding to the transgene upregulation.
Fig 2. Gene set enrichment analysis of Met/Cys-deprived HeLa cells.
Differentially expressed genes between three time points of starvation (15-30-72 h) and controls were selected based on a P value <0.05 and a fold change of at least 2, leading to a total of 996 upregulated, and 1037 downregulated genes. The enrichment analysis was performed separately for up and down regulated genes, using the EnrichR tool and the KEGG (A) and REACTOME (B, C) databases. Ranking is based on the combined score provided by EnrichR, and categories are displayed up to 20 items with an Adjusted P value <0.05. No significant categories were found with upregulated genes against the KEGG database. All data are shown in S1 File. The enrichment analysis using all differentially expressed genes together did not reveal any additional enriched process.
To characterize the pathway leading to the reactivation of silenced transgenes, we used HeLa-OA1 and HeLa-GFP cells, as described . In addition, to test cell types relevant for AA metabolism, such as liver and muscle, we generated clones of HepG2 human hepatoma and C2C12 mouse skeletal muscle cells, stably transfected with plasmids for OA1 and GFP transgenes, respectively (HepG2-OA1 and C2C12-GFP cells; endogenous OA1 is not expressed in any of these cell types). In all cases, the integrated transgenes are under the control of the CMV promoter in the context of a pcDNA3.1 plasmid, are partially silenced, and can be efficiently upregulated by HDAC inhibitors (trichostatin A, TSA; ref.  and S3A, S3B and S4A Figs), indicating that their expression is controlled at least in part by epigenetic mechanisms, as previously described .
To establish whether the reactivation response results from the shortage of specific AAs only, such as Met/Cys, or it is triggered by any AA deprivations, we cultured HeLa-OA1, HeLa-GFP, HepG2-OA1 and C2C12-GFP cells for 24–48 hours with a battery of media deprived of EAAs or semi-EAAs, including Met/Cys, Thr, Gln, Val, Leu, Tyr, Trp, Lys, and His. As negative controls, cells were cultured in full medium, carrying the entire AA complement, and in a medium deprived of Ala, a non-essential AA. The expression of the transgene transcript was then evaluated by RT-qPCR. As shown in Fig 3, and in S3C and S4B Figs, most EAA-deficiencies induced reactivation of the OA1 or GFP transgenes in all four cell lines, with the notable exception of Trp deprivation, which consistently resulted in no or minimal reactivation of the transgenes. Indeed, despite some variability, Met/Cys deficiency, but also Thr, Val, Tyr, and His deprivation always gave an efficient response, while Leu, Gln and Lys elicited evident responses in some cases, but not in others. Depletion of Phe gave results comparable to Tyr deprivation, however it significantly altered multiple reference genes used for normalization and therefore was eventually omitted from the analysis (not shown). Finally, in the above experiments we used a combined Met/Cys deficiency, to avoid the potential sparing of Met by Cys  and for consistency with our previous studies . Nevertheless, the analysis of single Met or Cys starvation, both at the protein and transcript levels, revealed an exclusive role of Met deprivation in transgene reactivation, consistent with the notion that Cys is not an EAA (S3D and S3E Fig).
Fig 3. EAA deprivation induces reactivation of silent transgenes in HeLa and HepG2 cells.
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in various AA-deprived media for 48 h and 24 h, respectively, compared to full medium. Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. control (full medium = 1). *P<0.05, **P<0.01, ***P<0.001 (one way ANOVA, followed by Dunnett’s post-test vs. full medium).
Collectively, these results indicate that transgene reactivation by EAA starvation is reproducible with most EAAs, shared by different cell types (epithelium, liver, and skeletal muscle), and conserved in different mammalian species (human, mouse).
mTORC1 inhibition and GCN2 activation trigger the best-known signaling pathways responding to AA starvation . We previously showed that inhibition of mTORC1 is not sufficient to reproduce transgene reactivation in HeLa cells . By contrast, the involvement of GCN2 and the ISR, including the downstream effectors ATF4 and CHOP, has never been tested. In addition, this pathway has been typically assessed in transient assays, lasting for a few hours, which may not be comparable with the prolonged starvation conditions necessary to reactivate the transgene expression (at least 15–24 h). Thus, we tested whether CHOP expression was upregulated upon incubation of HeLa-OA1, HepG2-OA1 and C2C12-GFP cells in media deprived of different EAAs for 24–48 h.
As shown in Fig 3 and S4B Fig, we found that CHOP expression is increased in all EAA-starvation conditions, but not in the absence of Ala, in all tested cell lines. Similar, yet less pronounced, results were obtained with ATF4, consistent with the notion that activation of this transcription factor is mainly mediated by translational upregulation (not shown) [15, 26]. However, the upregulation of CHOP does not parallel quantitatively that of the transgene, neither appears sufficient to induce it. In fact, CHOP is highly upregulated even upon Trp starvation, which consistently results in no or minimal reactivation of the transgenes (compare CHOP with OA1 or GFP expression; Fig 3 and S4B Fig). Thus, while the ISR appears widely activated upon EAA starvation, the upregulation of its downstream effector CHOP only partly correlates with transgene reactivation and may not be sufficient to induce it.
The activation of the ISR upon AA starvation suggests that GCN2 may be involved in the transgene reactivation response. Therefore, we tested whether direct pharmacological activation of this kinase is sufficient to trigger the transgene reactivation similarly to starvation. In addition, we used pharmacological inhibitors of mTOR to corroborate previous negative results in HeLa cells  in the other cell lines under study. To this aim, HeLa-OA1 or GFP, HepG2-OA1 and C2C12-GFP cells were cultured in the presence of different concentrations of PP242 (mTOR inhibitor) or L-Histidinol (GCN2 activator, inhibiting tRNAHis charging by histidyl-tRNA synthetase), either alone or in combination for 24 h, compared to Met/Cys-deprived and full medium. As shown in Fig 4 and S5 Fig, while inhibition of mTORC1 consistently leads to minor or no effects, in agreement with previous findings , treatment with L-Histidinol results in efficient reactivation of the transgene in HepG2-OA1 and C2C12-GFP cells, but not in HeLa cells.
Fig 4. mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells.
Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium. Mean ± SEM of 4 (A) or 3 (B) independent experiments. Data are expressed as fold change vs. control (full medium = 1). *P<0.05, **P<0.01, ***P<0.001 (one way ANOVA, followed by Dunnett’s post-test vs. full medium). PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively.
Specifically, L-Histidinol is not effective in HeLa-OA1 and HeLa-GFP cells, either alone or in combination with PP242 (Fig 4A and S5A Fig), or by using different concentrations of the drug, with or without serum (not shown). In these cells, L-Histidinol appears also unable to trigger the ISR, as indicated by lack of CHOP upregulation, possibly due to their different sensitivity to the drug. These findings are consistent with previous reports, describing the use of L-Histidinol in HeLa cells in conditions of low His concentration in the culture medium , which would resemble AA starvation in our system and therefore may not be applicable. Thus, even though the amount of the amino alcohol was adapted to exceed 20 to 80 times that of the amino acid, as described , HeLa cells may be resistant or able to compensate.
In contrast, in other cell types, L-Histidinol has been utilized in regular DMEM, to mimic the AA response triggered by DMEM lacking His [48, 49]. Consistently, in HepG2-OA1 cells, L-Histidinol is sufficient to elicit extremely high levels of transgene reactivation, and its combination with PP242 results in additive or even synergistic effects, possibly due to an indirect effect of mTOR inhibition on GCN2 activity (Fig 4B) [50, 51]. Similarly, C2C12-GFP cells efficiently reactivate the transgene upon treatment with L-Histidinol, but not PP242 (S5B Fig). However, differently from HepG2-OA1 cells, simultaneous treatment of C2C12-GFP cells with L-Histidinol and PP242 does not lead to synergistic effects. Consistent with stimulation of the ISR, CHOP and to a minor extent ATF4 are upregulated by L-Histidinol in both cell lines, yet their expression levels show only an incomplete correlation with those of the transgene (Fig 4B, S5B Fig, and not shown).
The finding that GCN2 activation by L-Histidinol is sufficient to reactivate the transgenes in both HepG2-OA1 and C2C12-GFP cells pointed to this kinase, and to the downstream ISR, as the pathway possibly involved in the EAA starvation response. Thus, we investigated whether the ISR is sufficient to trigger upregulation of the OA1 transgene in HepG2-OA1 cells by pharmacological means. As CHOP expression does not correspond quantitatively and is not sufficient to induce transgene reactivation, we tested the role of the core upstream event of the ISR, namely the phosphorylation of eIF2α , which can be induced by pharmacological treatments, independent of GCN2 (Fig 5A). To this aim, we used Salubrinal, a specific phosphatase inhibitor that blocks both constitutive and ER stress-induced phosphatase complexes against eIF2α, thereby increasing its phosphorylation . We found that, while the ISR is activated upon Salubrinal treatment, as shown by increased CHOP expression, it does not induce OA1 transgene reactivation (Fig 5B).
Fig 5. The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells.
(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR. In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture). (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium. Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). *P<0.05 (paired two-tailed Student’s t-test vs. control). (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM). Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). **P<0.01, ***P<0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h. Mean ± range of two experiments. Data are expressed as fold change vs. control (w/o HisOH = 1, top; control siRNA = 1, bottom). *P<0.05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated).
To test whether the ISR is necessary to trigger the transgene response to L-Histidinol, we used the chemical compound ISRIB, which inhibits the activation of the ISR, even in the presence of phosphorylated eIF2α, likely by boosting the activity of the guanine-nucleotide exchange factor (GEF) for eIF2α, namely eIF2B [53, 54]. HepG2-OA1 cells were stimulated with L-Histidinol, either in the presence or absence of ISRIB. As shown in Fig 5C, while the expression of CHOP is inhibited by ISRIB, as expected, the reactivation of the OA1 transgene is not affected. In addition, knockdown of the closest eIF2α downstream effector ATF4 by siRNAs does not interfere with the reactivation of the OA1 transgene by L-Histidinol (Fig 5D). Together, these data suggest that eIF2α phosphorylation and the downstream ISR pathway are neither sufficient nor necessary to induce transgene reactivation.
To definitively establish if GCN2 is necessary to trigger the transgene reactivation response to EAA starvation, we directly suppressed its expression by CRISPR/Cas9-mediated knock-out (KO). We generated three independent GCN2-KO clones from the parental HeLa-OA1 cell line, by using three different guide RNAs, two against exon 1 (clones 183#11 and 185#5), and one against exon 6 (clone 239#1) of the GCN2 gene. Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone 183#11, and on both alleles of exon 6 in clone 239#1; by contrast, clone 185#5 showed multiple alleles in exon 1, consistent with the presence of two cell populations, and was not characterized further at the genomic level (S6 Fig). None of these clones express GCN2 at the protein level, as shown by immunoblotting (Fig 6A). To test the GCN2-KO cells for their ability to respond to EAA starvation, parental HeLa-OA1 cells and the three GCN2-KO clones were cultured in media deprived of Met/Cys or Thr (corresponding to the most effective treatments in this cell line; see Fig 3A) for 24–48 h and transgene expression was assessed by RT-qPCR. We found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, thus excluding that this kinase is necessary for the response to EAA starvation in HeLa-OA1 cells (Fig 6B and 6C).
Fig 6. GCN2 knockout does not interfere with transgene reactivation in HeLa cells.
(A) Immunoblotting of protein extracts from the HeLa-OA1 parental cell line and GCN2-KO clones 183#11, 185#5 and 239#1, immunodecorated with anti-GCN2 antibody. Arrow, GCN2 specific band. Ponceau staining was used as loading control. (B, C) Relative transgene (OA1) mRNA abundance in HeLa-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or Thr (C) deprived medium for 24 h or 48 h, respectively, compared to full medium. Mean ± SD of 3 technical replicates from 1 experiment. Data are expressed as fold change vs. control (full medium = 1). Since independent clones may display variable reactivation responses (e.g. due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates.
Similarly, we generated GCN2-KO clones from the parental HepG2-OA1 cell line by the same strategy. By using a guide RNA against exon 1 of the GCN2 gene, we obtained three independent GCN2-KO clones, namely E23, F22 and F27. Genomic characterization confirmed the presence of mutations on both alleles of exon 1 of the GCN2 gene in clone F27 (S7 Fig) and all three clones showed a very low amount—if any—of residual GCN2 protein, compared to the original HepG2-OA1 cell line (Fig 7A). To assess the ability of GCN2-KO cells to reactivate the transgene upon starvation, we cultured parental HepG2-OA1 cells and the three GCN2-KO clones in media deprived of Met/Cys or His (corresponding to the most effective treatments in this cell line; see Fig 3B) for 24 h, and evaluated the transgene expression by RT-qPCR. As shown in Fig 7B and 7C, we found that the reactivation of the OA1 transgene is neither abolished, nor reduced by KO of GCN2, as in HeLa cells. To further confirm this result, we knocked-down GCN2 by RNA interference (RNAi), and incubated the cells with or without L-Histidinol for 6 h. As shown in Fig 8, treatment of HepG2-OA1 cells with L-Histidinol results in efficient transgene reactivation, even upon significant GCN2 downregulation, both at the mRNA and protein levels. Taken together, these data strongly support the conclusion that GCN2 is not necessary for transgene reactivation in response to EAA starvation, either in HeLa or in HepG2 cells.
Fig 7. GCN2 knockout does not interfere with transgene reactivation in HepG2 cells.
(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody. Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27. Arrow, GCN2 specific band. For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs. parental cell line (= 1). (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium. Mean ± SD of 3 technical replicates from 1 experiment.

Answer: No, it is not necessary.